10,000 Matching Annotations
  1. Sep 2023
    1. Determiner (D) – Also known as determinative. Goes with a noun and specifies something about that noun (but doesn’t quite describe it the way an adjective does.) Articles are one type of determiners (a, the, an) but demonstratives (this cat, these shoes) that go with nouns, possessive ‘pronouns’ like my, your, her (with nouns), possessive nouns like ‘Mike’s’, quantifiers with nouns (many, most, some), numerals with nouns (one cat, seventeen cats, zero cats) and the question word which with a noun are all also determiners. Determiners are always part of noun phrases and come before any adjectives describing the head noun. Examples of determiners: a, the, seventeen, my, her, many, all, most, no (in we have no bananas), John’s

      So what i'm understanding per the example words in this paragraph is that determiners always and only go before a noun.

    1. 'Have pity on your father's grey head; have pity on your infant son. Offerthe sacrifice for the welfare of the emperors.

      this takes a lot of strength to object to

    2. We drew close to one another and began to let our fists fly. My opponent tried to get hold of my feet, but Ikept striking him in the face with the heels of my feet. Then I was raised up into the air and I began topummel him without as it were touching the ground. Then when I noticed there was a lull, I put my twohands together linking the fingers of one hand with those of the other and thus I got hold of his head. He fellflat on his face and I stepped on his head

      She won against the Egyptian

    1. By reminding readers of the ideas you’re responding to, return sentencesensure that your text maintains a sense of mission and urgency from start tofinish.

      Sense of meeting when repeating claims. Also gets stuck in the reader's head.

    1. Ichabod’s flimsy garments fluttered in the air, as he stretched his long lank body away over his horse’s head, in the eagerness of his flight.

      Flutters in the wind as he leans forward over his horse's head, desperate to escape the pursuing spectre.

    1. On the federal level, children’s education in their earliest years is the province of the Department of Health and Human Services, which runs Head Start and other early-childhood programs through its Administration for Children and Families

      I did not know this was happening.

    1. n 2016, the Intensive Care Society proposed a bundle called “Recommended bundleof Interventions for the prevention of VAP”, including elevation of head of bed, daily seda-tion vacation and assessment of readiness to extubate, use of subglottic secretion drainage,avoidance of scheduled ventilator circuit changes, oral hygiene without chlorhexidine andPUD prophylaxis (only for high-risk patients), without mentioning DVT prophylaxis.

      By performing these interventions, we can help decrease the patient's risk for developing VAP. This can also lead to decreased lengths of mechanical ventilation. Elevating the head of the bed is crucial for preventing respiratory distress as this provides optimal positioning for lung expansion which works these muscles as prevents further medical problems. - GL, MI, ES

    1. It's not going to help this, but-- because it looks like it gets smaller as it goes down. But they are in fact the same size. They're the same size. The illusion works. And I know they're the same size because my assistant did it for me.

      I thought this was a very good example to show what bias is that it's almost like an illusion you create in your head you think is real when that is not the case.

    2. Names are actually an important part of our identities

      Our name is the first thing we learn that is associated with something. The association is us, your name is apart of you from the moment you can understand any part of language. Whenever you hear your name called you automatically respond with turning your head and listening, our names are very much apart of our identity.

    3. We've never experienced him looking like this, and so we don't expect it. We create in effect, a self-fulfilling prophecy.

      I believe that this is a powerful statement as we have expectations about everything in life. From people to places and to relationships. As society, not many of us are willing to go in blind to anything. Fear is the unknown and many of us do not understand that. We expect things to go as planned in our head. When we meet someone certain attributes may be different but people are relatively the same. As we have experience meeting people and certain expectations. It is hard to change our thought pattern about this.

    1. “Why would you come in here, sit for an hour, and not eat?

      It is surprising that the man came into Chuck's Donuts and did not eat any of the apple fritter. It is very intriguing and makes the reader contemplate what could be stirring inside of his head. This scene portrays Kayley's obervant and curious nature. She pays very close attention to the customer's every move and small detail. Does she analyze each person who comes into her father's store? As a side-note, I also wonder why they are awake so early as well. It is interesting to see the differences between Kayley and Tevy's interests, and it does make sense given that they are four years apart. The reader can observe that Tevy is much less hyperfocused on the shop and their guests, and even shoots down many of her sister's comments because she is consumed by her studies. The differences in characteristics exagerrates the age gap between the two sisters. Perhaps Kayley creates her own amusement out of understanding the customers because her sister is very preoccupied with her workload.

    1. Because the United States isn’t the king or general of the status quo coalition, it’s the ‘team captain.’ If it proves to be a bad team captain, the team may well choose a new captain, or disband altogether, with catastrophic implications for American interests.

      I can't imagine another 'team captain' suddenly forthcoming. If Britain got its head out of its ass and regained that imperial edge, then maybe, or if China suddenly underwent a democratic revolution and reentered the world stage as a liberal power.

    1. Hoekstra, a Shell man and a McKinsey man in charge of EU climate policy?
      • for: climate change policy hypocrisy, fossil fuel lobby, EU climate policy, Hoekstra, Fox guarding the henhouse, Linked In post
      • comment

        • What does it say about the EU's authenticity to deal with the global boiling crisis when they put a fox in charge of the henhouse?
        • this seems awfully similar to the choice for positioning an oil man to head COP28.
        • The fossil fuel lobby is EXTREMELY busy in the opaque back end of politics. We need more light to shine and bring the back end fossil fuel lobby activity out of the shadows to pre-empt future leadership betrayals.
      • future research

        • uncover future fossil fuel lobby’s game plan and future attempts to coopt climate change policy leadership
        • needed in order to proactively preempt the next attempt at coopting climate leadership. It’s difficult when we are simply reacting
    1. Reviewer #3 (Public Review):

      The transition from planktonic to benthic depends upon several physical and chemical cues. Nitric oxide (NO) is known as a critical player in the induction of larval metamorphosis in several invertebrates. Although NO is a widespread signalling molecule in a broad range of organisms regulating key physiological processes, internal regulatory mechanisms studies are scarce. While the UV sensing in larvae of the annelid Platynereis dumerilii using ciliary photoreceptors has been studied, the neuronal signalling mechanism remains unknown. In this study, Kei Jokura et al. investigated how annelid Platynereis dumerilii larvae detect UV sensing and modulate swimming behaviour through nitric oxide feedback. Using existing resources of Platynereis larval connectome/volume EM data, they identified NOS-expressing interneurons within the ciliary photoreceptors circuit (cPRCs). They demonstrated that NO is produced in cPRCs during UV/violet stimulation by using a fluorescent NO-reporter line. Further, they demonstrated that Nitric oxide signalling mediates UV-avoidance behaviour by using NOS-mutant larvae. Finally, they mapped out the signalled mechanisms of the cPRC circuit using published spatially mapped single-cell transcriptome data of Platynereis larvae, the Ca sensor lines, in situ HCR, and immunostaining. Additionally, by using their findings from Ca imagining data of cPRC, INNOS and INRGWa cells collected in wild-type, NOS knockout and NIT-GC2 morphant larvae, Kei Jokura et al. developed a mixed cellular-circuit-level mathematical model. However, my expertise in mathematical modelling is limited, so I cannot comment on this section.

      No doubt, the study has been conducted extensively. However, I have a few comments, please see below.

      Page 4: "In contrast, both two- and three-day-old homozygous NOS-mutant larvae showed a strongly diminished UV avoidance response (Figure 3A, B and Figure 3-figure supplement 1B, C)." Instead of using subjective terms like "strongly," it would be more relevant to provide statistical values. However, I could not locate any means of statistical analysis on larval behaviour. Can the authors indicate the statistical values for all behaviour studies?

      Page 5: "(D) Vertical displacement in 30 sec bins of wild type and mutant (NOSΔ11/Δ11 and NOSΔ23/Δ23) three-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top." The error bars for WT are too long at the end of the experiment. It is not clear how the authors decided to use this time frame. Did the authors try carrying this out for an extended time period? How did the authors decide on 120 seconds as the time frame for exposure? Authors should provide data on larval behaviour for an extended time.

      Page 13: "During the UV response, prototroch cilia beat slower than trunk cilia, resulting in a head-down stable state ('rear-wheel drive'). In contrast, during the pressure response prototroch cilia beat faster than trunk cilia, leading to a head-up orientation ('front-wheel drive'). Testing this hypothesis will require biophysical experiments and mathematical modelling." Authors should carry out ciliary beating analysis under UV light in the current study with NOS mutant larvae. Since the pressure and UV detection systems are closely related, comparing the difference in ciliary beating is important to demonstrate this hypothesis. Further, did the authors check the Ca sensor GCaMP6s under pressure conditions?

      Page 18: "strips. One strip contained UV (395 nm) LEDs (SMB1W-395, Roithner Lasertechnik) and the other infrared (810 nm) LEDs (SMB1W-810NR-I, Roithner Lasertechnik)." Authors should test larval swimming behaviour at different wavelengths. Even though they are performed in previous work, the experiment with different wavelengths is necessary to be conducted in NOS mutant larvae in parallel with a control. This will confirm that NOS is principally associated with UV. Further, to demonstrate that this mechanism is associated with ciliary movement, authors need to provide this evidence.

  2. inst-fs-iad-prod.inscloudgate.net inst-fs-iad-prod.inscloudgate.net
    1. Talent renders the whole idea of rehearsal meaningless; when you findsomething at which you are talented, you do it (whatever it is) until yourfingers bleed or your eyes are ready to fall out of your head. Even when noone is listening (or reading, or watching), every outing is a bravuraperformance, because you as the creator are happy. Perhaps even ecstatic.That goes for reading and writing as well as for playing a musicalinstrument, hitting a baseball, or running the four-forty. The sort ofstrenuous reading and writing program I advocate — four to six hours aday, every day — will not seem strenuous if you really enjoy doing thesethings and have an aptitude for them; in fact, you may be following such aprogram already. If you feel you need permission to do all the reading andwriting your little heart desires, however, consider it hereby granted byyours truly
      1. Vivid detail. 2.The writer make me think about where am I investing my time. It does no matter how much time you spend doing what you love. For example: hobbies, own business, reading, writing ,sports, etc. You are not going to feel tired if you enjoy the what you do.
      2. The writer did effectively incorporated the vivid detail in the essay because when he wrote the story about his son Owen wants to learn how to play the saxophone, the details bring a picture of the story to my mind.
    2. left the Champion office and sat in a hot taxi and looked at Lagosinching past, the hawkers pressing sunglasses against the window, the busesspitting out thick gray smoke, the cars stuck bumper to bumper in traffic.“See this stupid man! He wan scratch me!” my taxi driver said, gesturingto the car beside us. Then he stuck his head out and cursed in rapid Yoruba.

      In this excerpt, the author uses vivid detail and dialogue as textual evidence to demonstrate elements of personal narrative. As the reader, I was able to imagine the sights and sounds the author experienced and it made me reflect on experiences I've had in life that were similar. I pictured myself sitting in my car on the busy streets of NYC during gridlock on a hot summer day feeling FRUSTRATED alongside many other frustrated drivers or in the back seat of the black and yellow taxi coming out of the airport and into the hustling metropolis of a very smoggy Santiago, Chile. The writer effectively incorporates these elements throughout the essay in an engaging well-structured manner therefore allowing the reader to envision and connect to the writers' words as they read along. I try to use these elements in my own work and writing because relatability and descriptive passages are highly effective ways to keep a reader captivated.

    3. I might have let out a sound, Imight have only shivered, but Kate noticed and asked if something waswrong.I pointed. “I knew him,” I said.Kate shook her head. “Oh, sorry, sorry. It was an operation at the bankjust across the road,” she said.I remember the splashes of blood on Nnamdi’s face, his head slumpedagainst the seat of the car; the blood was a deep gray in the black-and-whitephoto.
      1. Remembering how infatuated she was with him, took her down memory lane. The evidence from a personal narrative and how she vividly recalls her childhood sweetheart.
      2. This text allows the reader to see far beyond imagination, with different scenes and settings for the readers imagination. The relationship they had with one another; you can tell they were infatuated with each other. 3.While reading this I connected with the characters and understood the writer's point of view of a childhood love & an adult's heartbreak. 4.The writer used great dialogue which inserted me into the story. I could feel the love and passion she had for him. It also made me reflect on my childhood sweetheart.
    1. Vannevar Bush, "As We May Think," Atlantic Month1y, (July 1945).

      As We May Think

      From The Atlantic Monthly, July 1945: 101-108. Reprinted with permission. (c)1945, V. Bush.

      As Director of the Office of Scientific Research and Development, Dr. Vannevar Bush has coördinated the activities of some six thousand leading American scientists in the application of science to warfare. In this significant article he holds up an incentive for scientists when the fighting has ceased. He urges that men of science should then turn to the massive task of making more accessible our bewildering store of knowledge. For many years inventions have extended man's physical powers rather than the powers of his mind. Trip hammers that multiply the fists, microscopes that sharpen the eye, and engines of destruction and detection are new results, but the end results, of modern science. Now, says Dr. Bush, instruments are at hand which, if properly developed, will give man access to and command over the inherited knowledge of the ages. The perfection of these pacific instruments should be the first objective of our scientists as they emerge from their war work. Like Emerson's famous address of 1837 on "The American Scholar," this paper by Dr. Bush calls for a new relationship between thinking man and the sum of our knowledge. - The Editor

      This has not been a scientist's war; it has been a war in which all have had a part. The scientists, burying their old professional competition in the demand of a common cause, have shared greatly and learned much. It has been exhilarating to work in effective partnership. Now, for many, this appears to be approaching an end. What are the scientists to do next?

      For the biologists, and particularly for the medical scientists, there can be little indecision, for their war work has hardly required them to leave the old paths. Many indeed have been able to carry on their war research in their familiar peacetime laboratories. Their objectives remain much the same.

      It is the physicists who have been thrown most violently off stride, who have left academic pursuits for the making of strange destructive gadgets, who have had to devise new methods for their unanticipated assignments. They have done their part on the devices that made it possible to turn back the enemy. They have worked in combined effort with the physicists of our allies. They have felt within themselves the stir of achievement. They have been part of a great team. Now, as peace approaches, one asks where they will find objectives worthy of their best.

      I

      Of what lasting benefit has been man's use of science and of the new instruments which his research brought into existence? First, they have increased his control of his material environment. They have improved his food, his clothing, his shelter; they have increased his security and released him partly from the bondage of bare existence. They have given him increased knowledge of his own biological processes so that he has had a progressive freedom from disease and an increased span of life. They are illuminating the interactions of his physiological and psychological functions, giving the promise of an improved mental health.

      Science has provided the swiftest communication between individuals; it has provided a record of ideas and has enabled man to manipulate and to make extracts from that record so that knowledge evolves and endures throughout the life of a race rather than that of an individual.

      There is a growing mountain of research. But there is increased evidence that we are being bogged down today as specialization extends. The investigator is staggered by the findings and conclusions of thousands of other workers--conclusions which he cannot find time to grasp, much less to remember, as they appear. Yet specialization becomes increasingly necessary for progress, and the effort to bridge between disciplines is correspondingly superficial.

      Professionally our methods of transmitting and reviewing the results of research are generations old and by now are totally inadequate for their purpose. If the aggregate time spent in writing scholarly works and in reading them could be evaluated, the ratio between these amounts of time might well be startling. Those who conscientiously attempt to keep abreast of current thought, even in restricted fields, by close and continuous reading might well shy away from an examination calculated to show how much of the previous month's efforts could be produced on call. Mendel's concept of the laws of genetics was lost to the world for a generation because his publication did not reach the few who were capable of grasping and extending it; and this sort of catastrophe is undoubtedly being repeated all about us, as truly significant attainments become lost in the mass of the inconsequential.

      The difficulty seems to be, not so much that we publish unduly in view of the extent and variety of present-day interests, but rather that publication has been extended far beyond our present ability to make real use of the record. The summation of human experience is being expanded at a prodigious rate, and the means we use for threading through the consequent maze to the momentarily important item is the same as was used in the days of square-rigged ships.

      But there are signs of a change as new and powerful instrumentalities come into use. Photocells capable of seeing things in a physical sense, advanced photography which can record what is seen or even what is not, thermionic tubes capable of controlling potent forces under the guidance of less power than a mosquito uses to vibrate his wings, cathode ray tubes rendering visible an occurrence so brief that by comparison a microsecond is a long time, relay combinations which will carry out involved sequences of movements more reliably than any human operator and thousands of times as fast-- there are plenty of mechanical aids with which to effect a transformation in scientific records.

      Two centuries ago Leibnitz invented a calculating machine which embodied most of the essential features of recent keyboard devices, but it could not then come into use. The economics of the situation were against it: the labor involved in constructing it, before the days of mass production, exceeded the labor to be saved by its use, since all it could accomplish could be duplicated by sufficient use of pencil and paper. Moreover, it would have been subject to frequent breakdown, so that it could not have been depended upon; for at that time and long after, complexity and unreliability were synonymous.

      Babbage, even with remarkably generous support for his time, could not produce his great arithmetical machine. His idea was sound enough, but construction and maintenance costs were then too heavy. Had a Pharaoh been given detailed and explicit designs of an automobile, and had he understood them completely, it would have taxed the resources of his kingdom to have fashioned the thousands of parts for a single car, and that car would have broken down on the first trip to Giza.

      Machines with interchangeable parts can now be constructed with great economy of effort. In spite of much complexity, they perform reliably. Witness the humble typewriter, or the movie camera, or the automobile. Electrical contacts have ceased to stick when thoroughly understood. Note the automatic telephone exchange, which has hundreds of thousands of such contacts, and yet is reliable. A spider web of metal, sealed in a thin glass container, a wire heated to brilliant glow, in short, the thermionic tube of radio sets, is made by the hundred million, tossed about in packages, plugged into sockets--and it works! Its gossamer parts, the precise location and alignment involved in its construction, would have occupied a master craftsman of the guild for months; now it is built for thirty cents. The world has arrived at an age of cheap complex devices of great reliability; and something is bound to come of it.

      II

      A record, if it is to be useful to science, must be continuously extended, it must be stored, and above all it must be consulted. Today we make the record conventionally by writing and photography, followed by printing; but we also record on film, on wax disks, and on magnetic wires. Even if utterly new recording procedures do not appear, these present ones are certainly in the process of modification and extension.

      Certainly progress in photography is not going to stop. Faster material and lenses, more automatic cameras, finer-grained sensitive compounds to allow an extension of the minicamera idea, are all imminent. Let us project this trend ahead to a logical, if not inevitable, outcome. The camera hound of the future wears on his forehead a lump a little larger than a walnut. It takes pictures 3 millimeters square, later to be projected or enlarged, which after all involves only a factor of 10 beyond present practice. The lens is of universal focus, down to any distance accommodated by the unaided eye, simply because it is of short focal length. There is a built-in photocell on the walnut such as we now have on at least one camera, which automatically adjusts exposure for a wide range of illumination. There is film in the walnut for a hundred exposure, and the spring for operating its shutter and shifting its film is wound once for all when the film clip is inserted. It produces its result in full color. It may well be stereoscopic, and record with spaced glass eyes, for striking improvements in stereoscopic technique are just around the corner.

      The cord which trips its shutter may reach down a man's sleeve within easy reach of his fingers. A quick squeeze, and the picture is taken. On a pair of ordinary glasses is a square of fine lines near the top of one lens, where it is out of the way of ordinary vision. When an object appears in that square, it is lined up for its j picture. As the scientist of the future moves about the laboratory or the field, every time he looks at something worthy of the record, he trips the shutter and in it goes, without even an audible click. Is this all fantastic? The only fantastic thing about it is the idea of making as many pictures as would result from its use.

      Will there be dry photography? It is already here in two forms. When Brady made his Civil War pictures, the plate had to be wet at the time of exposure. Now it has to be wet during development instead. In the future perhaps it need not be wetted at all. There have long been films impregnated with diazo dyes which form a picture without development, so that it is already there as soon as the camera has been operated. An exposure to ammonia gas destroys the unexposed dye, and the picture can then be taken out into the light and examined. The process is now slow, but someone may speed it up, and it has no grain difficulties such as now keep photographic researchers busy. Often it would be advantageous to be able to snap the camera and to look at the picture immediately.

      Another process now in use is also slow, and more or less clumsy. For fifty years impregnated papers have been used which turn dark at every point where an electrical contact touches them, by reason of the chemical change thus produced in an iodine compound included in the paper. They have been used to make records, for a pointer moving across them can leave a trail behind. If the electrical potential on the pointer is varied as it moves, the line becomes light or dark in accordance with the potential.

      This scheme is now used in facsimile transmission. The pointer draws a set of closely spaced lines across the paper one after another. As it moves, its potential is varied in accordance with a varying current received over wires from a distant station, where these variations are produced by a photocell which is similarly scanning a picture. At every instant the darkness of the line being drawn is made equal to the darkness of the point on the picture being observed by the photocell. Thus, when the whole picture has been covered, a replica appears at the receiving end.

      A scene itself can be just as well looked over line by line by the photocell in this way as can a photograph of the scene. This whole apparatus constitutes a camera, with the added feature, which can be dispensed with if desired, of making its picture at a distance. It is slow, and the picture is poor in detail. Still, it does give another process of dry photography, in which the picture is finished as soon as it is taken.

      It would be a brave man who would predict that such a process will always remain clumsy, slow, and faulty in detail. Television equipment today transmits sixteen reasonably good pictures a second, and it involves only two essential differences from the process described above. For one, the record is made by a moving beam of electrons rather than a moving pointer, for the reason that an electron beam can sweep across the picture very rapidly indeed. The other difference involves merely the use of a screen which glows momentarily when the electrons hit, rather than a chemically treated paper or film which is permanently altered. This speed is necessary in television, for motion pictures rather than stills are the object.

      Use chemically treated film in place of the glowing screen, allow the apparatus to transmit one picture only rather than a succession, and a rapid camera for dry photography results. The treated film needs to be far faster in action than present examples, but it probably could be. More serious is the objection that this scheme would involve putting the film inside a vacuum chamber, for electron beams behave normally only in such a rarefied environment. This difficulty could be avoided by allowing the electron beam to play on one side of a partition, and by pressing the film against the other side, if this partition were such as to allow the electrons to go through perpendicular to its surface, and to prevent them from spreading out sideways. Such partitions, in crude form, could certainly be constructed, and they will hardly hold up the general development.

      Like dry photography, microphotography still has a long way to go. The basic scheme of reducing the size of the record, and examining it by projection rather than directly, has possibilities too great to be ignored. The combination of optical projection and photographic reduction is already producing some results in microfilm for scholarly purposes, and the potentialities are highly suggestive. Today, with microfilm, reductions by a linear factor of 20 can be employed and still produce full clarity when the material is re-enlarged for examination. The limits are set by the graininess of the film, the excellence of the optical system, and the efficiency of the light sources employed. All of these are rapidly improving .

      Assume a linear ratio of 100 for future use. Consider film of the same thickness as paper, although thinner film will certainly be usable. Even under these conditions there would be a total factor of 10,000 between the bulk of the ordinary record on books, and its microfilm replica. The Encyclopedia Britannica could be reduced to the volume of a matchbox. A library of a million volumes could be compressed into one end of a desk. If the human race has produced since the invention of movable type a total record, in the form of magazines, newspapers, books, tracts, advertising blurbs, correspondence, having a volume corresponding to a billion books, the whole affair, assembled and compressed, could be lugged off in a moving van. Mere compression, of course, is not enough; one needs not only to make and store a record but also be able to consult it, and this aspect of the matter comes later. Even the modern great library is not generally consulted; it is nibbled at by a few.

      Compression is important, however, when it comes to costs. The material for the microfilm Britannica would cost a nickel, and it could be mailed anywhere for a cent. What would it cost to print a million copies? To print a sheet of newspaper, in a large edition, costs a small fraction of a cent. The entire material of the Britannica in reduced microfilm form would go on a sheet eight and one-half by eleven inches. Once it is available, with the photographic reproduction methods of the future, duplicates in large quantities could probably be turned out for a cent apiece beyond the cost of materials. The preparation of the original copy? That introduces the next aspect of the subject.

      III

      To make the record, we now push a pencil or tap a typewriter. Then comes the process of digestion and correction, followed by an intricate process of typesetting, printing, and distribution. To consider the first stage of the procedure, will the author of the future cease writing by hand or typewriter and talk directly to the record? He does so indirectly, by talking to a stenographer or a wax cylinder; but the elements are all present if he wishes to have his talk directly produce a typed record. All he needs to do is to take advantage of existing mechanisms and to alter his language .

      At a recent World Fair a machine called a Voder was shown. A girl stroked its keys and it emitted recognizable speech. No human vocal chords entered into the procedure at any point; the keys simply combined some electrically produced vibrations and passed these on to a loudspeaker. In the Bell Laboratories there is the converse of this machine, called a Vocoder. The loud-speaker is replaced by a microphone, which picks up sound. Speak to it, and the corresponding keys move. This may be one element of the postulated system.

      The other element is found in the stenotype, that somewhat disconcerting device encountered usually at public meetings. A girl strokes its keys languidly and looks about the room and sometimes at the speaker with a disquieting gaze. From it emerges a typed strip which records in a phonetically simplified language a record of what the speaker is supposed to have said. Later this strip is retyped into ordinary language, for in its nascent form it is intelligible only to the initiated. Combine these two elements, let the Vocoder run the stenotype, and the result is a machine which types when talked to.

      Our present languages are not especially adapted to this sort of mechanization, it is true. It is strange that the inventors of universal languages have not seized upon the idea of producing one which better fitted the technique for transmitting and recording speech. Mechanization may yet force the issue, especially in the scientific field; whereupon scientific jargon would become still less intelligible to the layman.

      One can now picture a future investigator in his laboratory. His hands are free, and he is not anchored. As he moves about and observes, he photographs and comments. Time is automatically recorded to tie the two records together. If he goes into the field, he may be connected by radio to his recorder. As he ponders over his notes in the evening, he again talks his comments into the record. His typed record, as well as his photographs, may both be in miniature, so that he projects them for examination.

      Much needs to occur, however, between the collection of data and observations, the extraction of parallel material from the existing record, and the final insertion of new material into the general body of the common record. For mature thought there is no mechanical substitute. But creative thought and essentially repetitive thought are very different things. For the latter there are, and may be, powerful mechanical aids.

      Adding a column of figures is a repetitive thought process, and it was long ago properly relegated to the machine. True, the machine is sometimes controlled by a keyboard, and thought of a sort enters in reading the figures and poking the corresponding keys, but even this is avoidable. Machines have been made which will read typed figures by photocells and then depress the corresponding keys; these are combinations of photocells for scanning the type, electric circuits for sorting the consequent variations, and relay circuits for interpreting the result into the action of solenoids to pull the keys down.

      All this complication is needed because of the clumsy way in which we have learned to write figures. If we recorded them positionally, simply by the configuration of a set of dots on a card, the automatic reading mechanism would become comparatively simple. In fact, if the dots are holes, we have the punched-card machine long ago produced by Hollorith for the purposes of the census, and now used throughout business. Some types of complex businesses could hardly operate without these machines.

      Adding is only one operation. To perform arithmetical computation involves also subtraction, multiplication, and division, and in addition some method for temporary storage of results, removal from storage for further manipulation, and recording of final results by printing. Machines for these purposes are now of two types: keyboard machines for accounting and the like, manually controlled for the insertion of data, and usually automatically controlled as far as the sequence of operations is concerned; and punched-card machines in which separate operations are usually delegated to a series of machines, and the cards then transferred bodily from one to another. Both forms are very useful; but as far as complex computations are concerned, both are still in embryo.

      Rapid electrical counting appeared soon after the physicists found it desirable to count cosmic rays. For their own purposes the physicists promptly constructed thermionic-tube equipment capable of counting electrical impulses at the rate of 100,000 a second. The advanced arithmetical machines of the future will be electrical in nature, and they will perform at 100 times present speeds, or more.

      Moreover, they will be far more versatile than present commercial machines, so that they may readily be adapted for a wide variety of operations. They will be controlled by a control card or film, they will select their own data and manipulate it in accordance with the instructions thus inserted, they will perform complex arithmetical computations at exceedingly high speeds, and they will record results in such form as to be readily available for distribution or for later further manipulation. Such machines will have enormous appetites. One of them will take instructions and data from a whole roomful of girls armed with simple keyboard punches, and will deliver sheets of computed results every few minutes. There will always be plenty of things to compute in the detailed affairs of millions of people doing complicated things.

      IV

      The repetitive processes of thought are not confined, however, to matters of arithmetic and statistics. In fact, every time one combines and records facts in accordance with established logical processes, the creative aspect of thinking is concerned only with the selection of the data and the process to be employed, and the manipulation thereafter is repetitive in nature and hence a fit matter to be relegated to the machines. Not so much has been done along these lines, beyond the bounds of arithmetic, as might be done, primarily because of the economics of the situation. The needs of business, and the extensive market obviously waiting, assured the advent of mass-produced arithmetical machines just as soon as production methods were sufficiently advanced.

      With machines for advanced analysis no such situation existed; for there was and is no extensive market; the users of advanced methods of manipulating data are a very small part of the population. There are, however, machines for solving differential equations--and functional and integral equations, for that matter. There are many special machines, such as the harmonic synthesizer which predicts the tides. There will be many more, appearing certainly first in the hands of the scientist and in small numbers.

      If scientific reasoning were limited to the logical processes of arithmetic, we should not get far in our understanding of the physical world. One might as well attempt to grasp the game of poker entirely by the use of the mathematics of probability. The abacus, with its beads strung on parallel wires, led the Arabs to positional numeration and the concept of zero many centuries before the rest of the world; and it was a useful tool--so useful that it still exists.

      It is a far cry from the abacus to the modern keyboard accounting machine. It will be an equal step to the arithmetical machine of the future. But even this new machine will not take the scientist where he needs to go. Relief must be secured from laborious detailed manipulation of higher mathematics as well, if the users of it are to free their brains for something more than repetitive detailed transformations in accordance with established rules. A mathematician is not a man who can readily manipulate figures; often he cannot. He is not even a man who can readily perform the transformations of equations by the use of calculus. He is primarily an individual who is skilled in the use of symbolic logic on a high plane, and especially he is a man of intuitive judgment in the choice of the manipulative processes he employs.

      All else he should be able to turn over to his mechanism, just as confidently as he turns over the propelling of his car to the intricate mechanism under the hood. Only then will mathematics be practically effective in bringing the growing knowledge of atomistics to the useful solution of the advanced problems of chemistry, metallurgy, and biology. For this reason there will come more machines to handle advanced mathematics for the scientist. Some of them will be sufficiently bizarre to suit the most fastidious connoisseur of the present artifacts of civilization.

      V

      The scientist, however, is not the only person who manipulates data and examines the world about him by the use of logical processes, although he sometimes preserves this appearance by adopting into the fold anyone who becomes logical, much in the manner in which a British labor leader is elevated to knighthood. Whenever logical processes of thought are employed--that is, whenever thought for a time runs along an accepted groove--there is an opportunity for the machine. Formal logic used to be a keen instrument in the hands of the teacher in his trying of students' souls. It is readily possible to construct a machine which will manipulate premises in accordance with formal logic, simply by the clever use of relay circuits. Put a set of premises into such a device and turn the crank, and it will readily pass out conclusion after conclusion, all in accordance with logical law, and with no more slips than would be expected of a keyboard adding machine.

      Logic can become enormously difficult, and it would undoubtedly be well to produce more assurance in its use. The machines for higher analysis have usually been equation solvers. Ideas are beginning to appear for equation transformers, which will rearrange the relationship expressed by an equation in accordance with strict and rather advanced logic. Progress is inhibited by the exceedingly crude way in which mathematicians express their relationships. They employ a symbolism which grew like Topsy and has little consistency; a strange fact in that most logical field.

      A new symbolism, probably positional, must apparently precede the reduction of mathematical transformations to machine processes. Then, on beyond the strict logic of the mathematician, lies the application of logic in everyday affairs. We may some day click off arguments on a machine with the same assurance that we now enter sales on a cash register. But the machine of logic will not look like a cash register, even of the streamlined model.

      So much for the manipulation of ideas and their insertion into the record. Thus far we seem to be worse off than before--for we can enormously extend the record; yet even in its present bulk we can hardly consult it. This is a much larger matter than merely the extraction of data for the purposes of scientific research; it involves the entire process by which man profits by his inheritance of acquired knowledge. The prime action of use is selection, and here we are halting indeed. There may be millions of fine thoughts, and the account of the experience on which they are based, all encased within stone walls of acceptable architectural form; but if the scholar can get at only one a week by diligent search, his syntheses are not likely to keep up with the current scene.

      Selection, in this broad sense, is a stone adze in the hands of a cabinetmaker. Yet, in a narrow sense and in other areas, something has already been done mechanically on selection. The personnel officer of a factory drops a stack of a few thousand employee cards into a selecting machine, sets a code in accordance with an established convention, and produces in a short time a list of all employees who live in Trenton and know Spanish. Even such devices are much too slow when it comes, for example, to matching a set of fingerprints with one of five million on file. Selection devices of this sort will soon be speeded up from their present rate of reviewing data at a few hundred a minute. By the use of photocells and microfilm they will survey items at the rate of a thousand a second, and will print out duplicates of those selected.

      This process, however, is simple selection: it proceeds by examining in turn every one of a large set of items, and by picking out those which have certain specified characteristics. There is another form of selection best illustrated by the automatic telephone exchange. You dial a number and the machine selects and connects just one of a million possible stations. It does not run over them all. It pays attention only to a class given by a first digit, then only to a subclass of this given by the second digit, and so on; and thus proceeds rapidly and almost unerringly to the selected station. It requires a few seconds to make the selection, although the process could be speeded up if increased speed were economically warranted. If necessary, it could be made extremely fast by substituting thermionic-tube switching for mechanical switching, so that the full selection could be made in one one-hundredth of a second. No one would wish to spend the money necessary to make this change in the telephone system, but the general idea is applicable elsewhere.

      Take the prosaic problem of the great department store. Every time a charge sale is made, there are a number of things to be done. The inventory needs to be revised, the salesman needs to be given credit for the sale, the general accounts need an entry, and, most important, the customer needs to be charged. A central records device has been developed in which much of this work is done conveniently. The salesman places on a stand the customer's identification card, his own card, and the card taken from the article sold--all punched cards. When he pulls a lever, contacts are made through the holes, machinery at a central point makes the necessary computations and entries, and the proper receipt is printed for the salesman to pass to the customer.

      But there may be ten thousand charge customers doing business with the store, and before the full operation can be completed someone has to select the right card and insert it at the central office. Now rapid selection can slide just the proper card into position in an instant or two, and return it afterward. Another difficulty occurs, however. Someone must read a total on the card, so that the machine can add its computed item to it. Conceivably the cards might be of the dry photography type I have described. Existing totals could then be read by photocell, and the new total entered by an electron beam.

      The cards may be in miniature, so that they occupy little space. They must move quickly. They need not be transferred far, but merely into position so that the photocell and recorder can operate on them. Positional dots can enter the data. At the end of the month a machine can readily be made to read these and to print an ordinary bill. With tube selection, in which no mechanical parts are involved in the switches, little time need be occupied in bringing the correct card into use--a second should suffice for the entire operation. The whole record on the card may be made by magnetic dots on a steel sheet if desired, instead of dots to be observed optically, following the scheme by which Poulsen long ago put speech on a magnetic wire. This method has the advantage of simplicity and ease of erasure. By using photography, however, one can arrange to project the record in enlarged form, and at a distance by using the process common in television equipment.

      One can consider rapid selection of this form, and distant projection for other purposes. To be able to key one sheet of a million before an operator in a second or two, with the possibility of then adding notes thereto, is suggestive in many ways. It might even be of use in libraries, but that is another story. At any rate, there are now some interesting combinations possible. One might, for example, speak to a microphone, in the manner described in connection with the speech-controlled typewriter, and thus make his selections. It would certainly beat the usual file clerk.

      VI

      The real heart of the matter of selection, however, goes deeper than a lag in the adoption of mechanisms by libraries, or a lack of development of devices for their use. Our ineptitude in getting at the record is largely caused by the artificiality of systems of indexing. When data of any sort are placed in storage, they are filed alphabetically or numerically, and information is found (when it is) by tracing it down from subclass to subclass. It can be in only one place, unless duplicates are used; one has to have rules as to which path will locate it, and the rules are cumbersome. Having found one item, moreover, one has to emerge from the system and re-enter on a new path.

      The human mind does not work that way. It operates by association. With one item in its grasp, it snaps instantly to the next that is suggested by the association of thoughts, in accordance with some intricate web of trails carried by the cells of the brain. It has other characteristics, of course; trails that are not frequently followed are prone to fade, items are not fully permanent, memory is transitory. Yet the speed of action, the intricacy of trails, the detail of mental pictures, is awe-inspiring beyond all else in nature.

      Man cannot hope fully to duplicate this mental process artificially, but he certainly ought to be able to learn from it. In minor ways he may even improve, for his records have relative permanency. The first idea, however, to be drawn from the analogy concerns selection. Selection by association, rather than by indexing, may yet be mechanized. One cannot hope thus to equal the speed and flexibility with which the mind follows an associative trail, but it should be possible to beat the mind decisively in regard to the permanence and clarity of the items resurrected from storage.

      Consider a future device for individual use, which is a sort of mechanized private file and library. It needs a name, and, to coin one at random, "memex" will do. A memex is a device in which an individual stores all his books, records, and communications, and which is mechanized so that it may be consulted with exceeding speed and flexibility. It is an enlarged intimate supplement to his memory.

      It consists of a desk, and while it can presumably be operated from a distance, it is primarily the piece of furniture at which he works. On the top are slanting translucent screens, on which material can be projected for convenient reading. There is a keyboard, and sets of buttons and levers. Otherwise it looks like an ordinary desk.

      In one end is the stored material. The matter of bulk is well taken care of by improved microfilm. Only a small part of the interior of the memex is devoted to storage, the rest to mechanism. Yet if the user inserted 5000 pages of material a day it would take him hundreds of years to fill the repository, so he can be profligate and enter material freely.

      Most of the memex contents are purchased on microfilm ready for insertion. Books of all sorts, pictures, current periodicals, newspapers, are thus obtained and dropped into place. Business correspondence takes the same path. And there is provision for direct entry. On the top of the memex is a transparent platen. On this are placed longhand notes, photographs, memoranda, all sorts of things. When one is in place, the depression of a lever causes it to be photographed onto the next blank space in a section ~_ the memex film, dry photography being employed

      There is, of course, provision for consultation of the record by the usual scheme of indexing. If the user wishes to consult a certain book, he taps its code on the keyboard, and the title page of the book promptly appears before him, projected onto one of his viewing positions. Frequently-used codes are mnemonic, so that he seldom consults his code book; but when he does, a single tap of a key projects it for his use. Moreover, he has supplemental levers. On deflecting one of these levers to the right he runs through the book before him, each page in turn being projected at a speed which just allows a recognizing glance at each. If he deflects it further to the right, he steps through the book 10 pages at a time; still further at 100 pages at a time. Deflection to the left gives him the same control backwards.

      A special button transfers him immediately to the first page of the index. Any given book of his library can thus be called up and consulted with far greater facility than if it were taken from a shelf. As he has several projection positions, he can leave one item in position while he calls up another. He can add marginal notes and comments, taking advantage of one possible type of dry photography, and it could even be arranged so that he can do this by a stylus scheme, such as is now employed in the telautograph seen in railroad waiting rooms, just as though he had the physical page before him.

      VII

      All this is conventional, except for the projection forward of present-day mechanisms and gadgetry. It affords an immediate step, however, to associative indexing, the basic idea of which is a provision whereby any item may be caused at will to select immediately and automatically another. This is the essential feature of the memex. The process of tying two items together is the important thing.

      When the user is building a trail, he names it, inserts the name in his code book, and taps it ~out on his keyboard. Before him are the two items to be joined, projected onto adjacent viewing positions. At the bottom of each there are a number of blank code spaces, and a pointer is set to indicate one of these on each item. The user taps a single key, and the items are permanently joined. In each code space appears the code word. Out of view, but also in the code space, is inserted a set of dots for photocell viewing; and on each item these dots by their positions designate the index number of the other item.

      Thereafter, at any time, when one of these items is in view, the other can be instantly recalled merely by tapping a button below the corresponding code space. Moreover, when numerous items have been thus joined together to form a trail, they can be reviewed in turn, rapidly or slowly, by deflecting a lever like that used for turning the pages of a book. It is exactly as though the physical items had been gathered together from widely separated sources and bound together to form a new book. It is more than this, for any item can be joined into numerous trails.

      The owner of the memex, let us say, is interested in the origin and properties of the bow and arrow. Specifically he is studying why the short Turkish bow was apparently superior to the English long bow in the skirmishes of the Crusades. He has dozens of possibly pertinent books and articles in his memex. First he runs through an encyclopedia, finds an interesting but sketchy article, leaves it projected. Next, in a history, he finds another pertinent item, and ties the two together. Thus he goes, building a trail of many items. Occasionally he inserts a comment of his own, either linking it into the main trail or joining it by a side trail to a particular item. When it becomes evident that the elastic properties of available materials had a great deal to do with the bow, he branches off on a side trail which takes him through textbooks on elasticity and tables of physical constants. He inserts a page of longhand analysis of his own. Thus he builds a trail of his interest through the maze of materials available to him.

      And his trails do not fade. Several years later, his talk with a friend turns to the queer ways in which a people resist innovations, even of vital interest. He has an example, in the fact that the outraged Europeans still failed to adopt the Turkish bow. In fact he has a trail on it. A touch brings up the code book. Tapping a few keys projects the head of the trail. A lever runs through it at will, stopping at interesting items, going off on side excursions. It is an interesting trail, pertinent to the discussion. So he sets a reproducer in action, photographs the whole trail out, and passes it to his friend for insertion in his own memex, there to be linked into the more general trail.

      VIII

      Wholly new forms of encyclopedias will appear, ready-made with a mesh of associative trails running through them, ready to be dropped into the memex and there amplified. The lawyer has at his touch the associated opinions and decisions of his whole experience, and of the experience of friends and authorities. The patent attorney has on call the millions of issued patents, with familiar trails to every point of his client's interest. The physician, puzzled by a patient's reactions, strikes the trail established in studying an earlier similar case, and runs rapidly through analogous case histories, with side references to the classics for the pertinent anatomy and histology. The chemist, struggling with the synthesis of an organic compound, has all the chemical literature before him in his laboratory, with trails following the analogies of compounds, and side trails to their physical and chemical behavior.

      The historian, with a vast chronological account of a people, parallels it with a skip trail which stops only on the salient items, and can follow at any time contemporary trails which lead him all over civilization at a particular epoch. There is a new profession of trail blazers, those who find delight in the task of establishing useful trails through the enormous mass of the common record. The inheritance from the master becomes, not only his additions to the world's record, but for his disciples the entire scaffolding by which they were erected.

      Thus science may implement the ways in which man produces, stores, and consults the record of the race. It might be striking to outline the instrumentalities of the future more spectacularly, rather than to stick closely to methods and elements now known and undergoing rapid development, as has been done here. Technical difficulties of all sorts have been ignored, certainly, but also ignored are means as yet unknown which may come any day to accelerate technical progress as violently as did the advent of the thermionic tube. In order that the picture may not be too commonplace, by reason of sticking to present-day patterns, it may be well to mention one such possibility, not to prophesy but merely to suggest, for prophecy based on extension of the known has substance, while prophecy founded on the unknown is only a doubly involved guess.

      All our steps in creating or absorbing material of the record proceed through one of the senses--the tactile when we touch keys, the oral when we speak or listen, the visual when we read. Is it not possible that some day the path may be established more directly?

      We know that when the eye sees, all the consequent information is transmitted to the brain by means of electrical vibrations in the channel of the optic nerve. This is an exact analogy with the electrical vibrations which occur in the cable of a television set: they convey the picture from the photocells which see it to the radio transmitter from which it is broadcast. We know further that if we can approach that cable with the proper instruments, we do not need to touch it; we can pick up those vibrations by electrical induction and thus discover and reproduce the scene which is being transmitted, just as a telephone wire may be tapped for its message.

      The impulses which flow in the arm nerves of a typist convey to her fingers the translated information which reaches her eye or ear, in order that the fingers may be caused to strike the proper keys. Might not these currents be intercepted, either in the original form in which information is conveyed to the brain, or in the marvelously metamorphosed form in which they then proceed to the hand?

      By bone conduction we already introduce sounds into the nerve channels of the deaf in order that they may hear. Is it not possible that we may learn to introduce them without the present cumbersomeness of first transforming electrical vibrations to mechanical ones, which the human mechanism promptly transforms back to the electrical form? With a couple of electrodes on the skull the encephalograph now produces pen-and-ink traces which bear some relation to the electrical phenomena going on in the brain itself. True, the record is unintelligible, except as it points out certain gross misfunctioning of the cerebral mechanism; but who would now place bounds on where such a thing may lead?

      In the outside world, all forms of intelligence, whether of sound or sight, have been reduced to the form of varying currents in an electric circuit in order that they may be transmitted. Inside the human frame exactly the same sort of process occurs.

      Must we always transform to mechanical movements in order to proceed from one electrical phenomenon to another? It is a suggestive thought, but it hardly warrants prediction without losing touch with reality and immediateness.

      Presumably man's spirit should be elevated if he can better review his shady past and analyze more completely and objectively his present problems. He has built a civilization so complex that he needs to mechanize his records more fully if he is to push his experiment to its logical conclusion and not merely become bogged down part way there by overtaxing his limited memory. His excursions may be more enjoyable if he can reacquire the privilege of forgetting the manifold things he does not need to have immediately at hand, with some assurance that he can find them again if they prove important.

      The applications of science have built man a well-supplied house, and are teaching him to live healthily therein. They have enabled him to throw masses of people against one another with cruel weapons. They may yet allow him truly to encompass the great record and to grow in the wisdom of race experience. He may perish in conflict before he learns to wield that record for his true good. Yet, in the application of science to the needs and desires of man, it would seem to be a singularly unfortunate stage at which to terminate the process, or to lose hope as to the outcome.

    1. The Burial of the Dead

      In the "Ethnography" text, the author cites Mr. Basevi's interesting point: that graves are not "receptacles for the dead, but refuges for the living." He also suggests that graves weren't always separated from the living spaces, and that people were actually buried underground because that was where people lived. I am interested in how this connects to the portrayal of nature in the Wasteland. Eliot calls April the "cruellest month, breeding lilacs out of dead ground". It is a strong juxtaposition–in the way a grave is a "refuge for the living", this muddy, deadness is a refuge for growth and newness. This led me to realize the many ways humans are reminded of their mortality. One of the most subtle, and I would argue the most haunting because of its subtlety, are the reminders of one's expendability. In the same way that no matter how harsh a winter gets, a flower will always end up peaking its head out of the ground come April, there will always be a cycle of "new people", or youth, to replace the older generations. The ego does not enjoy realizing its own superfluousness, so it is for these reasons that we damn the youth for their "newness", and April for having the audacity to grow new flowers and new grass in the very ground which is a graveyard for last-year's life.

    1. This is the purpose of ethically appropriate research with careful oversight. The ban does not change the need for discussion. If anything, it brings the debate back to the reality of patients seeking care for diseases that currently have no cure.

      Menke is making the point that banning ethical gene-editing research is the equivalent of putting your head in the sand.

    1. I became adept at turning wheels in my head and at makingchains of cause and effect: "This one turns this way so that mustturn that way s o . . . "

      I feel this is how learning looks like for some. They are given an idea or a concept which gets their "gears" turning to analyze it and make sense of the new information. I can see this in my son who is 2 as he manipulates his puzzles to make sense. You can see the hears turning in his head as he sticks his tongue out to understand the complexity of the puzzle. Scholars are the same as new information is given to them. I am one who has to process and marinate on new information before it has become part of me. This allows scholars to take it from short term memory to long term memory.

    1. The shimmering head and shoulders of a woman blink into existence above the arena floor. Electrical currents course over her metal teeth, and her form stutters and flickers as she speaks in a strange language.

      La tête et les épaules scintillantes d'une femme clignotent en apparaissant au-dessus du sol de l'arène. Des courants électriques parcourent ses dents métalliques, et sa forme bégaie et scintille alors qu'elle parle dans une langue étrange.

    1. he much marvelled that you would send me a servant to the Company; he saith I had been better knocked on the head. And indeed so I find it now, to my great grief and misery; and [I] saith that if you love me you will redeem me suddenly

      Did this guy's parents sell him into indentured servitude? And now he is straight up asking them to buy him out of indentured servitude because things are so bad. It sounds like they might not be able to afford it though

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this work, the authors are trying to satisfy a real need in MR safety, when concerns can arise about the thermal increase due to metallic materials in patients carrying orthopedic implants. The "MR conditional" labeling of the implant obtained by ASTM in-vitro tests may help to plan the MR scan, but it is normally limited to a single specific MR sequence and a B0 value, and it is not always available. The adoption of an in-silico simulation testbed overcomes this limitation, providing a fast and reliable prediction of temperature increase from RF, in real-life scan conditions on human-like digital models. The FDA is pushing this approach.

      Strengths:<br /> The presented in-silico testbed looks valuable and validated. It is based on the widely available Visible Human Project (VHP) datasets, and the testbed is available online. The approval of the testbed by the FDA as a medical device development tool (MDDT) is a good premise for the large-scale adoption of this kind of solution.

      Weaknesses:<br /> There are a couple of limitations in the study that must be clearly highlighted to the readers.

      While the RF-related heating is very well modeled, the gradients-related heating is out of the scope of this paper and not considered. Readers must be warned that RF causes only a part of the heating, and literature is reporting cases where also gradient switching can contribute, as correctly mentioned in this work. A cautious attitude should consider this as a significant limitation of the study.

      Moreover, the way the implant is embedded in the VHP model is shortly documented in the materials and methods and mostly focuses on implant registration on bone tissue. It is not clear how to manage the empty space and the soft tissue stretching/reshaping generated by the simulated surgery (for example, by the cut of the femoral head in total hip arthroplasty). It is reported by literature that the level of accuracy in the simulated surgery can impact in some cases (RF vs. gradients heating, massive vs. thin or elongated implants) on temperature predictions.

  3. inst-fs-iad-prod.inscloudgate.net inst-fs-iad-prod.inscloudgate.net
    1. Sir Francis Head, an English traveler and a Governor-General ofCanada, tells us that "in both the northern and southernhemispheres of the New World, Nature has not only outlined herworks on a larger scale, but has painted the whole picture withbrighter and more costly colors than she used in delineating andin beautifying the Old World.... The heavens of America appearinfinitely higher, the sky is bluer, the air is fresher, the coldis intenser, the moon looks larger, the stars are brighter thethunder is louder, the lightning is vivider, the wind isstronger, the rain is heavier, the mountains are higher, therivers longer, the forests bigger, the plains broader." Thisstatement will do at least to set against Buffon's account ofthis part of the world and its productions.

      it sounds like once more of the world was discovered, the land that had already been known and the newly learned lands both seemed brighter and just overall better than before. Possibly because things started to piece together and connect as they found that the world was much more than they originally thought.

  4. moodle.lynchburg.edu moodle.lynchburg.edu
  5. viewingblackgirlhood.com viewingblackgirlhood.com
    1. The lead photograph of the story waspeculiarly cropped: The queen had no head

      hypersexualization of black women and black bodies. Removing a head from a woman is dehumanizing and insinuates all she has to offer is her body.

    1. These offerings, when made by one of the heads of a family, serve for the whole.

      when the head of a family makes an offering, it is considered to cover the entire family.

    1. Within this corporate body, the individuals are united in such a way that they are transformed into members (metaphor to limbs, hands, legs, etc.) and Christ was their head, i.e., the logos: the creative, ordering wisdom.

      Bishop Baron...

    1. In at least 100 words, please explain the process you go through when you write a paper. What steps do you take to move from beginning, middle, and end of a writing assignment?(required)

      When I write a paper I tend to plan out a small rough draft in my head before writing but I do not think about it for too long as I will get distracted and off track. I then begin to write while loosely following my plan which seems to work for me because when I keep on writing without thinking of a strict rubric my writing flows better. I introduce whatever topic I am going to write about with some added background info as well. After that I introduce my subclaims while inserting evidence backed up with explanations until I reach my conclusions.

    1. When KFKB was given an upgrade to 5,000 watts while the Kansas City Star’s application to take its station to equal power was denied, the newspaper launched an exposé of Brinkley’s medical franchise. Their investigation was buttressed by the ongoing public accusations of medical quackery against Brinkley made by Dr. Morris Fishbein, head of the AMA, which was beginning its successful drive for the professionalization of the practice of medicine. Soon the FRC reversed its previously tolerant stance, and in late 1929 revoked Brinkley’s license, charging that he was in fact operating a point-to-point service for commercial purposes and not a proper broadcasting station in the public interest.

      This interests me as it doesn't necessarily prove or disprove my original theory that Brinkley may have been a quack doctor.

    1. Thefirst evidence of cultivated plants and of sedentary commu-nities appears roughly 12,000 years ago. Until then—that isto say for ninety-five percent of the human experience onearth—we lived in small, mobile, dispersed, relatively egali-tarian, hunting-and-gathering bands.

      The fact that the first evidence of life in communities was 12,000 years ago blows my mind. It is so crazy to me for two reasons. One being the way we are able to figure out, and date this back to that period. The process of dating old artifacts and finding out information about what the environment was like in these old communities is amazing. This part of the text also makes me question my understanding on the human past because when I think about time in the grand scheme of things , 12,000 years is an extremely long time. When I look back in time, 200 maybe even 100 years ago, everything is just so different, the way people acted, the way things were. I just can't wrap my head around how and what human life was like then.

  6. moodle.colgate.edu moodle.colgate.edu
    1. functional Magnetic Resonance Imaging (fMRI)
      • signal extracted from MRI scanner
      • giant magnet
      • Head coil: largely to help with the resolution of the anatomical scale (MRI image)
      • Surface coil: purpose is to boost the resolution of the functional scale (put behind a certain part of the head as almost a magnifying glass)
    1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      We would like to thank all reviewers for taking the time to evaluate our manuscript fairly and critically. Many helpful suggestions and discussion points were raised. One important group of comments raised concerns whether our proposed timer and counter models were the appropriate conceptual framework to discuss nuclear multiplication in schizogony, whether they were mutually exclusive, and whether other alternatives should be considered. These comments were instrumental for us to uncover some inconsistencies in our previous modeling approach. In the new manuscript, we now define the counter and timer models much more rigorously in the context of Plasmodium cell division. Based on these refined models we now provide a new statistical analysis that goes beyond the previous analysis, significantly improving the statistical support for our conclusions. Details are given in the following individual replies.

      Reviewer #1 (Evidence, reproducibility and clarity):

      Summary

      Malaria parasites replicating in human red blood cells show a striking diversity in the number of progeny per replication cycle. Variation in progeny number can be seen between different species of malaria parasites, between parasite isolates, even between different cells from the same isolate. To date, we have little understanding of what factors influence progeny number, or how mechanistically it is controlled. In this study, the authors try to define how the mechanism that determines progeny number works. They propose two mechanisms, a 'counter' where progeny number is determined by the measurement of some kind of parasite parameter, and a 'timer' where parasite lifecycle length would be proportional to progeny number. Using a combination of long-term live-cell microscopy and mathematical modelling, the authors find consistent support for a 'counter' mechanism. Support for this mechanism was found using both Plasmodium falciparum, the most prominent human malaria parasite, and P. knowlesi, a zoonotic malaria parasite. Of the parameters measured in this study, the only thing that seemed to predict progeny number was parasite size around the onset of mitosis. The authors also found that during their replication inside red blood cells, malaria parasites drastically increase their nuclear to cytoplasmic ratio, a cellular parameter remains consistent in the vast majority of cell-types studied to date.

      Major Comments

      It is stated a few times in this study that P. knowlesi has an ~24 hour lifecycle, and while this is the case for in vivo P. knowlesi, it was established in the study when P. knowlesi A1-H1 was adapted to human RBCs (Moon et al., 2013) that this significantly extended the lifecycle to ~27 hours, which should be made clear in the text. As much of this study revolves around lifecycle length and timing, the authors should consider some of their findings with the context that in vitro adaption can significantly alter lifecycle length.

      The reviewer raises an important point that we didn’t discuss for P. knowlesi. We now mention this directly in the introduction chapter (line 67) and in the discussion (lines 470ff). We are aware that P. knowlesi takes about 27 hours in the lab, which was also communicated by the Moon lab. We now cite relevant studies again in this context. We further address the issue of modified cell cycle time in vitro in the discussion in the sense that absolute values must be taken with caution and the focus of this study is about the relative ratio and correlation between the different cell cycle metrics.

      • The dichotomous distinction between 'timer' and 'counter' as mutually exclusive mechanisms seems to be a drastic oversimplification. Considering the drastic variation we see in merozoite number across species, between isolates, and between cells, it seems much more likely that there are factors controlled by both time-sensed and counter-sensed mechanisms that both influence progeny number.

      The study of progeny regulation in malaria parasites is very much in the early stages. We can agree that our models are simplifications, as is the case with all models. Our choice of just the two models timer and counter was driven by the number of cellular parameters we measure, i.e., duration of division phase and progeny number. These data essentially allow us to test the two competing models we presented. As we quantify more and more cellular parameters, based on the quantitative live cell imaging protocols established here, we will be able to test more complex cell cycle models. With our current data, we believe more complex models are not warranted.

      However, this valuable criticism, in conjunction with related remarks by other reviewers, made us reevaluate the constraints of our model more precisely. We noticed that the criteria used in the previous version in the manuscript contained unnecessary additional assumptions. Briefly, the previous counter model also required that final merozoite number was tightly controlled, while the previous timer model required the growth rate to be tightly controlled. These side assumptions were not made explicit in the manuscript and could bias the support towards one or the other model.

      We now improved the modeling approach substantially by removing implicit side assumptions, and clearly defining timer and counter models in terms of their correlations. The refined formulation of the timer posits that between individual parasites the target duration and the nuclear multiplication rate vary in a statistically independent way; while in a counter, target number and nuclear multiplication rate are statistically independent. We now explain this extended analysis in more detail in the introduction (lines 86ff). We also now more clearly state the dichotomous nature of the model (line 488). A new results paragraph (lines 213ff) and an entirely new Fig. 2 (and Fig. S4) contains the model predictions and statistical comparison between the models.

      This more rigorous treatment showed that including the variance of the multiplication rate was critical to allow a clean discrimination between the models. Also, with the sole exception of P.knowlesi H2B, where no model was clearly favored (Fig. 2G-H,K), the timer model was found to be inconsistent with the data, while the counter was clearly favored. Our new goodness-of-fit analysis also showed that although the counter is strongly simplified, it produced adequate fits, demonstrating that potential model refinements would need to be justified by new, more extensive data.

      It is also important to consider that the degree of variation in merozoite number could rather be an expression of varying growth conditions and does not directly predict which of the proposed models are true. For instance, a counter where the target merozoite number varies strongly depending on growth conditions, would be consistent with all available data. It is an interesting question for future work whether a counter would indeed describe growth across different isolates.

      The biological reality of growth regulation is certainly complex, and the counter model will likely need to be refined in the future, which we acknowledge in a corresponding statement in the discussion (lines 491ff). Nevertheless, we find it encouraging that a simple model can explain the vast majority of our data very well.

      Additionally, the only parasite parameter measured in this study, size at time of first nuclear division, explained only a small proportion of the variance observed in merozoite number.

      It is indeed the case that amongst the measured parasite parameters i.e. schizont stage duration, nuclear volume, and cell size we only found the latter to correlate with the final progeny number. We did not aim to imply that all variation in progeny number is explained by cell size. It is likely that a putative counter relies on a set of factors, which are somehow linked to cell size. In addition, intrinsic stochasticity in nuclear growth is likely to contribute to final merozoite number variability, which is included in our models via a variable growth rate. Defining the actual limiting factor or combination of factors will be an exciting challenge for the future studies building on this one.

      • For modelling of a timer-based mechanism, the designation of t0 is subjective. The authors chose the time of first nuclear division as their t0. It is possible that a timer-based mechanism could not be supported based on this model the chosen t0 differs from when the "parasite's timer" starts. For example, t could also have been designated as the time from merozoite invasion (t0) to egress (tend). It would be unreasonable to suggest the authors repeat experiments with a longer time-frame to address this, but this possibility should be discussed as a limitation of the model. It may also be possible to develop a different model where t0 = merozoite invasion and tend = egress, and test this model against the data already collected in this study.

      This is a valid point. We indeed, considered the time point of invasion as the other relevant time point in the IDC for a possible timer. Due to necessary compromises in imaging protocols between acquisition length, temporal, and spatial resolution we have not been able yet to combine full-length IDC measurements with quantification of progeny number. Given the choice, however, between time point of invasion and the onset of nuclear division as starting point for a potential timer we would still favor the latter: An argument can be made that a timer that regulates offspring number would be more accurate when activated at the moment of the relevant cellular events rather than “running” for a very prolonged growth phase before any “decision” concerning parasite replication. We are still convinced that the entry into the schizont stage, which we analyze here, marks an important cell cycle transition point that has been highlighted in many different studies. As suggested, we now discuss the limitations of our selection of t0 in the text (lines 146ff).

      • The calculation of the multiplication rate is confusingly defined. In Figure 1 it is stated that it is "...based on t and n", which would imply that the multiplication rate is the number of merozoites formed per hour of schizogony, which would give an average value of ~2 for P. falciparum and ~1.5 for P. knowlesi. The averages rate values shown, however, are in the range of 0.15-3. The authors should clarify how these values were determined.

      Thank you for pointing out the need for more clarity. Since the nuclear multiplication, similar to e.g. cell population growth, follows an exponential law, the multiplication rate used (lambda) is in fact a logarithmic growth rate. Therefore, it occurs in the exponent (not as a coefficient) in the exponential growth function ( ), which explains the range. We now mention this more explicitly in the results (lines 163ff).

      • In Figure 2, the time from tend until egress is calculated, and this is interpreted as the time required for segmentation. In the Rudlaff et al., 2020 study cited in this paper, it is shown that segmentation starts before the final round of nuclear divisions are complete. Considering this, the time from tend until egress is not an appropriate proxy for segmentation time. The authors should consider rewording to something akin to "time from final nuclear division until egress" to more accurately reflect these data.

      Thank you for indicating our imprecise use of the nomenclature. Indeed, some essential segmentation-associated structures such as rhoptries and subpellicular microtubules are clearly forming before the last division. We were referring to “segmentation” as the time window where actual ingression of the plasma membrane occurs between nuclei with the concurrent formation of more prominent IMC-associated sub-pellicular microtubules between nuclei (as in Fig. 1A last panel). We can, however, agree that consistently using the term “merozoite formation” is more adequate here. We have now corrected the terminology according to the suggestions of the reviewer (lines 271ff).

      • There is a significant discrepancy between the data in Figure 5 and Supplementary Figure 8. In Supplementary Figure 8, the authors establish that culturing parasites in media diluted 0.5x has a marginal effect on parasite growth, with no discernible change in parasitaemia over 96 hours. By contrast, in Figure 5a the parasitaemia of parasites cultured in 0.5x diluted media is approximately 5-fold lower than those in 1x media. The authors should explain the significant discrepancy between these results.

      The reviewer correctly points out a difference in parasitaemia between two parasite culture experiments, shown in Figs 5a (now 6A) and S8 (now S11), respectively. There were several differences in the experimental setup used in the two experiments that could explain this discrepancy. In Fig. 5a the parasites were synchronized to early ring stages while in Fig. S8 we used asynchronous cultures (maybe with a slight majority of late stages). One could speculate that by the time the synchronized ring stage culture reached egress the effect of nutrient depletion, which started at t = 0 h is more pronounced. This effect could have been exacerbated by the more frequent media change of 24 h in Fig. 5a vs 48h in Fig. S8. Lastly, the starting parasitemia was differently set being higher at around 0.5% in the Fig. 5a while only 0.2% in Fig. S8. Possibly a lack of nutrient is “felt less” by the culture at lower parasitemias. Generally, in Fig. S8 we were more focused on highlighting the difference between 1x/0.5x and the more diluted conditions on the long-term culture and to show that continuous culture is actually possible in 0.5x medium. We have now expanded the legends to highlight those differences more clearly.

      • In Supplementary Figure 4, the mask on the cell at t0 shows two distinct objects, but it seems very unlikely that they are two distinct nuclei as they vary approximately 5-fold in diameter. The authors should provide more detail on how their masking was performed for their volumetric analysis. Specifically, whether size thresholds were also applied during object detection.

      Thank you for requesting clarification here. Fig S4 (now S7) shows only one z-slice (not a projection) of the entire image stack, to illustrate how the thresholding approach was performed on every single image slice. The two objects in the shown cell are indeed two nuclei, but because they are not in the same z-plane appear to be of different size. In particular, only a slice of the upper part of the nucleus on the lower right is visible in the shown slice. Throughout the study, volume determination was realized by adding up the individual slices, as is explained in detail in the Materials and Methods sections. We have now added a more explanation in the figure legend to clarify the procedure.

      Minor Comments

      • Line 45-48 mentions that merozoite number influences growth rate and virulence, but the corresponding reference (Mancio-Silva et al., 2013) only discusses the relationship between merozoite number and growth rate, not virulence.

      We thank the reviewer for requesting this distinction. Merozoite number and virulence have not been correlated in vivo so far. Certainly, because one can’t retrieve late-stage P. falciparum parasites from patients, but maybe partly because merozoite number has not gotten significant attention as a metric in the previous decades. Even if merozoite number is intuitively connected to growth rate which might causes higher parasitemia which is in turn linked to more severe disease outcome it is important to emphasize that those are certainly not equivalent. We have therefore removed the statement about virulence (line 48).

      • Line 59 states that a 48 hour lifecycle is a baseline from which in vitro cultured parasites deviate. Clinical isolates also show variation in lifecycle length and so it is more accurate to just say that 48 hours is an average, rather than a baseline.

      The word “baseline” has been changed to “average” (line 61).

      • Line 63 cites a study for the lifecycle length of P. knowlesi (Lee et al., 2022), but there seems to be no mention of lifecycle length in this reference

      This reference was meant to serve as an introductory review article to research in P. knowlesi. Actually, to the knowledge of the authors, there is no study presenting quantitative data showing that the in vitro cycle of P. knowlesi is actually around 27 h. Our lab experience is however coherent with a 27 h cycle, which was confirmed by personal communication by the Moon lab. We now also cite in the next sentence the inaugural P. knowlesi adaptation publication (Moon et al. 2013) showing some time course data indicating the duration of the IDC to be around ~27h (lines 67ff).

      • If I am interpreting Figure 3B correctly, this is essentially a paired analysis where the same erythrocytes are measured twice, once at t0 and once at tend. If this is the case, this data may be better represented with lines that connect the t0 and tend values.

      Yes, these are the same erythrocytes measured twice. We have modified Figure 3 (now Fig. 4) accordingly.

      • Figure 3A seems to imply that to calculate diameter of the erythrocytes, three measurements were made and averaged for each cell. I think this is a nice way to get a more accurate erythrocyte diameter, but if this is the case, it should be specified in the figure legend or methods.

      This is already described in the figure legend (line 305).

      • In Figure 4I it is shown that in P. falciparum merozoite number doesn't correlate with nucleus size, but for P. knowlesi in Supplementary Figure 7c, a significant anticorrelation is observed. The authors should state this in the text and discuss this discrepancy.

      Contrary to all other graphs, visual inspection of the distribution of data points in Fig. S10C shows that it contains two outlier data points at the bottom right. Those two specific points are also responsible for the significant anticorrelation. We did not filter or remove any quantification results but also didn’t have sufficient confidence in this data distribution (which is further based on the segmentation of the Histone2B not on an NLS mCherry signal) to make substantial claims about anticorrelation. Because we considered it informative we still decided to show it in the supplements. We now briefly mention the issues with the data set and its interpretation in the text (lines 350ff).

      • The authors show that merozoite number roughly correlates with cell size at t0 but it would be interesting to see whether cell size at tend also corresponds with cell size at t0. This might help answer whether the cell is larger because it has more merozoites, or whether it has more merozoites because it is larger.

      Plotting parasite cell volume at t0 against cell volume at tend (as well as between t-2 and tend) indeed shows a positive correlation (see below). While it is an interesting thought we concluded after some discussion that no convincing causal relationship between cell size and merozoite number can be inferred based on this analysis. Since we consider the possible statement that cells that are bigger in the beginning are also bigger in the end unavailing, we decided not to include the data.

      • I don't feel that "nearly identical" is an appropriate summary of erythrocyte indices in Supplementary Figure 9, considering there is a statistically significant increase in mean cell volume. I think it is unlikely that this change is consequential, and performing these haematology analyses is a nice quality control step, but this change should be stated in the text.

      In the modified text we now express the significant change in MCV in terms of percentage, which is around 1.2% (line 381).

      • In Supplementary Figure 8, parasitaemia only increases ~2-fold compared to >5-fold the previous two cycles. It seems likely that at the final timepoint on this graph the parasites are starting to crash, and therefore it may be best to end the graph with the 96 hour timepoint.

      The reviewer suggests that cultures at those parasitemias might not be in perfect health. Our Giemsa stains did not show signs of an unhealthy culture and kept growing. It was, however, important for us to show that cultures can be maintained in culture over a prolonged period of time in 0.5x medium, even when resulting in reduced growth, while this was not possible with lower dilutions. Therefore, we would like to keep the data point. We have added a cautionary comment in the legend.

      • The error bars in Figure 5C aren't easily visible, moving them in front of the datapoints may help their visibility.

      Error bars were moved in front of the data points.

      • In Figure 6D & E, the y-axis labels should be changed to whole integers as all the values in the graph are whole numbers.

      We have changed the y-axis labels accordingly.

      • My interpretation of Figure 6 C-E, is that these are the same cells measured at three time points (t-2, t0 and tend). If this is the case, 6C is missing the cell that has a merozoite number of 8, which is presumably why the y-axes are not equalised for the three graphs.

      It is correct that the same cells are displayed in all three plots, with the exceptions of three cells in 6C (for the timepoint t-2), which are missing for the following reasons: 1) it was not possible to determine the volume at this respective timepoint due to technical issues or 2) the cell was already just before t0 at the start of the movie so that t-2 had already passed. We now note this in the figure legend and have also equalized the y-axes (now Fig. 7C-E).

      Reviewer #1 (Significance):

      In the asexual blood-stage of their lifecycle, malaria parasites replicate through a process called schizogony. During schizogony an initially mononucleated parasite undergoes multiple asynchronous rounds of mitosis followed by nuclear division without cytokinesis, producing a variable number of daughter nuclei. Parasites then undergo a specialised cytokinesis, termed segmentation to where nuclei are packaged into merozoites that go on to invade new host cells. While nucleus, and therefore merozoite, number are known to be varied between cells, across isolates, and across species, little is known about the mechanisms regulating merozoite number. In this study, the authors use live-cell microscopy to understand how parasites determine their progeny number. They suggest that parasites regulate their progeny number using a 'counter' mechanism, which would respond to the size or concentration of a cellular parameter, as opposed to a 'timer' mechanism. Long-term live-cell microscopy experiments using malaria parasites are extremely technically challenging, and the authors should be commended for their efforts in this regard. While I agree that the data generated from these experiments are technically sound, I have some reservations expressed above about the interpretation of some of these results. I would strongly encourage the authors to consider rewording some of their interpretations taking into account some of the caveats listed above. I would also consider fitting/testing an additional mathematical model where the time-frame proposed for the 'timer' mechanism begins following merozoite invasion.

      We thank the reviewer for the appreciation of our work and hope we have sufficiently reworked the manuscript based on the comments listed above. Furthermore, we think the improved model statement and analysis improves the clarity of our conclusions. Indeed, we would like to test additional models including the full IDC once, as mentioned above, we are technically able to generate these data.

      This work is of specific interest to anybody who grows malaria parasites, as the dynamics of their growth is obviously important to understand. Further, this work is of interest more generally to cell biologists who study the regulation of progeny number or cell size. I have no experience with the application of mathematical modelling to understand biological systems, and so I cannot comment on the interest of this work to that field.

      Reviewer #2 (Evidence, reproducibility and clarity):

      This is a solid study that further characterises the dynamics of nuclear division in Plasmodium falciparum and P. knowlesi. Of two, among potentially several, models for how the number of daughter nuclei, and thus parasites - (called merozoites in this genus), are one that posits nuclei divide until a fixed timer ends, and one that posits that nuclei divide to reach a fixed number that is defined by a cellular counter. I find some practical difficulties in definitive measurement of either model, one issue with the former is that experimental definition of the start of the timer is problematic - we may define the starter's gun (eg by the first nuclear division) but it isn't necessary that the cell is using that same start time.

      We are pleased that the Reviewer found our study ‘solid’. Concerning the timer model, we agree that the selection of the starting point is a critical aspect of this study, as also Reviewer 1 pointed out. We selected this particular “t0” because the entry into the mitotic phase marks an important cell cycle transition. Several studies have suggested a “schizogony entry checkpoint” might be active just before (Matthews et al, 2018; Voß et al, 2023; van Biljon et al, 2018; McLean & Jacobs-Lorena, 2020). Once cells are committed to the schizont stage they are less responsive to stimuli. Alternatively, the timepoint of erythrocyte invasion could be a legitimate starting point. Due to necessary compromises in our imaging protocol between acquisition length, temporal, and spatial resolution we have not been able yet to combine full-length IDC measurements with quantification of progeny number, and therefore we leave exploration of an earlier timer start for future work. Within the confines of the model comparison in the current study, we think the selected t0 is already highly informative. We now explain the selection and limitations more explicitly in the text (line 144ff).

      Additionally, as the authors confirm here, being sure when that first nuclear division has occurred is particularly tricky with Plasmodium parasites, in part because the first few nuclei seem to clump together, preventing one from unambiguously calibrating the first division.

      The Reviewer is concerned about difficulties with precise reporting of the time point of first nuclear division. We suspect there was a misunderstanding here. In the text (line 137) we had written the following:

      “Although separating individual nuclei after the first two rounds of division was challenging due to their spatial proximity, the improvements in resolution and 3D image analysis allowed us to count the final number of nuclei routinely and reliably at the transition into the segmenter stage.”

      To clarify, when analyzing 3D image stacks produced by the LSM900 Airyscan the first nuclear division can consistently and unambiguously be detected. In anaphase the nuclei are pushed apart quite substantially before getting a bit closer together afterwards (see e.g. Fig. 1B and C). Hence the precision of the detection is only limited by the 30 min interval of the time lapse. Later, at the four nuclei stage, crowding makes distinction more difficult. In the final segmenter stage, the reorganization and condensation of nuclei makes reliable counting possible again. We have now reformulated the quoted sentence for more clarity (lines 137ff).

      Furthermore, getting decent replicate numbers is hard because of the difficulties of time lapse microscopy, and most Plasmodium studies (including this one) suffer from low enough numbers that it isn't always clear whether the numbers support one model over another.

      The reviewer points out the difficulty of obtaining enough replicates in Plasmodium time-lapse studies. We agree that depending on technology, sufficient replicates can be challenging. In the present study we obtained Ns between 25 and 35 for all conditions in P. falciparum and P. knowlesi from three independent replicas. To gain confidence in the conclusions from a limited, but not austere, data, it is essential to 1) reduce model complexity to a minimum and 2) perform stringent statistical analysis including accounting for small-sample variation. Motivated by this concern of the Reviewer and a similar point raised by Reviewer 1, we have revisited our modeling approach in the revised manuscript. This led us to a corrected, more rigorous definition of what precisely we mean by ‘counter’ and ‘timer’ models: The timer posits that between individual parasites the target duration and the nuclear multiplication rate and vary in a statistically independent way, while in a counter target number and nuclear multiplication rate are statistically independent. With no further adjustable parameters, the two models are thus both mutually exclusive and minimal. Although biological reality is likely to be more complex, we feel that these minimal models are adequate for the amount and resolution of our current, state-of-the art data. The general result remained the same: The counter model is strongly preferred in almost all our experiments data (new Fig. 2), with the sole exception of P. knowlesi H2B, where indeed more data may be needed to come to a clear conclusion. Furthermore, we have taken care to scrutinize these conclusions accounting for goodness-of-fit for the respective sample size N. This analysis showed, surprisingly, that the counter model was sufficient to account for the data: the real dataset was as similar to the counter prediction as synthetic, counter-generated data. We hope that this improved statistical analysis can help the reader judge the robustness of our conclusions.

      Nonetheless, several recent studies, particularly a study from the same institute (Klaus et al., 2022) employing timelapse imaging of nuclei, and timing the nuclear division of parasites, finds poor correlation between the duration of "schizogeny" (although perhaps using a different definition to the one used by the parasite) and the final number or merozoites. They therefore argue that there is poor evidence for a timer, and conclude by elimination that a counter must exist instead. A review by some of the authors of that study and some of this current study (Voß et al 2023), also concludes that the data from Klaus and colleagues "strongly support" a counter model. This current study also concludes that a counter model controls final nuclear/merozoite number in P. falciparum and P. knowlesi. This much at least is not particularly novel given the recent work on this topic, although the addition of the P. knowlesi data is interesting and consistent with the prior P. falciparum work.

      Our present work, indeed, does confirm the previous report of a counter over a timer, through a more targeted approach. While Klaus et al. used timing data of first nuclear cycle vs. the full duration, we now provide, thanks to an improvement microscopy setup and protocol, simultaneous measurements of timing and final progeny number, i.e. counting of merozoites/nuclei. While the preference for a counter model is not fundamentally novel, the additional information that the counter model holds in different strains, conditions and species is, in our opinion, not trivial and points to some degree of evolutionary conservation. We also demonstrate here that the counter model is not only preferred over the timer, it also fits the data adequately, so that it can be considered ‘correct’ at this level of complexity. Another, possibly more important, value of this study lies in the quantitative and time-resolved assessment of multiple important parasite metrics such a cell volume and nuclear volume together with merozoite number at the single cell level. Although descriptive, this has not been achieved in Plasmodium until now.

      As above, the authors concede that it is difficult to determine with strong confidence when the first nuclear division has occurred, so it may well be that there is substantial noisiness in the time that they define schizogeny to commence. If that were the case, this would contribute to the poor correlation observed between schizogeny duration and number of merozoites produced, so this could be an important confounding experimental factor. This deserves some more discussion by the authors.

      Concerning the confidence with which we identify the first nuclear division we could hopefully clarify in the section above that our precision is only limited by the time resolution of the acquired time-lapse. Therefore, the uncertainty about the start time is not particularly high, and moreover, can expected to affect timer and counter (via the growth rate) to a similar degree. We see no unfair advantage for the counter for this reason.

      Alternative methods to count absolute DNA content (rather than trying to count individual nuclei) might be useful ways of independently confirming this phenomenon. Alternative possibilities for what constitutes the "start" of a possible timer are also warranted - it could be for example, the first division of one of the other organelles.

      This is an interesting suggestion. Next generation fluorogenic DNA dyes have been used by us and the Ganter group (Simon et al. 2021, Klaus et al. 2022, Wenz et l. 2023) to assess DNA content of single cells over time. Our experience shows that there are some caveats to using these Hoechst based dyes, some of which we discussed in the aforementioned publications. While they allow some reasonable absolute quantification of DNA content for the very first S-Phase (and subsequent nuclear division), in later stages only relative quantification can be achieved. One underlying reason is the apparent increase of dye permeability, and therefore higher intensity, at late schizont stages. This issue is exacerbated by the asynchronous DNA replication of multiple nuclei. Further, nuclear division itself can be delayed or even inhibited when increasing the concentration of the dye, which suggest an impact on cell physiology (well documented for Hoechst based dyes in other organisms). When reaching the segmenter stage, the resulting variance in fluorescent intensity would make it challenging to assign a reliable number of nuclei required for analysis, a problem that does not occur when counting individual nuclei. Taken together, unfortunately, all these confounding factors make DNA content analysis in live single cells for the entire schizont stage unachievable at this point.

      These and previous authors in any case conclude that a counter model must exist through exclusion of a timer model. I am less convinced that the evidence discounting the timer is conclusive, and that a straight counter model is the only alternative. Indeed I am unconvinced by the suitability of this strictly dichotomous two-model system to categorise the division of unicellular eukaryotes, and these theories are not universally held to be sufficient to describe division.

      We thank the Reviewer for this insightful comment. As already detailed above, we have clarified and corrected our model definitions in the revised manuscript. Further, we want to make the important distinction between organisms, including unicellular ones that undergo binary fission and the ones like Plasmodium that use schizogony. Our model, although inspired by model organisms, is tailored to a multinucleated division mechanism, and clearly defined within those boundaries. The timer and counter models we consider are defined by their correlation structures. They are at two extremes of a continuum of models which could be characterized, for instance, by the ratio of correlations (growth rate - nuclear number) vs. (growth rate – duration) as an additional parameter. As the reviewer points out, excluding the timer model is not equivalent to proving the counter model, and indeed a partially correlated model, or a more complex model entirely, could yield a better fit. However, within the realm of models without additional parameters, and which are testable with the available data, only timer and counter remain, as different timer start points are not experimentally accessible. Importantly and somewhat surprisingly, the counter model also gave a fit that is as good as can be reasonably expected for the experimental sample size (new Fig. 2). So, we maintain that within the current experimental constraints, the counter model is the only viable option for almost all our tested conditions. The observation that in H2B-GFP expressing P. knowlesi parasites no clear distinction can be made between the models, indeed, suggest that the reality of multiplication rate regulation is more complex and may be limited by different constraints in different growth regimes. We now state these limitations and the room for further model adjustments with more data in the Discussion section.

      Nonetheless, if a counter exists, what is being counted that determines the final number? The authors consider that this might be a physical object or resource inside the parasite, or an extrinsic/extracellular resource. They investigate this by comparing the final cell number to a number of factors. First, the authors investigate the size of the RBC (by musing the diameter as an indicator)- little information is given about the source of the blood used, but it appears to be from a single donor of unknown age, who has approximately typical variance in RBC diameter (at least, after manipulation and storage). The authors observe little correlation between these variables.

      We share the curiosity of the reviewer about what might be “counted” by the parasite. This shall be the subject of future studies, and our present study provides the necessary basis for asking this question and defines a framework to investigate it. Concerning the size of the host cell, the blood used was from a different donor for each of the replicas, which we now specify in the figure legend (line 302). No significant difference between the RBC diameters between the donors was observed. A correlation between RBC diameter and progeny number was indeed not observed.

      Second the authors measure parasite size at the onset of schizogeny, and find that bigger parasites result in more daughter merozoites early in schizogeny (perhaps not surprising, given the earlier mentioned technical problems with measuring the first few steps of schizogeny), but that this different initial cell size doesn't result in a different final merozoite number, or as they describe it "not quite significant anymore". Previous p values were taken as cause for rejecting the timer hypothesis and the timer model. In this case the authors instead interpret the data as suggesting "that the setting of the counter might correlate with parasite cell size". This is inconsistent statistical and analytical handling, and highlights the earlier potential pitfall of rejecting timer-based models based on not gathering data that statistically show a correlation. This needs reworking to highlight that these data are inherently noisy, difficult to measure accurately, and aren't necessarily going strongly reveal a trend even where one biologically exists, and that this ought not be used as grounds for confident rejection of a model.

      The Reviewer raises concerns about the consistency of the statistical interpretation of our data. We care deeply about the well-foundedness of our conclusions and hope to eliminate these concerns in the following. First, we hope that the issue about the “technical problems” in measuring the first division has been solved in our response to previous comments. Next, to clarify an apparent misunderstanding: As stated in the text (lines 329ff) and shown in now Fig. 5D-E, cell size at onset of nuclear division or 2 hours prior does significantly correlate with final merozoite number. The lack of significant p-value (0.08) only pertains to the correlation of cell size at the end of the schizont stage (tend) with merozoite number (now Fig. 5F). We have removed the unfortunate wording “not quite significant anymore” in that context. Finally, regarding potential mechanisms, a potential counter must be set before the first nuclear division is completed because only that way it can be set independent of the speed of nuclear multiplication. This observation gives the statistically significant correlation of volume at the onset of division and progeny number its relevance. We have reformulated the marked sentence for more clarity (lines 331ff). Furthermore, we point out that our rejection of the timer is now based on a revisited statistical analysis (Fig. 2), which is no longer based on a simple correlation between final number and duration, as detailed above.

      Finally, the authors grow the parasites in dilute media, and find that they produce fewer daughter parasites. This is anecdotally unsurprising, as most Plasmodium laboratories are aware that sub-optimal growth conditions result in less healthy schizonts with fewer viable merozoites (and lower magnitudes of single-cycle expansion), but is nonetheless an important result that highlights explicitly how much this occurs in the specific conditions of dilute media. Given the lack of investigation of exactly which nutrient, carbon source, or combination thereof leads to the reduced merozoite number, it is unclear if or how much this is relevant to the scenario of a natural infection and realistic levels of that nutrient in a human or primate parasite environment.

      As rightfully pointed out by the reviewer suboptimal growth conditions affecting parasite growth and multiplication rate have been shown in many instances. The number of studies that actually quantify a reduction in merozoite number under different growth conditions is certainly much lower (Brancucci et al. 2017 (lipids), Mancio-Silva et al. 2017 (calorie-restriction in mice), Tinto-Font et al. 2022 (temperature) come to mind). What our study adds to this body of literature is to which extent duration of the schizont stage and cell volume are affected in relation to progeny number at the single cell level. Importantly, we wanted to test whether the counter model still holds under these more adverse conditions, which we found to be the case. Along the lines of the work on calorie restriction and the likely implication of isoleucine in the process investigated in the laboratory of Maria Mota, it will be exciting to identify a “limiting factor” in future studies. Indeed, any study done in complete RPMI culture medium can be questioned regarding its physiological relevance and we added a sentence addressing this aspect in the discussion (lines 514ff). Yet, our medium dilution experiments suggest that at least to some degree an extracellular resource is implicated, which makes sense from a biological function point-of-view.

      Minor issues

      The manuscript confuses the terms "less" and "fewer". Fewer should be used for countable nouns (fewer daughter cells, fewer nuclei, fewer merozoites), less for uncountable nouns (e.g. less speed, less volume).

      Thank you for pointing this out. The words have been replaced accordingly.

      I didn't understand lines 93-95; "This excluded a timer and thereby confirmed a counter as the mechanism regulating termination of nuclear multiplication (Klaus et al., 2022). A direct correlation between duration of schizont stage and merozoite number is, however, still missing." If I understand the first sentence concludes that there ought not be a direct correlation between schizont duration and merozoite number, but the second sentence, says that that correlation is "however" missing. Isn't this expected? Perhaps reword for clarity?

      Thank you for requesting clarification here. The exclusion of the timer by Klaus et al. 2022 was based on the correlation between duration of the first nuclear division cycle and the total duration of all nuclear replication phases. At no point did Klaus et al. count merozoites in live single cells, which was mainly due to lower spatial resolution of their images (M. Ganter, personal communication). Therefore, they could not directly assess the relation between progeny number and schizont stage duration, which we now report for the first time. The sentence was supposed to convey that this type of data was missing and was now reformulated for more clarity (line 114).

      Lines 104

      "We further uncover that throughout schizogony P. falciparum infringes on the otherwise ubiquitously constant N/C-ratio (Cantwell and Nurse, 2019)" This seems obvious to me, and not something uncovered by this study. In most of the numerous apicomplexans that divide by endoschizogeny, the cells achieve a near final size considerably before the final rounds of nuclear division so the N/C ratio must not remain constant - this is a direct corollary of many previous descriptions and not a novel finding of this study, and this claim here should be made more modest.

      We understand the point raised by the reviewer but still think that our claim is justified due to several aspects. There are examples of eukaryotic cells that undergo multinucleated stages during division were the N/C-ratio is constant (Dundon et al. 2016, Cantwell and Nurse, 2019), while we are not aware of any counter-example in the literature. Studies have also shown that e.g. certain mutant yeast that fail to undergo cytokinesis will increase their volume by factor of up to 16 alongside the still replicating and growing nucleus maintain the N/C-ratio (Neumann et al. 2007, Jorgensen et al. 2007). This demonstrates the tremendous plasticity that cells can reveal with respect to nucleus and cell size regulation. Until the contrary was shown, it was conceivable that nuclear compaction, which does occur (Fig. 5H), compensates for the increase in nuclear number while the cell volume is only increasing slightly. Importantly, we are not aware of any literature where nuclear volume has been quantified for blood stage Plasmodium. Cell volume quantifications remain limited to modelling and the study by Waldecker et al., which provides a few datapoints throughout the IDC. Whether this finding is expected or not, formally speaking, our claim is justified, but for more clarity we replace “uncover” with “demonstrate”. We also introduce the N/C-ratio as cellular parameter in P. falciparum pointing out another divergent aspect of its biology and might in the future understand the functional implication of this usually constant ratio, which is still unclear.

      Dundon SE, Chang SS, Kumar A, Occhipinti P, Shroff H, Roper M, Gladfelter AS. Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium. Mol Biol Cell. 2016 Jul 1;27(13):2000-7.

      Neumann FR, and Nurse P. Nuclear size control in fission yeast. J. Cell Biol. 2007; 179: 593–600. pmid:17998401

      Jorgensen P, Edgington NP, Schneider BL, Rupeš I, Tyers M & Futcher B Molecular Biology of the Cell 18 (2007) The size of the nucleus increases as yeast cells grow.

      Helena Cantwell, Paul Nurse; A homeostatic mechanism rapidly corrects aberrant nucleocytoplasmic ratios maintaining nuclear size in fission yeast. J Cell Sci; 132 (22)

      I lack specialist statistical knowledge to comment on the statistical analyses performed on the correlation data, and in particular, whether the high p values for t-Tests for correlation are sufficient to support the argument that there is not a correlation, and whether these observations are sufficiently powered to robustly test that hypothesis.

      We are confident that our reworked model analysis, as explained above, now sufficiently supports our hypotheses.

      Reviewer #2 (Significance):

      The manuscript purports to find a counting mechanism that determines parasite merozoite numbers, and that this coutner is set by an externally provided and diffusible resource. Many nutrients are in excess in normal culture media, but not all. If that counted nutrient(s) were normally in excess in the bloodstream, it could hardly be said to be the factor that is counted and that therefore defines merozoite number. Conversely, if the amount of that nutrient were increased in normal media, would parasites make even more merozoites? Further, if the "counted" item is a freely diffusible compound in the media, it should be equally accessible to each parasite in a culture condition, and isn't a reasonable explanation for the variable merozoite numbers in the normal media conditions. To me, it is unsurprising that parasites that are healthy and well fed are able to produce more merozoites, but I don't see this as being the same as support for a counter model where the parasite senses and counts a set number of merozoites to produce in response to a specific external counter. I think the shoehorning of this phenomenon into a paradigm used to describe some other eukaryotes may not be appropriate, and that the rejection of one overly simplistic timer model should not automatically lead to us dichotomously accepting a simple counter method as the alternative. The authors need to do more to either identify a countable input whose gradual increase leads to a predictable and gradual increase in merozoite number, to show that they do use a counter, or provide substantially more caveats to their argument that the parasites are using a counter based on an externally provided resource to determine merozoite number.

      The reviewer comments on the feasibility of a counter mechanism based on an externally provided and diffusible resource. In fact this is a limited view of how a counter may arise and not the one we subscribe to. Rather, while a resource may be diffusible in the medium, it would need to be consumed during schizogony, and insufficiently replenished, in order to enable counting by dilution in the host cell. Furthermore, the reviewer has doubts that the fact that “healthy and well fed […] produce more merozoites” implies “support for a counter model”. We fully agree, and we argue in the manuscript that it is the correlations between schizogony durations and merozoite counts that support a counter model.

      As we have argued above, the two alternative models we consider are inspired by paradigm from other eukaryotes, but their definitions in the present context are simple enough for them to be considered natural minimal models of schizogony. As the simplest imaginable phenomenological models of multiplication control, we find it natural to compare them, and we hope our new introductory section introduces them appropriately now. Naturally, we hope to expand on this simple model in the future and identify more precisely the limiting resources and describe a more direct response.

      Audience - relatively specialised - likely interested audience would combine apicomplexan cell biologists, as well as theorists of cell division mechanism

      Advance - limited - confirms phenomenon also described by other researchers in their institute, and extends to another related organism.

      We would like to add that the present data are the first quantitative joint measurements of schizogony dynamics and outcome in P.falciparum and knowlesi. They allowed for the first time a direct correlation of duration and merozoite number, thereby accessing the question of growth control head on. Further they provide a quantitative reference of several key cellular parameters for anybody studying asexual blood stage parasites.

      Reviewer #3 (Evidence, reproducibility and clarity):

      Summary:

      Stürmer and colleagues used super-resolution time-lapse microscopy to probe the mechanism regulating the number of merozoites produced by a single cell in Plasmodium falciparum and P. knowlesi. The authors conclude the followings-

      1. P. knowlesi has similar duration of schizont stage to P. falciparum, although having a 24 h intraerythrocytic developmental cycle (IDC) to 48 h of P. falciparum.
      2. Nuclear multiplication dynamics suggests a counter mechanism of division- which is further suggested by a significant relation of merozoite numbers with schizont size at the onset of division.
      3. Nutritional deprivation caused increase in nuclear volume and decrease in merozoite number. For the most part, the experiments that are presented in this manuscript support the conclusion of the authors. The data are presented in a concise and clear manner. However, some clarification and a couple of experiment (listed below) would improve this manuscript.

      Major comments:

      1. The authors generated at least 3 transgenic lines for this study, But the did not present any genetic validation of the lines in the manuscript. For completeness, I recommend to provide genetic validation (either pcr genotyping or whole genome sequencing) of the lines that were generated and used in this study in the supplement.

      Our study exclusively used episomal expression of the respective fluorescent reporter (H2B-GFP, NLS-mCherry, and cytoplasmic GFP). As is customary in the field resistance to selection drugs and distinct fluorescent signals are assumed to sufficiently validate the presence of the plasmids. We now added the schematic maps of the plasmids in a new Fig. S1 to make our approach more visually clear.

      1. In the H2B-GFP lines, the authors episomally GFP-tagged histone 2B to label the nuclear chromatin for both P. falciparum and P. knowlesi. This provides a very useful parasite line which enables the live time-lapse microscopy. Using these parasite lines, the authors first show that despite having a 24 h IDC in P. knowlesi vs 48 h in P. falciparum, both these parasites have a similar duration of the schizont stage (8.s vs 9.4 h). My concern here is whether this GFP-tagging is influencing the growth dynamics as in slowing down the P. knowlesi parasites. However, if that was the case authors should have seen that for P. falciparum too. Also, for the P. falciparum parasites that episomally express cytosolic GFP and Nuclear mCherry have a higher number of merozoites compared to the H2B-GFP P. falciparum and the authors speculate this is probably because of not tagging Histone 2B. Given this, it is important to show that none of the H2B-GFP parasites show any significant fitness cost due to GFP tagging of histone. I recommend a simple experiment to compare the multiplication rate of H2B-GFP lines to the parental lines in identical growth conditions. This suggested experiment was described in PMID: 35164549 to determine fitness cost of knockout lines. This experiment is vital for validation of the H2B-GFP lines and subsequent interpretation of the data that were presented in this manuscript.

      We thank the reviewer for this excellent suggestion. To validate our lines further we now have carried out multiplication rate measurements similar to the one described in the designated publication for all the used lines alongside their parental strains (Fig. S2). We found no significant differences in between the wild type and the episomally expressing parasite lines (lines 131ff), which gives us confidence that episomal expression of tagged proteins do not significantly alter growth dynamics in these cases.

      1. The authors used the microtubule live cell dye SPY555-Tubulin in P. falciparum to validate the findings presented in 1D and 1E. They did not do that for P. knowlesi. If there is no unsurmountable technical difficulty, I suggest doing the same with P. knowlesi. This will also address the concern that I have pointed out in #1.

      Thank you for this suggestion. We have now generated the requested data with P. knowlesi, added it to what is now Supplemental Figure 3 and included it in our new analysis (Fig. 2I-J). The numerical values align well with the observations made when measuring schizont stage dynamics with the H2B-GFP expressing P. knowlesi line (line 158). A notable difference is that the Tubulin data strongly support the (refined) counter model, while the H2B data alone allow no distinction.

      1. The data in Figure 3 shows that merozoite number does not depend on host cell diameter. My question here is, were these data collected using different donor blood? Or were this measured from different biological replicate? These are not clear from the writing. I am not sure about whether blood from various donor would have on the data, however, different preparation of the cells across various biological replicate will have some effect on host cell diameter hence on data. State if these were collected from independent biological replicates and about the donor blood.

      The data results where indeed collected from three independent biological replicates using different donor blood batches. This is now stated in the figure legend. The batches displayed no difference in RBC diameter.

      1. It is interesting to see that nutrient-limited conditions increase average nuclear volume but less merozoite numbers. In this experiment, as I understand, complete media was diluted 0.5x, which basically diluted every component of the media by half. From this experiment I can see nutritional deprivation as a whole having an effect and supports the counter mechanism, it would be intriguing to see if there is any effect of a particular nutrient have any effect on progeny division. For example, parasites can be grown in amino acid deprived media (except isoleucine) which makes the parasites fully dependent on host cell amino acids. This sort of specific nutrient deprivation will probably allow the authors to probe for specific nutrients that plays role as counter mechanism factor.

      This is indeed a very exciting direction we would like to investigate in more detail in follow-up studies. Our aim for this study was to confirm that nutrient deprivation actually affects “counting” and to provide a workflow to investigate individual nutrients. In the meantime the Mota group, in a study we now cite in the discussion (lines 507ff), actually reported that isoleucine (and possibly methionine) levels are linked to progeny number. A follow-up on this topic using our strains and methodology is certainly worthwhile but requires more detailed analysis in the future.

      Minor comments:

      1. P. knowlesi is sometimes just written as knowlesi. Please, write P. Knowlesi.

      Has been corrected.

      1. Supplemental figure 1D, missing x-axis label.

      We added the x-axis label.

      1. In line 105, define N/C.

      Done.

      1. In line 205, I assume the authors mean episomally, not episomally.

      Thank you for pointing this out. We have replaced “ectopically” with “episomally” throughout the text.

      1. In line 275, Duration of Schizont stage was slightly....

      Has been corrected.

      1. All 'ml' or 'µl' should be 'mL' or 'µL'.

      Changes have been made.

      1. Define iRPMI.

      We added a definition (line 610).

      1. In line 475, replace 'as' with 'and'.

      Done.

      Reviewer #3 (Significance):

      The factors that regulate the number of progenies in malaria parasites remain unknown. While there are few previous studies attempting to answer the question, those studies were done on fixed stained cells. In this study, the authors used genetically modified fluorescent P. falciparum and P. knowlesi parasites that enable live microscopy. These parasites coupled with super-resolution time-lapse microscopy the authors attempt to investigate the mechanism(s) at play in regulating progeny division. This manuscript provides data to suggest that external resources might have some role in progeny division and supports the counter mechanism. More careful validation of the transgenic lines that were used to collect data presented needs to be more systematic and rigorous.

    1. Author Response

      The following is the authors’ response to the original reviews.

      eLife assessment

      This manuscript analyzes large-scale Neuropixels recordings from visual areas and hippocampus of mice passively viewing repeated clips of a movie and reports that neurons respond with elevated firing activities to specific, continuous sequences of movie frames. The important results support a role of rodent hippocampal neurons in general episode encoding and advance understanding of visual information processing across different brain regions. The strength of evidence for the primary conclusion is solid, but some technical limitations of the study were identified that merit further analyses.

      We thank the editors and reviews for the assessment and reviews. We have provided clarifications and updated the manuscripts to address the seeming technical limitations that are perhaps due to some misunderstanding, please see below. We provide additional results that isolate the contribution of pupil diameter, sharpwave ripple and theta power to show that movie tuning cannot be explained by these nonspecific effects. Nor are these mere time cells or some other internally generated patterns due to many differences highlighted below.

      Reviewer #1 (Public Review):

      Taking advantage of a publicly available dataset, neuronal responses in both the visual and hippocampal areas to passive presentation of a movie are analyzed in this manuscript. Since the visual responses have been described in a number of previous studies (e.g., see Refs. 11-13), the value of this manuscript lies mostly on the hippocampal responses, especially in the context of how hippocampal neurons encode episodic memories. Previous human studies show that hippocampal neurons display selective responses to short (5 s) video clips (e.g. see Gelbard-Sagiv et al, Science 322: 96-101, 2008). The hippocampal responses in head-fixed mice to a longer (30 s) movie as studied in this manuscript could potentially offer important evidence that the rodent hippocampus encodes visual episodes.

      We have now included citations to Gelbard-Sagiv et al. Science 2008 paper and many other references too, thank you for pointing that out. There are major differences between that study and ours.

      a. The movies used in previous study contained very familiar, famous people and famous events, and the experiment was about the patient’s ability to recall those famous movie episodes. In our case the mice had seen this movie clip only in two habituation sessions before.

      b. They did not look at the fine structure of neural responses below half a second whereas we looked at the mega-scale representations from 30ms to 30s.

      c. The movie clips in that study were in full color with audio, we used an isoluminant, black-and-white, silent movie clip.

      d. Their movie clips contained humans and was observed by humans, whereas our study mice observed a movie clip with humans and no mice or other animals.

      The analysis strategy is mostly well designed and executed. A number of factors and controls, including baseline firing, locomotion, frame-to-frame visual content variation, are carefully considered. The inclusion of neuronal responses to scrambled movie frames in the analysis is a powerful method to reveal the modulation of a key element in episodic events, temporal continuity, on the hippocampal activity. The properties of movie fields are comprehensively characterized in the manuscript.

      Thank you.

      Although the hippocampal movie fields appear to be weaker than the visual ones (Fig. 2g, Ext. Fig. 6b), the existence of consistent hippocampal responses to movie frames is supported by the data shown. Interestingly, in my opinion, a strong piece of evidence for this is a "negative" result presented in Ext. Fig. 13c, which shows higher than chance-level correlations in hippocampal responses to same scrambled frames between even and odd trials (and higher than correlations with neighboring scrambled frames). The conclusion that hippocampal movie fields depend on continuous movie frames, rather than a pure visual response to visual contents in individual frames, is supported to some degree by their changed properties after the frame scrambling (Fig. 4).

      Yes, hippocampal selectivity is not entirely abolished with scrambled movie, as we show in several figures (Figure 4d,g and Figure 4- figure supplement 6), but it is greatly reduced, far more than that in the afferent visual cortices. The fraction of tuned cells for scrambled movies dropped to 4.5% in hippocampus, which is close to the chance level of 3%. In contrast, in visual areas selectivity was still above 80%.

      Significant overlap between even and odd trials is to be expected for the tuned cells. Without a significant overlap, i.e. a stable representation, they will not be tuned. Despite this, the correlation between even and odd trials for the (only 4.5% of) tuned cells in the hippocampus was more than 2-fold smaller than (more than 80% of) cells in visual cortices. This strongly supports our hypothesis that unlike visual cortices, hippocampal subfields depended very strongly on the continuity of visual information. We have now clarified this in the main text.

      However, there are two potential issues that could complicate this main conclusion.

      One issue is related to the effect of behavioral variation or brain state. First, although the authors show that the movie fields are still present during low-speed stationary periods, there is a large drop in the movie tuning score (Z), especially in the hippocampal areas, as shown in Ext. Fig. 3b (compared to Ext. Fig. 2d). This result suggests a potentially significant enhancement by active behavior.

      There seems to be some misunderstanding here. There was no major reduction in movie tuning during immobility or active running. As we wrote in the manuscript, the drop in selectivity during purely immobile epochs is because of reduction in the amount of data, not reduction in selectivity per se. Specifically, as the amount data reduces, the statistical strength of tuning (z-scored sparsity) reduces. For example, if we split the total of 60 trials worth of data into two parts, the amount of data reduces to about half in each part, leading to a seeming reduction in selectivity in both halves. Figure 1-figure supplement 4c shows nearly identical tuning in all brain regions during immobility (red bars) and equivalent subsamples (yellow-orange) chosen randomly from the entire data, including mobility and immobility. We also show that the movie tuning persists in sessions with and without prolonged running behavior (Figure 1-figure supplement 7), as well as by splitting the data based on pupil dilation or theta power. Please see below for more details.

      Second, a general, hard-to-tackle concern is that neuronal responses could be greatly affected by changes in arousal or brain state (including drowsy or occasional brief slow-wave sleep state) in head-fixed animals without a task. Without the analysis of pupil size or local field potentials (LFPs), the arousal states during the experiment are difficult to know.

      In the revised manuscript we show that the behavioral state effects cannot explain movie tuning. Specifically:

      a. We compared sessions in which the mouse was mostly immobile versus sessions in which the mouse was mostly running. Movie tuned cells were found in both these cases (Figure 1-figure supplement 7).

      b. We detected and removed all data around sharp-wave ripples (SWR). Movie tuning was unchanged in the remaining data. (Figure 1-figure supplement 6).

      c. As a further control, we quantified arousal by two standard metrics. First within a session, we split the data into two groups, segments with high theta power and segments with low theta power. Significant movie tuning persisted in both.

      d. Finally, pupil dilation is another common method to estimate arousal, so data within a session were split into two parts: those with pupil dilation versus constriction. Movie tuning remained significant in both parts. See the new Figure 1-figure supplement 7.

      Many example movie fields in the presented raw data (e.g., Fig. 1c, Ext. Fig. 4) are broad with low-quality tuning, which could be due to broad changes in brain states. This concern is especially important for hippocampal responses, since the hippocampus can enter an offline mode indicated by the occurrence of LFP sharp-wave ripples (SWRs) while animals simply stay immobile. It is believed that the ripple-associated hippocampal activity is driven mainly by internal processing, not a direct response to external input (e.g., Foster and Wilson, Nature 440: 680, 2006). The "actual" hippocampal movie fields during a true active hippocampal network state, after the removal of SWR time periods, could have different quantifications that impact the main conclusion in the manuscript.

      We included the broadly tuned hippocampal neurons to demonstrate the movie-field broadening compared to those in visual areas. We now include more examples with sharp movie fields in the hippocampal regions (Figure 1a-d right column, 2d and h, Figure 1-figure supplement 5 and Figure 2-figure supplement 1). Further, as stated above, we detected sharp-wave ripples and removed one second of data around SWR. Movie tuning was unchanged in the remaining data. Thus, movie tuning is not generated internally via SWR (Figure 1-figure supplement 6). See also Figure 1-figure supplement 7 and Figure 2-figure supplement 8 and the response above.

      Another issue is related to the relative contribution of direct visual response versus the response to temporal continuity in movie fields. First, the data in Ext. Fig. 8 show that rapid frame-to-frame changes in visual contents contribute largely to hippocampal movie fields (similarly to visual movie fields).

      There seems to be some misunderstanding here. That figure showed that the frame-to-frame changes in the visual content had the highest effect on visual areas MSUA and much weaker in hippocampus (Extended Data Fig. 8, as per previous version, now Figure3-figure supplement 2). For example, the depth of modulation (max – min) / (max + min) for MSUA was 21% and 24% for V1 but below 6% for hippocampal regions. Similarly, the MSUA was more strongly (negatively) correlated with F2F correlation for visual areas (r=0.48 to 0.56) than hippocampal (0.07 to 0.3). Similarly, comparing the number of peaks or their median widths, visual regions showed stronger correlation with F2F, and largest depth of modulation than hippocampal regions, barring handful exceptions (like CA3 correlation between F2F and median peak duration). This strongly supports our claim that visual regions generated far greater response of the frame-to-frame changes in the movie than hippocampal regions.

      Interestingly, the data show that movie-field responses are correlated across all brain areas including the hippocampal ones.

      In Figure 3c we compared the MSUA responses with normalization between brain regions. Amongst the 21 possible brain region pairs, 5 were uncorrelated, 7 were significantly negatively correlated and 9 were significantly positively correlated.

      The changes in population overlap, number and widths of peaks are strongly correlated only between visual areas and some of the hippocampal region pairs. The correlation is much weaker for hippocampal-visual area pairs, but often significantly different from chance. This is quantified explicitly in the revised text Figure 3-figure supplement 2 with an additional correlation matrix at the right.

      This could be due to heightened behavioral arousal caused by the changing frames as mentioned above, or due to enhanced neuronal responses to visual transients, which supports a component of direct visual response in hippocampal movie fields.

      As shown in Figure 1-figure supplements 4,5,6 and 7 and described above, the effect of arousal as quantified by theta power of pupil diameter (or by accounting for running behavior or SWR occurrences) cannot explain the results in hippocampal areas and the correlations in multiunit responses are unrelated across many brain areas.

      Second, the data in Ext. Fig. 13c show a significant correlation in hippocampal responses to same scrambled frames between even and odd trials, which also suggests a significant component of direct visual response.

      This is plausible. The fraction of hippocampal cells which were significantly tuned for the scrambled presentation (4.5%) was close to chance level (3%), and this small subset of cells was used to compute the population overlap between even and odd trials in Figure 4-figure supplement 6 (Ext Fig. 13 with old numbering). As described above, this significant but small amount of tuning could generate significant population overlap, which is to be expected by construction.

      Is there a significant component purely due to the temporal continuity of movie frames in hippocampal movie fields? To support that this is indeed the case, the authors have presented data that hippocampal movie fields largely disappear after movie frames are scrambled. However, this could be caused by the movie-field detection method (it is unclear whether single-frame field could be detected).

      As described in the methods section, the movie-field detection algorithm had a resolution of 3.3ms resolution, which ensured that we could detect single frame fields. As reported, we did find such short movie fields in several cells in the visual areas. The sparsity metric used is agnostic to the ordering of the responses, and hence single frame field, and the resultant significant movie-tuning, if present, can be detected by our methods.

      Another concern in the analysis is that movie-fields are not analyzed on re-arranged neural responses to scrambled movie frames. The raw data in Fig. 4e seem quite convincing. Unfortunately, the quantifications of movie fields in this case are not compared to those with the original movie.

      We saw very few (3.6-4.9%) cells with significant movie tuning for scrambled presentation in the hippocampus. Hence, we did not quantify this earlier. This is now provided in new Figure 4-figure supplement 5. The amount of movie tuning for the scrambled presentation taken as-is, or after rearranging the frames is below 5% for all hippocampal brain regions and not significantly different between the two.

      Reviewer #2 (Public Review):

      Purandare and Mehta investigated the neural activities modulated by continuous and sequential visual stimuli composed of natural images, termed "movie-tuning," measured along the visuo-hippocampal network when the animals passively viewed a movie without any task demand. Neurons selectively responded to some specific parts of the movie, and their activity timescales ranged from tens of milliseconds to seconds and tiled the entire movie with their movie-fields. The movie-tuning was lost in the hippocampus but not in the visual cortices when the image frames were temporally scrambled, implying that the rodent hippocampus encoded the specific sequence of images.

      The authors have concluded that the neurons in the thalamo-cortical visual areas and the hippocampus commonly encode continuous visual stimuli with their firing fields spanning the mega-scale, but they respond to different aspects of the visual stimuli (i.e., visual contents of the image versus a sequence of the images). The conclusion of the study is fairly supported by the data, but some remaining concerns should be addressed.

      1) Care should be taken in interpreting the results since the animal's behavior was not controlled during the physiological recording.

      This was done intentionally since plenty of research shows that task demand (e.g., Aronov and Tank, Nature 2017) can not only modulate hippocampal responses but also dramatically alter them. We have now provided additional figures (Figure 1-figure supplement 6 and 7) where we quantified the effects of the behavioral states (sharp wave ripples, theta power and pupil diameter), as well as the effect of locomotion (Figure 1-figure supplement 4). Movie tuning remained unaffected with these manipulations. Thus, movie tuning cannot be attributed to behavioral effects.

      It has been reported that some hippocampal neuronal activities are modulated by locomotion, which may still contribute to some of the results in the current study. Although the authors claimed that the animal's locomotion did not influence the movie-tuning by showing the unaltered proportion of movie-tuned cells with stationary epochs only, the effects of locomotion should be tested in a more specific way (e.g., comparing changes in the strength of movie-tuning under certain locomotion conditions at the single-cell level).

      Single cell analysis of the effect of locomotion and visual stimulation is underway, and beyond the scope of the current work. As detailed in Figure 1-figure supplement 4, we have ensured that in spite of the removal of running or stationary epochs, as well as removal of sharp wave ripple events (Figure 1-figure supplement 6) movie tuning persists. Further, we now provide examples of strongly tuned cells from sessions with predominantly running or predominantly stationary behavior (Figure 1-figure supplement 7).

      2) The mega-scale spanning of movie-fields needs to be further examined with a more controlled stimulus for reasonable comparison with the traditional place fields. This is because the movie used in the current study consists of a fast-changing first half and a slow-changing second half, and such varying and ununified composition of the movie might have largely affected the formation of movie-fields. According to Fig. 3, the mega-scale spanning appears to be driven by the changes in frame-to-frame correlation within the movie. That is, visual stimuli changing quickly induced several short fields while persisting stimuli with fewer changes elongated the fields.

      Please note that a strong correlation between the speed at which the movie scene changed across frames was correlated with movie-field width in the visual areas, but that correlation was much weaker in the hippocampal areas (correlation values - (LGN +0.61, V1 +0.51, AM-PM +0.55 vs. DG +0.39, CA3 +0.58, CA1 +0.42, SUB +0.24). Please see Figure 3-figure supplement 2 and the quantification of correlation between frame-to-frame changes in the movie and the properties of movie fields.

      The presentation of persisting visual input for a long time is thought to be similar to staying in one place for a long time, and the hippocampal activities have been reported to manifest in different ways between running and standing still (i.e., theta-modulated vs. sharp wave ripple-based). Therefore, it should be further examined whether the broad movie-fields are broadly tuned to the continuous visual inputs or caused by other brain states.

      As shown in Figure 1-figure supplement 6, movie field properties are largely unchanged when SWR are removed from the data, or when the effect of pupil diameter or theta power were factored for (Figure 1-figure supplement 7).

      3) The population activities of the hippocampal movie-tuned cells in Fig. 3a-b look like those of time cells, tiling the movie playback period. It needs to be clarified whether the hippocampal cells are actively coding the visual inputs or just filling the duration.

      Tiling patterns would be observed when the maxima are sorted in any data, even for random numbers. This alone does not make them time cells. The following observations suggest that movie fields cannot be explained as being time cells.

      a. Time cells mostly cluster at the beginning of a running epoch (Pastalkova et al. Science 2008, MacDonald et al. Neuron 2011) and they taper off towards the end. Such large clustering is not visible in these tiling plots for movie tuned cells.

      b. Time fields become wider as the temporal duration progresses (Pastalkova et al. Science 2008, MacDonald et al. Neuron 2011) as the encoded temporal duration increases. This is not evident in any movie fields.

      c. Widths of movie fields in visual areas, and to a smaller extent in the hippocampal areas, were clearly modulated by the visual content, like the change from one frame to the next (F2F correlation, Figure 3-figure supplement 2).

      d. Tiling pattern of movie fields was found in visual areas too, with qualitatively similar pattern as hippocampus. Clearly, visual area responses are not time cells, as shown by the scrambled stimulus experiment. Here, neural selectivity could be recovered by rearranging them based on the visual content of the continuous movie, and not the passage of time.

      The scrambled condition in which the sequence of the images was randomly permutated made the hippocampal neurons totally lose their selective responses, failing to reconstruct the neural responses to the original sequence by rearrangement of the scrambled sequence. This result indirectly addressed that the substantial portion of the hippocampal cells did not just fill the duration but represented the contents and temporal order of the images. However, it should be directly confirmed whether the tiling pattern disappeared with the population activities in the scrambled condition (as shown in Extended Data Fig. 11, but data were not shown for the hippocampus).

      As stated above for the continuous movie, tiling pattern alone does not mean those are time cells. Further, tuning, and tiling pattern remained intact with scrambled movie in the visual cortices but not in hippocampus. We now added a new supplement figure – Figure 4-figure supplement 5 where we compared the movie tuning for scrambled presentation with and without rearranging the frames. Hippocampal tuning remains at chance levels.

      Reviewer #3 (Public Review):

      In their study, Purandare & Mehta analyze large-scale single unit recordings from the visual system (LGN, V1, extrastriate regions AM and PM) and hippocampal system (DG, CA3, CA1 and subiculum) while mice monocularly viewed repeats of a 30s movie clip. The data were part of a larger release of publicly available recordings from the Allen Brian Observatory. The authors found that cells in all regions exhibited tuning to specific segments of the movie (i.e. "movie fields") ranging in duration from 20ms to 20s. The largest fractions of movie-responsive cells were in visual regions, though analyses of scrambled movie frames indicated that visual neurons were driven more strongly by visual features of the movie images themselves. Cells in the hippocampal system, on the other hand, tended to exhibit fewer "movie fields", which on average were a few seconds in duration, but could range from >50ms to as long as 20s. Unlike the visual system "movie fields" in the hippocampal system disappeared when the frames of the movie were scrambled, indicating that the cells encoded more complex (episodic) content, rather than merely passively reading out visual input.

      The paper is conceptually novel since it specifically aims to remove any behavioral or task engagement whatsoever in the head-fixed mice, a setup typically used as an open-loop control condition in virtual reality-based navigational or decision making tasks (e.g. Harvey et al., 2012). Because the study specifically addresses this aspect of encoding (i.e. exploring effects of pure visual content rather than something task-related), and because of the widespread use of video-based virtual reality paradigms in different sub-fields, the paper should be of interest to those studying visual processing as well as those studying visual and spatial coding in the hippocampal system. However, the task-free approach of the experiments (including closely controlling for movement-related effects) presents a Catch-22, since there is no way that the animal subjects can report actually recognizing or remembering any of the visual content we are to believe they do.

      Our claim is that these are movie scene evoked responses. We make no claims about the animal’s ability to recognize or remember the movie content. That would require entirely different set of experiments. Meanwhile, we have shown that these results are not an artifact of brain states such as sharp wave ripples, theta power or pupil diameter (Figure1-figure supplement 6 and 7) or running behavior (Figure 1-figure supplement 4). Please see above for a detailed response.

      We must rely on above-chance-level decoding of movie segments, and the requirement that the movie is played in order rather than scrambled, to indicate that the hippocampal system encodes episodic content of the movie. So the study represents an interesting conceptual advance, and the analyses appear solid and support the conclusion, but there are methodological limitations.

      It is important to emphasize that these responses could constitute episodic responses but does not prove episodic memory, just as place cell responses constitute spatial responses but that does not prove spatial memory. The link between place cells and place memory is not entirely clear. For example, mice lacking NMDA receptors have intact place cells, but are impaired in spatial memory task (McHugh et al. Cell 1996), whereas spatial tuning was virtually destroyed in mice lacking GluR1 receptors, but they could still do various spatial memory tasks (Resnik et al. J. Neuro 2012).

      The experiments about episodic memory would require an entirely different set of experiments that involve task demand and behavioral response, which in turn would modify hippocampal responses substantially, as shown by many studies. Our hypothesis here, is that just like place cells, these episodic responses without task demand would play a role, to be determined, in episodic memory. We have emphasized this point in the main text (Ln 391-393 in the revised manuscript).

      Major concerns:

      1) A lot hinges on hinges on the cells having a z-scored sparsity >2, the cutoff for a cell to be counted as significantly modulated by the movie. What is the justification of this criterion?

      The z-scored sparsity (z>2) corresponds to p<0.03. This would mean that 3% of the results could appear by chance. Hence, z>2 is a standard method used in many publications. Another advantage of z-scored sparsity is that it is relatively insensitive to the number of spikes generated by a neuron (i.e. the mean firing rate of the neuron and the duration of the experiment). In contrast, sparsity is strongly dependent on the number of spikes which makes it difficult to compare across neurons, brain regions and conditions (See Supplement S5 Acharya et al. Cell 2016).

      To further address this point, we compared our z-scored sparsity measure with 2 other commonly used metrics to quantify neural selectivity, depth of modulation and mutual information (Figure 1-figure supplement 3). Comparable movie tuning was obtained from all 3 metrics, upon z-scoring in an identical fashion.

      It should be stated in the Results. Relatedly, it appears the formula used for calculating sparseness in the present study is not the same as that used to calculate lifetime sparseness in de Vries et al. 2020 quoted in the results (see the formula in the Methods of the de Vries 2020 paper immediately under the sentence: "Lifetime sparseness was computed using the definition in Vinje and Gallant").

      The definition of sparsity we used is used commonly by most hippocampal scientists (Treves and Rolls 1991, Skaggs et al. 1996, Ravassard et al. 2013). Lifetime sparseness equation used by de Vries et al. 2020, differs from us by just one constant factor (1-1/N) where N=900 is the number of frames in the movie. This constant factor equals (1-1/900)=0.999. Hence, there is no difference between the sparsity obtained by these two methods. Further, z-scored sparsity is entirely unaffected by such constant factors. We have clarified this in the methods of the revised manuscript.

      To rule out systematic differences between studies beyond differences in neural sampling (single units vs. calcium imaging), it would be nice to see whether calculating lifetime sparseness per de Vries et al. changed the fraction "movie" cells in the visual and hippocampal systems.

      As stated above, the two definitions of sparsity are virtually identical and we obtained similar results using two other commonly used metrics, which are detailed in Figure 1-figure supplement 3.

      2) In Figures 1, 2 and the supplementary figures-the sparseness scores should be reported along with the raw data for each cell, so the readers can be apprised of what types of firing selectivity are associated with which sparseness scores-as would be shown for metrics like gridness or Raleigh vector lengths for head direction cells. It would be helpful to include this wherever there are plots showing spike rasters arranged by frame number & the trial-averaged mean rate.

      As shown in several papers (Aghajan et al Nature Neuroscience 2015, Acharya et al., Cell 2016) raw sparsity (or information content) are strongly dependent on the number of spikes of a neuron. This makes the raw values of these numbers impossible to compare across cells, brain regions and conditions. (Please see Supplement S5 from Acharya et al., Cell 2016 for details). Including the data of sparsity would thus cause undue confusion. Hence, we provide z-scored sparsity. This metric is comparable across cells and brain regions, and now provided above each example cell in Figure 1 and Figure 1-figure supplement 2.

      3) The examples shown on the right in Figures 1b and c are not especially compelling examples of movie-specific tuning; it would be helpful in making the case for "movie" cells if cleaner / more robust cells are shown (like the examples on the left in 1b and c).

      We did not put the most strongly tuned hippocampal neurons in the main figures so that these cells are representative of the ensemble and not the best possible ones, so as to include examples with broad tuning responses. We have clarified in the legend that these cells are some of the best tuned cells. Although not the cleanest looking, the z-scored sparsity mentioned above the panels now indicates how strongly they are modulated compared to chance levels. Additional examples, including those with sharply tuned responses are shown in Figure 1-figure supplement 5 and Figure 2-figure supplement 1.

      4) The scrambled movie condition is an essential control which, along with the stability checks in Supplementary Figure 7, provide the most persuasive evidence that the movie fields reflect more than a passive readout of visual images on a screen. However, in reference to Figure 4c, can the authors offer an explanation as to why V1 is substantially less affected by the movie scrambling than it's main input (LGN) and the cortical areas immediately downstream of it? This seems to defy the interpretation that "movie coding" follows the visual processing hierarchy.

      This is an important point, one that we find very surprising as well. Perhaps this is related to other surprising observations in our manuscript, such as more neurons appeared to be tuned to the movie than the classic stimuli. A direct comparison between movie responses versus fixed images is not possible at this point due to several additional differences such as the duration of image presentations and their temporal history.

      The latency required to rearrange the scrambled responses (60ms for LGN, 74ms for V1, 91ms for AM/PM) supports the anatomical hierarchy. The pattern of movie tuning properties was also broadly consistent between V1 and AM/PM (Figure 2).

      However, all metrics of movie selectivity (Figure 2) to the continuous movie showed a consistent pattern that was the exact opposite pattern of the simple anatomical hierarchy: V1 had stronger movie tuning, higher number of movie fields per cell, narrower movie-field widths, larger mega-scale structure, and better decoding than LGN. V1 was also more robust to the scrambled sequence than LGN. One possible explanation is that there are other sources of inputs to V1, beyond LGN, that contribute significantly to movie tuning. This is an important insight and we have modified the discussion (Ln 315-325) to highlight this.

      Relatedly, the hippocampal data do not quite fit with visual hierarchical ordering either, with CA3 being less sensitive to scrambling than DG. Since the data (especially in V1) seem to defy hierarchical visual processing, why not drop that interpretation? It is not particularly convincing as is.

      The anatomical organization is well established and an important factor. Even when observations do not fit the anatomical hierarchy, it provides important insights about the mechanisms. All properties of movie tuning (Figure 2) –the strength of tuning, number of movie peaks, their width and decoding accuracy firmly put visual areas upstream of hippocampal regions. But, just like visual cortex there are consistent patterns that do not support a simple feed-forward anatomical hierarchy. We have pointed out these patterns so that future work can build upon it.

      5) In the Discussion, the authors argue that the mice encode episodic content from the movie clip as a human or monkey would. This is supported by the (crucial) data from the scrambled movie condition, but is nevertheless difficult to prove empirically since the animals cannot give a behavioral report of recognition and, without some kind of reinforcement, why should a segment from a movie mean anything to a head-fixed, passively viewing mouse?

      We emphasize once again that our claim is about the nature of encoding of the movie across these neurons. We make no claims about whether this forms a memory or whether the mouse is able to recognize the content or remember it. Despite decades of research, similar claims are difficult to prove for place cells, with plenty of counter examples (See the points above). The important point here is that despite any cognitive component, we see remarkably tuned responses in these brain areas. Their role in cognition would take a lot more effort and is beyond the scope of the current work.

      Would the authors also argue that hippocampal cells would exhibit "song" fields if segments of a radio song-equally arbitrary for a mouse-were presented repeatedly? (reminiscent of the study by Aronov et al. 2017, but if sound were presented outside the context of a task). How can one distinguish between mere sequence coding vs. encoding of episodically meaningful content? One or a few sentences on this should be added in the Discussion.

      Aronov et al 2017, found the encoding of an audio sweep in hippocampus when the animals were doing a task (release the lever at a specific frequency to obtain a reward). However, without a task demand they found that hippocampal neurons did not encode the audio sequence beyond chance levels. This is at odds with our findings with the movie where we see strong tuning despite any task demand or reward. These results are consistent with but go far beyond our recent findings that hippocampal (CA1) neurons can encode the position and direction of motion of a revolving bar of light (Purandare et al. Nature 2022). Please see Ln 373-382 for related discussion.

      These responses are unlikely to be mere sequence responses since the scrambled sequence was also fixed sequence that was presented many times and it elicited reliable responses in visual areas, but not in hippocampus. Hence, we hypothesize that hippocampal areas encode temporally related information, i.e. episodic content. We have modified the discussion to address these points.

      Reviewer #1 (Recommendations For The Authors):

      1) Are LFP data available in the data set? If so, can SWRs identified and removed to refine the quantification of movie fields?

      Done, see Figure 1-figure supplement 6.

      2) Can movie fields be analyzed in re-arranged neural responses (Fig. 4e) and compared to those in other cases already shown (Fig. 4b, c)?

      Done, even after rearrangement the strength of movie tuning for the scrambled presentation was low, and below 5% in all hippocampal regions. See Figure 4-figure supplement 5 for details.

      3) It seems the authors are not fully committed to a main conclusion in the present manuscript. The title and abstract seem to emphasize the similar movie responses across visual and hippocampal areas, but the introduction and discussion emphasize the episode encoding of hippocampal neurons. The writing could be more consistent and the main message could be clearer.

      Selective responses to the continuous movie showed similar patterns (prevalence of tuning, multi-peaked nature, relation with frame to frame changes in visual images) between visual and hippocampal regions. But the visual responses to scrambled presentation could be rearranged, and the latency for rearrangement increased from LGN to V1 to AM-PM. On the other hand, selectivity to the scrambled presentation was virtually abolished in hippocampus, and responses could not be rearranged to resemble the continuous movie sequences. To reconcile these differences, we have hypothesized here that the hippocampal responses are episodic in nature, and rely on temporal continuity, whereas the visual regions rely directly on the visual content in the images.

      4) Line #158: "Net movie-field discharges was also comparable across brain areas...". This statement is not supported by Fig. 2g, which shows a wide range of median values across brain areas.

      Thank you for pointing this out. The normalized firing in movie-fields used in that figure are within 3x between V1 and subiculum. We have modified the text to contrast this with the 10x difference between movie-field durations.

      5) Line #253: What the two numbers (87.8%, 10.6%) mean is unclear (mean or median values). These numbers also appear inconsistent with the mean+-se values in Fig. 4 legend.

      The numbers mentioned on Ln253, in the main text reflect the median visual continuity index, combining across cells from hippocampal or visual regions. On the other hand, values reported in the Fig 4 legend are for V1 and subiculum, which are the regions with smallest and largest visual continuity index, respectively. We have re-written the main text, and legends for better clarity.

      6) The Gelbard-Sagiv et al paper (Science 322: 96-101, 2008) could be cited and its relevance to the present study could be discussed.

      Done

      7) Are there neurons recorded from a non-visual sensory or motor cortical area in the same experiment? This may provide a key negative control for the non-specific modulation caused by behavioral states or visual transients.

      Owing to the nature of the experiments where the Allen Institute intended to study visual processing, we could not find any of the recorded brain regions without movie selectivity.

      8) The differences in hippocampal and visual move fields between active and stationary time periods could be explicitly quantified.

      We have shown several raster plots where the responses are quite similar during immobile and moving epochs. Our goal is to show that there is indeed comparable movie tuning when the animals is immobile versus any random state. Doing specific analysis of behavioral dependency is difficult because in many sessions the amount of time the mice ran in many sessions was very little. A thorough analysis overcoming these, and other challenges is beyond the scope of this paper.

      Reviewer #2 (Recommendations For The Authors):

      1) The methods to determine the boundaries of the movie-fields should be clarified, and the detected peaks and boundaries should be indicated in the relevant figures (e.g., Fig. 2c, 2d, and 2h) to help readers clearly understand how the movie-fields were defined and how the shapes of the movie-fields look like.

      Done.

      2) When testing the influence of locomotion on movie-tuning in Extended Data Fig. 3, a single cell-based analysis is further needed. For example, you need to check whether the z-scored sparsity within one cell varies or not depending on locomotion conditions (as in Extended Data Fig. 10a-c). In addition, it is recommended to exclude the cells significantly modulated by locomotion (e.g., running velocity) before defining the movie-tuned cells.

      We now show example cells from sessions with or without prolonged running bouts in Figure 1-figure supplement 7 that have strong movie selectivity. We have also assessed the effects of theta power and pupil dilation on movie tuning in that figure. A more thorough analysis of the combined effects of locomotion and movie tuning is underway, but beyond the scope of the current work.

      3) Regarding the time-cell-related issue raised in the public review, it would be nice if the authors confirm whether the tiling patterns of hippocampal subregions have been weakened by presenting the population activities for the scrambled condition as in the visual cortices in Extended Data Fig. 11a.

      We have clarified in the earlier responses, please see above.

      4) In Fig. 4 and Extended Data Fig. 3, the proportion of movie-tuned cells in the hippocampus seems to drop significantly after only a portion of trials under specific conditions were extracted. Although the authors addressed the stability issue by comparing the neural responses between even and odd trials, the concern about whether the movie-tuning is driven by a certain portion of trials still remains. To avoid such misunderstanding, as mentioned in comment no.2, tracking the changes in the z-scored sparsity of one cell between continuous and scrambled conditions should be provided. In addition, according to the methods, the scrambled condition was divided into two blocks of 10 trials each, possibly causing premature movie-tuned activities. Thus, it should be more appropriate to compare with the first 10 trials of each block in the continuous condition.

      Done.

      5) Explanations related to statistical analysis should be added to the methods sections. In Fig. 2a (and related figures with similar analysis), when comparing three or more groups, the Kruskal-Wallis test should be performed first to check whether there is a difference between the groups, and then pairwise comparisons should follow with adjusted p-values for multiple comparisons. Also, in Fig. 4b (and related figures), it seems that the K-S test was performed to test the changes in cell proportion by combining all brain regions, as far as I understand. However, it would be more appropriate to test the proportional changes by a Chi-square test within each region since the total numbers of cells should differ across the regions.

      Yes, we have used the KS test throughout the analyses, unless otherwise mentioned or appropriate.

      6) The labeling for firing rate is 'FR (sp/sec)' in Fig. 1, 2, and 4, but it is 'Firing rate (Hz)' in Fig. 3.

      This has been fixed now, and only Firing rate (Hz), is used throughout. Thank you for pointing this out.

      7) There is a typo in Extended Data Fig. 11b. "... across all tuned responses from (b)." It should be (a) instead of (b).

      Done

      Reviewer #3 (Recommendations For The Authors):

      While the study presents an interesting dataset and conceptual approach, there are ways in which the manuscript should be strengthened.

      Minor concerns:

      1) Related to point (5) above, what content did the hippocampal "movie fields" encode? It would add a substantive dimension to the paper if the authors included examples of what segments of the movie the cells responded to. Are there "pan left" cells, or "man gets in the car" cells? Or was it more arbitrary than that? What is an example of a movie feature lasting 50ms that is stably encoded by a mouse hippocampal neuron?

      We show example cells with very sharply tuned neural responses (Figure 2h). A thorough analysis of the visual content is in progress but beyond the scope of this paper.

      2) Line 24-seems like it should read "Consistent presentation of the movie..." , with "ly" dropped from "consistent".

      Done

      3) Line 43-seems to be missing the article "a", and should read "...despite strong evidence for A hippocampal role in...".

      We rewrote this sentence for better clarity

      4) Line 54-to clarify, the higher visual areas recorded were the anteromedial (AM) and posterior-medial (PM) areas? The text additionally indicates a "medio-lateral" extrastriate area, but there is no such area. Can the text be revised to clear this up?

      Sorry about this confusion, indeed we meant posterior-medial (PM). Thank you for pointing this out.

      5) Line 84, "rate" should be pluralized to "rates".

      Done

      6) Line 108- the extra "But" at the start of the sentence should be removed.

      Done

      7) Figure 2h-was there any particular arrangement for the cells in this sub-panel? If not, could they be grouped by sub-region (or proximity between sub-regions) so it appears less arbitrary?

      Done

      8) Extended data 2 figure legend for (b) is missing a "that": "Fraction of selective neurons that was significantly above chance.... Ranging from 7.1% in CA

      Done

      9) Line 144-145, there is an extra "and" in the sentence: ".... were typically neither as narrow AND nor as prominent...."

      Done

      10) Line 203-the first word in the line should be "frames" (plural).

      Done, thank you for pointing this out

      11) Line 281-in "...scrambled sequence"-"sequence" should be plural. It looks like the same is true in line 882, in the legend title for Extended Data Fig. 11.

      Since we only showed one scrambled sequence (which was repeated 20 times), we rewrote the relevant lines to be “the scrambled sequence”

      12) Line 923-the first sentence of the legend for Extended Data Fig. 14-to what data or study are the authors referring to in saying that "More than 50% of hippocampal place cells shut down during maze exploration."? This was confusing, please clarify.

      This reference has now been added.

    2. Reviewer #1 (Public Review):

      Taking advantage of a publicly available dataset, neuronal responses in both the visual and hippocampal areas to passive presentation of a movie are analyzed in this manuscript. Since the visual responses have been described in a number of previous studies (e.g., see Refs. 11-13), the value of this manuscript lies mostly on the hippocampal responses, especially in the context of how hippocampal neurons encode episodic memories. Previous human studies show that hippocampal neurons display selective responses to short (5 s) video clips (e.g. see Gelbard-Sagiv et al, Science 322: 96-101, 2008). The hippocampal responses in head-fixed mice to a longer (30 s) movie as studied in this manuscript could potentially offer important evidence that the rodent hippocampus encodes visual episodes.

      The analysis strategy is mostly well designed and executed. A number of factors and controls, including baseline firing, locomotion, frame-to-frame visual content variation, are carefully considered. The inclusion of neuronal responses to scrambled movie frames in the analysis is a powerful method to reveal the modulation of a key element in episodic events, temporal continuity, on the hippocampal activity. The properties of movie fields are comprehensively characterized in the manuscript.

      Comments on latest version:

      The new analysis on how behavioral states and hippocampal ripples impacted the tuning of movie fields makes the main finding substantially more convincing. Other relatively minor concerns on the methodology and interpretation are also improved. I do not have further concerns.

    3. Reviewer #3 (Public Review):

      In their study, Purandare & Mehta analyze large-scale single unit recordings from the visual system (LGN, V1, extrastriate regions AM and PM) and hippocampal system (DG, CA3, CA1 and subiculum) while mice monocularly viewed repeats of a 30s movie clip. The data were part of a larger release of publicly available recordings from the Allen Brian Observatory. The authors found that cells in all regions exhibited tuning to specific segments of the movie (i.e. "movie fields") ranging in duration from 20ms to 20s. The largest fractions of movie-responsive cells were in visual regions, though analyses of scrambled movie frames indicated that visual neurons were driven more strongly by visual features of the movie images themselves. Cells in the hippocampal system, on the other hand, tended to exhibit fewer "movie fields", which on average were a few seconds in duration, but could range from >50ms to as long as 20s. Unlike the visual system "movie fields" in the hippocampal system disappeared when the frames of the movie were scrambled, indicating that the cells encoded more complex (episodic) content, rather than merely passively reading out visual input.

      The paper is conceptually novel since it specifically aims to remove any behavioral or task engagement whatsoever in the head-fixed mice, a setup typically used as an open-loop control condition in virtual reality-based navigational or decision making tasks (e.g. Harvey et al., 2012). Because the study specifically addresses this aspect of encoding (i.e. exploring effects of pure visual content rather than something task-related), and because of the widespread use of video-based virtual reality paradigms in different sub-fields, the paper should be of interest to those studying visual processing as well as those studying visual and spatial coding in the hippocampal system.

      Comments on latest version:

      The revised manuscript by Purandare et al. has been improved with the inclusion of additional analyses and discussion, and the changes mainly satisfy the concerns raised in the initial version of the manuscript.

      Regarding the methods, it was particularly helpful that the authors took measures to consider the impact of different states of arousal (pupil diameter), mobility, and SWRs on the expression and significance of movie field tuning, considering the lack of a task structure or behavioral report. Relatedly, the additional metrics applied (information rate and depth of movie field modulation) substantiate the results as based on z-scored sparsity. The explanation of lifetime sparseness as used here vs. in the work of de Vries et al. 2020 was also helpful.

      The addition of more clearly tuned cells also helps the study feel more rooted in solid ground. For clarity, and consistency with the rest of the paper, it would be helpful to add the sparseness metrics above the newly added neural data in the Figure supplements.

      The Discussion also contains elements that help balance both it and the paper as a whole. It draws a clearer distinction between the representation of visual scenes rather than encoding the contents of episodic memory, clarifying that hippocampal neurons were more likely doing the former than the latter. It is also appreciated that the authors added discussion acknowledging that the cortical processing did not quite follow an apparent hierarchical order.

      As a last observation, though the authors assert in their rebuttal that analysis of the visual content encoded in the movie fields is beyond the scope of the study, this would add an interesting dimension to the work. Because, to my awareness, much less is known regarding how the visual and hippocampal systems in rodents encode visual information when the visual input is dynamic and chunked, as with movies. It would prove an interesting addition to the more extensive work on the processing of static visual scenes.

    1. Reviewer #1 (Public Review):

      In this paper, the authors attempt to overcome the "fundamental limitations" of Lempel-Ziv complexity by developing and testing a complexity estimator based on state-space modelling (CSER) that they argue allows higher temporal resolution and spectral decomposition of entropy directly. They test the performance of this approach using MEG, EEG, and ECoG data from monkeys and humans. Although in principle, these developments might be useful for those already using LZ complexity in their analyses, these developments ignore much of the non-LZ entropy community which has already developed related solutions to the same issues. It is thus not clear currently whether this approach is necessary or unique per se:

      • As the authors intimate, LZ is a relatively crude but efficient estimator; it leverages a simple binarization of time points above and below the time series mean to look at patterns (in turn disregarding the magnitude of the signal itself). The unique benefit of LZ in and of itself is not at all clear to this reviewer. It is nearly guaranteed that LZ will be extremely highly correlated with various other common measures of "discrete" entropy (especially permutation entropy, which ranks all time-series points prior to computing motifs/patterns rather than anchor anything by the mean (as does LZ), but nevertheless ignores the value range of the signal). The general appeal of the authors' intended developments to further improve LZ specifically would dramatically boost should they be able to make a case that LZ is somehow special, to begin with.

      • Beyond this, we can now turn to the authors' rationale for the LZ developments proposed. Despite the authors' statement in the abstract that LZ complexity is "the most widely used approach complexity of neural dynamics," to my knowledge, sample entropy (and its multiscale variant, MSE) is much more commonly used in cognitive neuroscience. Such measures of entropy already enjoy several benefits over LZ. First, the continuous magnitude of the signal is relevant in sample entropy (i.e., it is not discrete in the same way as LZ because the values of each data point matter prior to the estimation of patterns). This is important for people in that community because electrophysiologists/neuroimagers often assume the values of the signal to matter (e.g., for ERPs, the magnitude of power, etc.). Ignoring the magnitude of signal values altogether, as in LZ, is a somewhat dramatic choice, especially if the authors then end up arguing that the spectral decomposition of entropy itself is valuable (after signal value ranges have been ignored!). In any case, as far as I know, LZ has never been shown the be more sensitive than e.g., sample entropy/MSE in relation to any outcome variable, but perhaps the authors can provide evidence for this and argue what LZ should practically do that is unique. Second, the use of MSE more easily allows (although not without its challenges) to directly compare spectral power and single/multiscale entropy straight away, which has been done in quite some depth already without the need for a state-space model of any kind (e.g., Kosciessa et al., 2020, PLOS CB). Instead of using a standard spectral power approach and comparing to entropy, the authors propose the spectrally decompose CSER entropy time series directly. Why? What should this do over standard multi-scale entropy approaches (like MSE, which estimate "fast" and "slow" complexity dynamics), which do not require a Fourier? And if they already believe that the spectrum cannot capture entropy (hence rationalizing the use of LZ-type measures in general), why do they want to invoke spectral estimation assumptions into the estimation of entropy when they could just compare the standard spectrum to entropy to begin with, without any complex modelling in between? I just don't see the need for a lot of what is proposed here; the authors provide solutions to problems that (at least for several in this community) may not exist at all.

      • Figure 2: the authors show results descriptively comparing LZ and CSER, but without comparing the two measures directly. The patterns overall look extremely similar; why not correlate the values from the two measures in each dataset to make a case for what CSER is adding here? By eye test, it appears they will be extremely highly correlated, which leaves the reader wondering what CSER (with all of its model complexities and assumptions) has added.

      • On the logic of and evidence for the use of CSER: The use of a state space model to allow estimation of "prediction errors" appears to be akin to a latent autocorrelation model with a lag/step size of 1 time-point, and trained only on prestim baseline data. When a successive time point is "deviant" from that autocorrelative function, the authors argue that this provides a measure of instantaneous entropy. This seems simple at first glance, but it is very difficult for this reviewer to wrap their head around. This approach anchors stim-related entropy estimation to prestim entropy for every subject, disallowing the direct comparison of values across subjects during the stimulus phase itself. This does not directly provide a measure of instantaneous task-related entropy, but a mixture of pre and post stim sources based on a state-space model. Does it need to be this complicated? Why does a simple window-based function not suffice to generate temporal dynamics of entropy without coupling the task-based signal to the prestim period? There are many such approaches already existing in the field.

      • Figure 3: The authors show that gamma-band CSER is the most sensitive. Isn't it true that this is the exact inverse of the dominance of typical spectral effects under such conditions (that across the literature in psychedelics, sleep, and anaesthesia, there are dominant shifts in low-frequency spectral power)? Although low-frequency power is expected to be a dominant determinant of entropy in the entire signal (see Kosciessa et al., 2020, PLOS CB), something else appears to be happening here. At face value, because gamma is the spectral band with the lowest power in every imaging modality we know of, there is inherently less repeatability/autocorrelation in that same signal, which necessarily should produce more "prediction error/instantaneous entropy" in any condition. When the authors then take the "mean difference" of gamma-based entropy values from each of the two conditions in each sample, any condition-based shift in entropy should inherently be easier to detect. In any case, why not simply show these CSER spectral results next to a standard spectrum over the same conditions and then directly compare the unique utility of e.g., gamma power to CSER gamma? And if you compute something like the percent change between conditions for each spectral band, do you maintain gamma dominance?

    1. Reviewer #3 (Public Review):

      Summary:<br /> In the current manuscript, Dekraker and colleagues have demonstrated the ability to align hippocampal subfield parcellations across disparate 3D histology samples that differ in contrast, resolution, and processing/staining methods. In doing so, they validated the previously generated Big-Brain atlas by comparing across seven different ground-truth subfield definitions. This is an impressive effort that provides important groundwork for future in vivo multi-atlas methods.

      Strengths:<br /> DeKraker and colleagues have provided novel evidence for the tremendously complicated curvature/gyrification of the hippocampus. This work underscores the challenge that this complicated anatomy presents in our ability to co-register other types of hippocampal data (e.g. MRI data) to appropriately align and study a structure in which the curvature varies considerably across individuals.

      This paper is also important in that it highlights the utility of using post-mortem histological datasets, where ground truth histology is available, to inform our rigorous study of the in vivo brain.

      This work may encourage readers to consider the limitations of the current methods that they currently use to co-register and normalize their MRI data and to question whether these methods are adequate for the examination of subfield activity, microstructure, or perfusion in the hippocampal head, for example. Thus the implications of this work could have a broad impact on the study of hippocampal subfield function in humans.

      Weaknesses:<br /> As the authors are well aware, hippocampal subfield definitions vary considerably across laboratories. For example, some neuroanatomists (Ding, Palomero-Gallagher, Augustinack) recognize that the prosubiculum is a distinct region from subiculum and CA1 but others (e.g. Insausti, Duvernoy) do not include this as a distinct subregion. Readers should be aware that there is no universal consensus about the definition of certain subfields and that there is still disagreement about some of the boundaries even among the agreed upon regions.

    1. Reviewer #2 (Public Review):

      Summary: Franke et al. characterize the representation of color in the primary visual cortex of mice and how it changes across the visual field, with a particular focus on how this may influence the ability to detect aerial predators. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet were presented in random combinations. Using a clustering approach, a set of functional cell-types were identified based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have varying spatial distributions in V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

      Strengths:<br /> The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

      Weaknesses:<br /> While the study presents solid evidence a few weaknesses exist, including the size of the dataset, clarity regarding details of data included in each step of the analysis and discussion of caveats of the work. The results presented here are based on recordings of 3 mice. While the number of neurons recorded is reasonably large (n > 3000) an analysis that tests for consistency across animals is missing. Related to this, it is unclear how many neurons at each stage of the analysis come from the 3 different mice (except for Suppl. Fig 4). Finally, the paper would greatly benefit from a more in depth discussion of the caveats related to the conclusion drawn at each stage of the analysis. This is particularly relevant regarding the caveats related to using spike triggered averages to assess the response preferences of ON-OFF neurons, and the conclusions drawn about the contribution of retinal color opponency.

      The authors provide solid evidence to support an asymmetric distribution of color opponent cells in V1 and a reduced color contrast representation in lower light levels. Some statements would benefit from more direct evidence such as the integration of upstream visual signals for color opponency in V1.

      Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

  7. ellahodgetts.substack.com ellahodgetts.substack.com
    1. Upon doing so, the AI robot seemed to counter all of the ideas I had originally had in my head and I felt like my outline was starting to lack depth and nuance. It really got in my head and made me lack confidence.

      This has been my experience exactly re: ChatGPT. Great for boilerplate, not so great for anything else.

  8. accessmedicine-mhmedical-com.libaccess.lib.mcmaster.ca accessmedicine-mhmedical-com.libaccess.lib.mcmaster.ca
    1. Pregnancy: Trauma physiologic changes relevant (CVS, Pulm, heme) HR increase 10-15, BP decreased in trimester 1/2 and increases slightly in 3 Volume up by 8L: relative anemia therefore more blod loss toelrated up to 35%. Increase in tidal volume and minute vent but decreased FRC so overall lower resting pco2 and resp alkalosis. Can desat fast esp in intubation. Also, fetus more suceptible to hypoxemia than mom. Patients also more coagulable: Increased levels of factors VII, VIII, IX, X, XII and have decreased fibrinolytic activity Also may have WC of 20k normal. LES decreased tone so more aspiration risk. LFT often deranged with known billiary stasis. Ureters may be compressed with hydro

      Pearl: DPL needs to be done supraumbilically and directed to the head. Chest tubes go higher as well.

      During Trauma: Place patient in left lateral decub. FHR monitoring initialy with fast and look at enough amniotic fluid then via FHR monitor. FHT over 160 and below 120 signify dsitress. If maternal shock and/or impending arrest c section inidcated. Or if significant fetal distress or known uterine injury. Determine fetal age via fundal hieght or history. 20 at umbo or 40 at xipphy.

      for CT; rads can damage furing preimplant, organogensis 3-16 weeks, and after 16. Safe dose of rads <5 rad. CT abdo 3.5. Shield abdomen with a lead apron whever possible.

      Pelvic # in pregnancy can cause severe bleed given uterine and RP vein dilation. Bullet wounds high risk to fetus while stabs less so.

      Any pregnant patient with ISS>9 35 weeks or more preggo or symptomatic with uterine contractions, monitor for 24. otherwise monitor and dc from ED>

    1. Homestead Act, which allowed male citizens (or those who declared their intent to become citizens) to claim federally owned lands in the West. Settlers could head west, choose a 160-acre surveyed section of land, file a claim, and begin “improving” the land by plowing fields, building houses and barns, or digging wells, and, after five years of living on the land, could apply for the official title deed to the land. Hundreds of thousands of Americans used the Homestead Act to acquire land.

      How did they choose? Who would get the land and who would not? Surely there wasn’t enough land for everyone right?

    1. Save Share Tweet EmailJames Barnesis a psychotherapist, lecturer and writer with a background in psychoanalysis and philosophy. He has a psychotherapy practice in Exeter, UK, and sees clients remotely.Edited by Christian JarrettSyndicate this idea Save Share Tweet EmailFor Donald Winnicott, your psyche isn’t just in your head – it emerges from your relationships with others and the world

      for: human INTERbeing, human INTERbeCOMing, DH, Deep Humanity

    1. Electroencephalography (EEG) is one technique for studying brain activity. This technique uses at least two and up to 256 electrodes to measure the difference in electrical charge (the voltage) between pairs of points on the head. These electrodes are typically fastened to a flexible cap (similar to a swimming cap) that is placed on the participant’s head. Figure 2.3.12.3.1\PageIndex{1} shows a patient wearing such a cap. From the scalp, the electrodes measure the electrical activity that is naturally occurring within the brain. They do not introduce any new electrical activity.

      how eeg works

    1. People live in the present. They plan for and worry about the future.

      I know it's the first line of the text, but I don't think all people live in the present so to speak. I find myself constantly looking back to the past, repeating history in my head as to what I did wrong/what I could've done better. Other people, live in the future, using the present as only a means to reach the reality which they think they deserve. Depending on your mindset, I think you could live in the past, the present, or the future. I just think it's important to note that if someone's past is deeply personal and constantly on their mind, it may merge into their present.

    1. group cohesion.

      Group cohesion is an attractiveness to one goal among the members of the group. For example the Pittsburgh Steelers, had a hard time grasping their group cohesion. According to britannica. com " Rooney watched the Steelers struggle in 1950's and the 60's until their fortunes turned around with the arrival of head coach Chuck Noll in 1969." With this encouragement from Chuck Noll the Steelers began to become more successful in their franchise eventually leading to win six Super Bowl titles. Art Rooney a Pittsburgh resident founded the franchise in 1933 ( britannica. com).

    1. However, we do not usually expect to experience agency within a narrative environment.

      This is a curious statement, because when I read books, for example, I always felt like, as a reader, I did have agency. Despite the story itself being laid out by the author, I was able to imagine a lot of details, especially imagery, in my head, which is something the author cannot control-they can try, but it is impossible to fully control what happens in a readers' head when they read your work.

    1. This study has several limitations. Firstly, due to the intrinsic limitation of DTI in the regions with complex fiber heterogeneity, we used the SS3T-CSD algorithm to estimate the fiber orientation distributions for whole-brain tractography. We applied the most used DTI measurements to study the aging pattern in the CC subregions. Advanced diffusion models with multi-shell protocol should be used to capture the microstructure (Raffelt et al., 2017). Secondly, the CC masks were generated by segmenting the T1 images and registered to the DWI images, the multi-modal registration is challenging because DTI is susceptible to both affine/linear (e.g., eddy-current and head motion) and non-linear echo planar image field distortions. The registration results in each step were all visually inspected and showed their accuracy. Finally, there were relatively small samples in groups from 21–50 years and 81–90 years, which may bias the results for our imaging measures. In this work, we tried to analyze as much as possible data from our center and therefore we combined two imaging datasets with identical imaging scanner and protocols. Further study should recruit more participants to explore the microstructural changes of the CC during the earlier lifespan.Go to:

      درمورد محدودیت های کار گفته: 1ـ تعداد سابجکتها برای بازه های مختلف سنی یکسان نبوده 2ـ سن پایین رو نداشتن و زیر 20 خوبه که به کار اضافه بشه 3ـ برای حل پیچیدگی مسیرهای عصبی از یک الگوریتم و whole brain استفاده کرده. خوب نفهمیدم؟؟!! 4ـ ماسک cc رو از T1 برداشت کردن و بعد به نیتیو رجیستر کردن که خب این باعث بروز خطا میشه.(اگر من از T1 همون سابجکت بردارم أیا دقیقن مثل همون نیتیو هست یا باید من هم رجیستر کنم؟) 5ـ دیتاشون multi shell نبوده. ولی HCP هست و HARDI محسوب میشه.

    1. Self-Care Ideas Self-Care For The Mind 1. Start a compliments file. Document the great things people say about you to read later. 2. Scratch off a lurker on your to-do list, something that’s been there for ages and you’ll never do. 3. Change up the way you make decisions. Decide something with your heart if you usually use your head. Or if you tend to go with your heart, decide with your head. 4. Go cloud-watching. Lie on your back, relax, and watch the sky. 5. Take another route to work. Mixing up your routine in small ways creates new neural pathways in the brain to keep it healthy. 6. Pay complete attention to something you usually do on autopilot, perhaps brushing your teeth, driving, eating, or performing your morning routine. 7. Goof around for a bit. Schedule in five minutes of “play” (non-directed activity) several times throughout your day. 8. Create a deliberate habit, and routinize something small in your life by doing it in the same way each day—what you wear on Tuesdays, or picking up the dental floss before you brush. 9. Fix a small annoyance at home that’s been nagging you—a button lost, a drawer that’s stuck, a light bulb that’s gone. 10. Punctuate your day with a mini-meditation with one minute of awareness of your thoughts, feelings, and sensations; one minute of focused attention on breathing; and one minute of awareness of the body as a a whole. 11. Be selfish. Do one thing today just because it makes you happy. 12. Do a mini-declutter. Recycle three things from your wardrobe that you don’t love or regularly wear. 13. Unplug for an hour. Switch everything to airplane mode and free yourself from the constant bings of social media and email. 14. Get out of your comfort zone, even if it’s just talking to a stranger at the bus stop. 15. Edit your social media feeds, and take out any negative people. You can just “mute” them; you don’t have to delete them.   Self-Care For The Body 1.Give your body ten minutes of mindful attention. Use the body scan technique to check in with each part of your body. 2. Oxygenate by taking three deep breaths. Breathe into your abdomen, and let the air puff out your stomach and chest. 3. Get down and boogie. Put on your favorite upbeat record and shake your booty. 4. Stretch out the kinks. If you’re at work, you can always head to the bathroom to avoid strange looks. 5. Run (or walk, depending on your current physical health) for a few minutes. Or go up and down the stairs three times. 6. Narrow your food choices. Pick two healthy breakfasts, lunches, and dinners and rotate for the week. 7. Activate your self-soothing system. Stroke your own arm, or if that feels too weird, moisturize. 8. Get to know yourself intimately. Look lovingly and without judgment at yourself naked. (Use a mirror to make sure you get to know all of you!) 9. Make one small change to your diet for the week. Drink an extra glass of water each day, or have an extra portion of veggies each meal. 10. Give your body a treat. Pick something from your wardrobe that feels great next to your skin. 11. Be still. Sit somewhere green, and be quiet for a few minutes. 12. Get fifteen minutes of sun, especially if you’re in a cold climate. (Use sunscreen if appropriate.) 13. Inhale an upbeat smell. Try peppermint to suppress food cravings and boost mood and motivation. 14. Have a good laugh. Read a couple of comic strips that you enjoy. (For inspiration, try Calvin and Hobbes, Dilbert, or xkcd.) 15. Take a quick nap. Ten to twenty minutes can reduce your sleep debt and leave you ready for action.   Self-Care For The Soul   1. Imagine you’re your best friend. If you were, what would you tell yourself right now? Look in the mirror and say it. 2. Use your commute for a “Beauty Scavenger Hunt.” Find five unexpected beautiful things on your way to work. 3. Help someone. Carry a bag, open a door, or pick up an extra carton of milk for a neighbour. 4. Check in with your emotions. Sit quietly and just name without judgment what you’re feeling. 5. Write out your thoughts. Go for fifteen minutes on anything bothering you. Then let it go as you burn or bin the paper. 6. Choose who you spend your time with today. Hang out with “Radiators” who emit enthusiasm and positivity, and not “Drains” whose pessimism and negativity robs energy. 7. Stroke a pet. If you don’t have one, go to the park and find one. (Ask first!) 8. Get positive feedback. Ask three good friends to tell you what they love about you. 9. Make a small connection. Have a few sentences of conversation with someone in customer service such as a sales assistant or barista. 10. Splurge a little. Buy a small luxury as a way of valuing yourself. 11. Have a self-date. Spend an hour alone doing something that nourishes you (reading, your hobby, visiting a museum or gallery, etc.) 12. Exercise a signature strength. Think about what you’re good at, and find an opportunity for it today. 13. Take a home spa. Have a long bath or shower, sit around in your bathrobe, and read magazines. 14. Ask for help—big or small, but reach out. 15. Plan a two-day holiday for next weekend. Turn off your phone, tell people you’ll be away, and then do something new in your own town.  Source: https://tinybuddha.com/blog/45-simple-self-care-practices-for-a-healthy-mind-body-and-soul/

      Self-Care Ideas Self-Care For The Mind

      1. Start a compliments file. Document the great things people say about you to read later.

      2. Scratch off a lurker on your to-do list, something that’s been there for ages and you’ll never do.

      3. Change up the way you make decisions. Decide something with your heart if you usually use your head. Or if you tend to go with your heart, decide with your head.

      4. Go cloud-watching. Lie on your back, relax, and watch the sky.

      5. Take another route to work. Mixing up your routine in small ways creates new neural pathways in the brain to keep it healthy.

      6. Pay complete attention to something you usually do on autopilot, perhaps brushing your teeth, driving, eating, or performing your morning routine.

      7. Goof around for a bit. Schedule in five minutes of “play” (non-directed activity) several times throughout your day.

      8. Create a deliberate habit, and routinize something small in your life by doing it in the same way each day—what you wear on Tuesdays, or picking up the dental floss before you brush.

      9. Fix a small annoyance at home that’s been nagging you—a button lost, a drawer that’s stuck, a light bulb that’s gone.

      10. Punctuate your day with a mini-meditation with one minute of awareness of your thoughts, feelings, and sensations; one minute of focused attention on breathing; and one minute of awareness of the body as a a whole.

      11. Be selfish. Do one thing today just because it makes you happy.

      12. Do a mini-declutter. Recycle three things from your wardrobe that you don’t love or regularly wear.

      13. Unplug for an hour. Switch everything to airplane mode and free yourself from the constant bings of social media and email.

      14. Get out of your comfort zone, even if it’s just talking to a stranger at the bus stop.

      15. Edit your social media feeds, and take out any negative people. You can just “mute” them; you don’t have to delete them.

      Self-Care For The Body

      1.Give your body ten minutes of mindful attention. Use the body scan technique to check in with each part of your body.

      1. Oxygenate by taking three deep breaths. Breathe into your abdomen, and let the air puff out your stomach and chest.

      2. Get down and boogie. Put on your favorite upbeat record and shake your booty.

      3. Stretch out the kinks. If you’re at work, you can always head to the bathroom to avoid strange looks.

      4. Run (or walk, depending on your current physical health) for a few minutes. Or go up and down the stairs three times.

      5. Narrow your food choices. Pick two healthy breakfasts, lunches, and dinners and rotate for the week.

      6. Activate your self-soothing system. Stroke your own arm, or if that feels too weird, moisturize.

      7. Get to know yourself intimately. Look lovingly and without judgment at yourself naked. (Use a mirror to make sure you get to know all of you!)

      8. Make one small change to your diet for the week. Drink an extra glass of water each day, or have an extra portion of veggies each meal.

      9. Give your body a treat. Pick something from your wardrobe that feels great next to your skin.

      10. Be still. Sit somewhere green, and be quiet for a few minutes.

      11. Get fifteen minutes of sun, especially if you’re in a cold climate. (Use sunscreen if appropriate.)

      12. Inhale an upbeat smell. Try peppermint to suppress food cravings and boost mood and motivation.

      13. Have a good laugh. Read a couple of comic strips that you enjoy. (For inspiration, try Calvin and Hobbes, Dilbert, or xkcd.)

      14. Take a quick nap. Ten to twenty minutes can reduce your sleep debt and leave you ready for action.

    1. making, temple priest, storyteller, interpreterofsigns, chanter, dancer, farmer andfisherman. Sometimes a kiiula or seer was consulted who would examine the child'sbody and be able to discern from its body shape and head shape the 'anoofthe child.The kiiula could, from this investigation, see the possible vocation to which the childwould be suited to and the future success or failureofthat child in that 'oihana.

      I am learning a lot about the Hawaiian culture and I think Artisa skillfully and fluently wrote this thesis.

    1. He raised again the jug regretfully And shook his head, and was again alone. There was not much that was ahead of him, And there was nothing in the town below– Where strangers would have shut the many doors That many friends had opened long ago.

      This poem explores an individual struggling with change and isolation, connecting to Charlotte Perkins Gilman's exploration of women's roles and constraints in a changing society, as seen through the narrator in "The Yellow Wallpaper." Both Robinson and Gilman highlight the tension between conformity to societal norms and the longing for a sense of personal identity. The thought of strangers shutting the doors friends would have opened is sad - there is a longing for connection that can no longer be filled as people grow more separate.

    1. Author Response

      Evaluation Summary:

      The manuscript shows that retinal ganglion cell light responses in awake mice differ substantially from those under two forms for anesthesia and previously attained ex vivo recordings. This difference is central to our understanding of how ganglion cell responses relate to behavior. There are a few technical issues and issues about how the work is presented that could be strengthened.

      We thank the reviewers for their constructive comments. We have addressed all the issues, and added substantially more data and analysis results in the revised manuscript, further supporting our findings that awake responses are larger, faster, and more linearly decodable in the mouse retina than those responses under anesthesia or ex vivo.

      Reviewer #1 (Public Review):

      This paper compares output signals from the mouse retina in three conditions: awake mice, anaesthetized mice, and isolated retinas. The paper reports substantial differences, particularly between awake and either of the other conditions. Retinal signaling has been well studied using ex vivo preparations, with an assumption that the findings from those studies can be carried over to how the retina operates in vivo. The results from this paper at a minimum indicate a need to be cautious about that assumption. There are several technical issues that need testing or further explanation, and several issues about the presentation that could be clarified.

      Spike sorting

      The paper does not describe any control analyses that test for contamination in spike sorting. These are needed to evaluate the work.

      We have reported the details of our spike sorting procedure in the revised manuscript (Data Analysis section in Methods and Figure 1). In short, single-units were identified by clustering in principal component space, followed by manual inspection of spike waveform (triphasic as expected from axonal signals; e.g., revised Figure 1F-H; Barry, 2015) as well as auto- and cross-correlograms (minimal inter-spike interval above 1 ms for a refractory period; e.g., revised Figure 1I-K). A small fraction of visually responsive cells (20/282, awake; 21/325, isoflurane; 1/103, FMM) had a small fraction of interspike intervals below 2 ms; but, whether or not including them in the analysis did not affect our main conclusions.

      Light levels

      The paper argues that differences in light level cannot account for the results. According to the methods, light levels were about two-fold higher at the retina in array recordings as compared to the front of the eye for in vivo recordings. The main text indicates that they differ less, it's not clear why the numbers in the main text and methods are different. Aside from this issue, this comparison does not consider the loss of light between the front of the eye and the retina. It is crucial that the paper provide a more detailed description of light levels. This should include converting those light levels to units that include the spectral output of the light source used (e.g. to isomerizations per rod or cone per second).

      The maximum light intensity of our in vivo setup was 31.3 mW/m2 (with 15.9 mW for UV LED and 15.4 mW/m2 for blue LED). Following the suggestion by the reviewer, we calculated the photon flux on the mouse retina in vivo by taking into account the loss of light by the eye optics. In short, assuming 50% and 68% transmittance at 365 nm and 454 nm, respectively (Jacobs & Williams 2007), the pupil size of 1 mm and the retinal diameter of 4 mm with the stimulus covering 73° in azimuth and 44° in elevation, we obtained the photon flux on the mouse retina in vivo as 3.81×103 and 6.64×103 photons/s/μm2 for UV and blue light, respectively. Assuming a total photon collecting area of 0.2 μm² for cones and 0.5 μm² for rods (Nikonov et al. 2006), and a relative sensitivity of rods, S- and M-cones to be [UV, Blue]=[25, 60], [90, 0], [25, 60]%, respectively (Jacobs & Williams 2007), we then estimated the photoisomerization (R) rate as: 2.5×103 R/rod/s, 0.7×103 R/S-cone/s, and 1.0×103 R/M-cone/s.

      In contrast, the maximum light intensity of the in vitro set up was 36 mW/m2 as reported in Vlasiuk and Asari (2021). The photon flux on the isolated retina was then estimated to be around 9×104 photons/s/μm2 (under the assumption that the white light from a CRT monitor is centered around 500 nm). Assuming the sensitivity of rods, S- and M-cones to be 40, 2 and 40%, respectively, we then obtained 4×104 R/rod/s, 2×103 R/S-cone/s, and 4×104 R*/Scone/s.

      Thus, the light intensity level was about ten times larger for the in vitro recordings than for the in vivo recordings. The amount of light reaching the retina in the awake condition should also be somewhat smaller than that under anesthesia due to pupillary reflexes. Past studies suggest that the darker the stimulus is, the slower the kinetics is and the smaller the response is for RGCs in an isolated retina (Wang et al 2011). Thus, the light intensity difference cannot simply account for the higher firing and faster kinetics in the awake condition than ex vivo or in the anesthestized condition.

      We have revised the manuscript accordingly.

      Comparison with other work

      The authors accurately point out that there is not much prior work on retinal outputs in awake animals. The paper, however, minimally describes the work that does exist. The Hong et al. (2018) paper, in particular, should be discussed. There are several differences between the results of that paper and the present paper. These include the fraction of recorded cells that are DS cells, and the maintained firing rates (though this does not appear to be studied systematically in Hong et al.).

      In the discussion section of the revised manuscript, we clarified connections to the existing studies on the retinal activity in vivo. To our knowledge, none of the past studies provided descriptive statistics on the awake RGC response properties (Hong et al., 2018; Schroeder et al., 2020; Sibille et al., 2022). Nevertheless, consistent with our study, we can see high baseline activity in the reported examples from C57BL6 mice (Figure 3C, Schroeder et al. 2020; Figure S7h, Sibille et al. 2022).

      Hong et al (2018), in contrast, reported somewhat different as pointed out by the reviewer. Firstly, they found a relatively low baseline activity in RGCs of albino CD1 mice. We think that this is likely due to general impairments of the vision/retina associated with albinism. While equipped with normal electroretinogram signals, CD1 mice showed no optomotor response and a reduced number of rods (Abdeljalil et al 2005; Brown et al 2007). This suggests a certain level of retinal dysfunction in these mice. Secondly, Hong et al (2018) reported a higher fraction of direction-selective RGCs in their recordings (>50% at a DS index threshold of 0.3). This is even higher than one would expect from anatomical and physiological studies ex vivo on BL6 mice (about a third; Sanes and Masland, 2015; Baden et al., 2016; Jouty et al 2013). Besides the effect of albinism, we think that this overrepresentation of DS cells in Hong et al (2018) arose as a consequence of the low baseline activity. As discussed above, the higher the baseline activity, the lower the DS/OS index by definition (Eq.(3) in Methods). Indeed we found much more cells with high DS/OS index values in our anesthetized data than in awake ones (42-54% vs 17% at an index value threshold of 0.15; Figure 2), even though these recordings were done in the same experimental set up.

      A related issue is that there are a few comparisons of ex vivo RGC responses with behavioral sensitivity. Smeds et al. (2019) is one example. More generally, the long-standing observation that dark-adapted sensitivity approaches limits set by Poisson fluctuations in photon absorption, and that prior RGC measurements are consistent with this result, is hard to explain if the RGCs are firing at high spontaneous rates under these conditions. RGC responses will certainly change with light level, but this merits discussion in the paper.

      As the reviewer pointed out, the retina may employ different coding principles under different light levels. In a scotopic condition, ex vivo studies reported a high tonic firing rate for OFF RGC types (~50 Hz, OFF sustained alpha cells in mice; Smeds et al 2019; ~20 Hz, OFF parasol cells in primates; Ala-Laurila and Rieke, 2014), while a low tonic firing for ON cell types (<1Hz for both ON sustained alpha in mice and ON parasol in primates). These ON cells were shown to be responsible for light detection by firing in the silent background, hence compatible with the sparse feature detection strategy. In contrast, our recordings were done in a high mesopic / low photopic range where both rods and cones are supposedly active. Unlike the scotopic condition with rod vision, we then found high firing in awake recordings in general, indicating that no visual feature can be readily detectable as brief firing events in the silent background. To explore the implications of such firing patterns on visual coding, we took a modelling approach in the revised manuscript. We found that a latency-based temporal code was not preferable in the awake condition (Figure 7); and that a linear decoder worked significantly better with the population responses in the awake condition to capture the presented random fluctuation of the light intensity (Figure 8). While we have not tested any behavioural relevance in our study besides correlation to locomotion/pupil size, it is then possible that the retina may work in different modes under different light intensity regimes (Tikidji-Hamburyan et al 2015).

      We clarified these points in the revised discussion section.

      Sampling bias

      The paper argues that sampling bias is not likely to contribute substantially to the results because of the wide variety of cell types recorded (line 431). This does not seem like a particularly strong argument, especially given the large degree of overlap in the distributions of most quantities across preparations. The argument about many cell types could be made more strongly if the distributions were completely separated, but that is not the case.

      We cannot deny the presence of a sampling bias in our datasets, and as the reviewer pointed out, we made comparisons only at a population level, but not at the level of individual cells or cell-types. However, the anesthetized and awake recordings were done with the same recording setup and techniques, and thus subject to the same sampling bias. Hence, the difference in the RGC response properties between these conditions cannot be explained by the sampling bias per se.

      Sensitivity

      The firing rates in response to 10% contrast sinusoids are quite low, as are the maximal firing rates for high contrast sinusoids. Relatedly, the modulation produced by the noise stimuli, particularly for the array recordings, is weak. This raises concerns about the health of some of the preparations.

      To our knowledge, in vivo contrast responses reported here were comparable to ex vivo data in previous reports (mouse, Jouty et al 2018, Pearson and Kerschensteiner 2015; rat, Jensen 2017, 2019). Likewise, the static nonlinearity and its upper bound for ex vivo responses were comparable between this study and previous reports (Santina et al. 2013; Kerschensteiner et al 2008; Cantrell et al 2010; Trapani et al 2022).

      We also examined batch effects in the response to the noise stimuli. We found certain variabilities across preparations in each recording condition, but not to the extent to discard any particular data as an obvious outlier (Figure 6 – figure supplement 1). While it is difficult to tell the health status of preparations retrospectively, we thus believe that the effects were negligible.

      Efficient coding

      Sparse firing is not a universal property of retinal ganglion cell responses. Primate midget RGCs, for example, have pretty high maintained firing rates as shown in many past studies. Mouse RGCs have also been reported to operate in a mode similar to the high firing rate On cells reported here (Ke et al. 2014). A more balanced discussion of this past work is needed.

      As the reviewer pointed out, some retinal ganglion cells show high firing under certain conditions. In a scotopic condition, for example, OFF cells have high firing rates, while ON cells fire virtually nothing unless a light stimulus is presented (Ke et al 2014; Smeds et al 2019). At the behavoural level, a single-photon detection above chance level nevertheless relies on the information from the ON but not the OFF pathway (Smeds et al 2019). Thus, the sparse coding framework still works as a valid strategy here, if not universal.

      This is, however, very different from what we report here. In a high-mesopic/low-photopic light level, we found a general increase of firing across all cell categories in the awake condition, compared to the anesthetized or ex vivo recordings (Figures 3 and 6). While this lowers information transfer rate (bits/spike; Figure 7), we found that the awake responses were more linearly decodable than the responses in the other conditions (Figure 8). We also ran a simulation and showed that a latency-based temporal code is not preferable for the awake responses (Figure 7 – figure supplement 1). These results suggest that the retina in awake condition is in favor of a rate code, though we have not tested all light levels or any behavioural relevance here.

      We clarified these points in the revised manuscript.

      Role of eye movements

      Could eye movements be at least partially responsible for the differences in response properties? Specifically, small fixational eye movements might produce a constantly varying input that could modulate firing.

      As described above (Essential Review item #2), eye movements were rarely observed during the head-fixed awake recordings. Eliminating those events from the analysis did not change our overall conclusions, and thus their contributions should be minimal in this study. It should also be noted that we mainly used full-field stimulation, and thus microsaccades should not substantially affect the amount of light impinging on the retina. We clarified these points in the revised manuscript.

      Reviewer #2 (Public Review):

      The technical achievements presented in the manuscript represent a tour de force, as optical tract recordings in awake mice have only rarely been done before. The substantial number of neurons recorded in both awake and anaesthetized conditions form a precious and worldwide unique dataset. However, since the recordings represent a non-standard approach, it would be, in my view, highly beneficial to show more details about the success of the method. How did the authors post-hoc identify electrode contacts located in the optical tract, how did the spike waveforms look like, what were the metrics of spike sorting quality, etc.

      We added more details about our recording and analysis methods in the revised manuscript. Below are answers to the reviewer’s specific questions:

      • The probe was coated with a fluorescent dye (DiI stain) and its location was verified histologically after the recordings (Figure 1E).

      • Spike waveforms typically had a triphasic shape (e.g., Figure 1F-H) as expected from axonal signals (Barry, 2015).

      • Single-units were identified by clustering in principal component space, followed by manual inspection of spike shape as well as auto- and cross-correlograms. Most units had a minimum interspike interval above 2 ms (93%, awake; 94%, isoflurane; 99%, FMM); and no units had the interspike intervals below 1 ms for a refractory period (e.g., Figure 1I-K), except for 1 (out of 103) for FMM-anesthetized recordings.

      We then selected visually responsive cells (SNR>0.15; see Eq.(1) in Methods) for the analyses.

      The authors go a long way in characterising the functional response properties of the recorded neurons and relating them to previous ex-vivo recordings. Based on the responses they find, the authors claim that they identified "... a new response type [which] likely emerged due to high baseline firing in awake mice". Regarding this claim, how do the authors rule out that it corresponds to any of the previously described cell types? For instance, the very sharp transient or brief modulations by the contrast part of the stimulus might have been missed in previous classifications based on calcium responses (e.g. Baden et al. 2016), where a number of cell types seem to respond equally strong to grey and white and have an elevated response throughout the sinusoidal modulation of contrast. I acknowledge that the authors touch upon the possibility that the newly described OFFsuppressive ON cells correspond to a known cell type in the discussion, but I would recommend changing the phrasing of the results to avoid potential misunderstandings.

      We agreed with the reviewer and revised the manuscript accordingly. Here we have two possibilities. Firstly, as the reviewer pointed out, this kind of response dynamics could be overlooked previously because of a difference in the recording modality (Ca imaging; Baden et al 2016) or clustering methods (Jouty et al 2019). Secondly, these cells may belong to one of the cell-types described in the past ex vivo studies, but exhibited distinct response dynamics in vivo as an emerging property of the awake condition. This is an interesting topic to pursue in future studies.

      The manuscript makes the interesting suggestion that "the retinal output characteristics [...] observed in vivo, [...] provide a completely different view on the retinal code". Given that this conclusion would change the way we should think about and do retinal neuroscience, in my view, the authors should take a few more steps to quantitatively demonstrate the implications of their findings on retinal coding, e.g. how much lower is the information transmitted per spike, how much does a temporal code based on spike timing suffer with the latencies observed in vivo. If the authors could quantify through computational modelling approaches the consequences of the observed differences, they might also be able to revise their title / main message, i.e. that "Awake responses SUGGEST inefficient dense coding in the mouse retina".

      To explore functional implications of our findings, we performed three more analyses as suggested by the reviewer. Specifically,

      1) We showed that the information transmitted per spike was significantly lower in awake condition, while the total information rate was comparable (Figure 7).

      2) We tested the performance of a linear decoder applied on the firing rate in response to full-field noise, and showed that it worked significantly better for the awake population responses (Figure 8).

      3) We simulated RGC responses to a full-field contrast change at different intensities in different conditions, and showed that a latency coding did not work well with awake responses, compared to ex vivo or anesthetized responses (Figure 7 – figure supplement 1).

      These results strengthened our conclusion that awake response dynamics were different from anesthetized or ex vivo responses, all arguing against the sparse efficient coding principles at least at a light level we examined. We nevertheless kept the title as is because we have not explored the retinal coding properties per se. Our main claim stays on the visual response characteristics of retinal outputs in awake mice.

      Reviewer #3 (Public Review):

      The manuscript by Boissonnet, Tripodi, and Asari compares retinal ganglion cell (RGC) light responses in awake mice (recorded in the optic nerve) with those under two forms for anaesthesia and previously attained ex vivo recordings. This is a well motivated study looking at a question that is really critical to the field.

      The presentation is generally clear and compelling. My suggestions are relatively minor and aimed at improving an already very strong article.

      1) More cells in the awake condition would help strenghten the conclusions. Only 51 cells are reported, and mouse RGCs comprise more than 40 different types. The authors are well aware of the possible confound of sampling bias, and the best way to mitigate this issue in this experimental paradigm is simply to record more cells. The anesthsia conditions each have about 100 cells, which is better.

      We made substantially more recordings in the awake condition, reaching 282 cells (in 15 animals) in total in the revised manuscript. This does not yet allow for a full cell-type classification as in the past ex vivo studies. Nevertheless, we did our best to broadly classify visual responses, and showed that the overall conclusions remained the same: awake RGCs had higher baseline firing and faster response kinetics in general. For details, see above our response to the Essential Revision item #1.

      2) It took me longer than it should have (had to look up the previous paper cited) to figure out that the ex vivo comparison data were recorded at 37{degree sign}C. This is an important detail since most ex vivo recordings are at 32{degree sign}C. The authors should make this clear in the text and perhaps say something in the Discussion about comparisons to the larger body of literature of ex vivo studies at 32{degree sign}.

      We are aware that most ex vivo studies on the retina were performed at 32 °C, which is lower than physiological body temperature (37 °C). However, the temperature of the ocular surface is around 37 °C (Vogel et al 2016), suggesting that the retina should operate at 37 °C in vivo. This is why we decided to perform ex vivo experiments at 37 °C in our previous study (Vlasiuk and Asari, 2021), allowing us to make a fair comparison between the ex vivo and in vivo recordings.

      We clarified the point in the revised manuscript.

      3) Direction and orientation selectivity should be separated in Fig. 2 and not combined into the confusing term "motion sensitive." Motion sensitivity has another meaning in the literature for RGCs that respond preferentially to moving over static stimuli without direction or orientation preference (Kuo et al., 2016; Manookin et al., 2018)

      We agree with the reviewer. In the revised manuscript, we separated the direction and orientation selective cells (Figure 2), and avoided the term “motion sensitive.”

      4) While I am certainly sympathetic to the argument that the RGC spike code is "inefficient" in the sense that it does not conform to efficient coding theory (ETC), I think it's oversimplified to claim that the present data is a key argument against ETC. Plenty of ex vivo data has already shown ETC to be incomplete at best, and misguided at worst, since it includes the implicit assumption that image reconstruction is the retina's objective function (or even that the experimenter has any idea what that objective function is). For example, OFF sustained alpha (OFF delta in guinea pig) RGCs are not quite sparse feature detectors even ex vivo, and they seem to be optimized to transmit contrast with high SNR (Homann and Freed, 2017). In general, the enormous coverage factor of the RGC population seems to make ETC untenable to begin with, as discussed in (Schwartz, 2021) and elsewhere. I realize that there are still people attached to simplistic forms of ETC as a key principle of retinal computatiion, so I am not asking for the authors to completely remove this angle. Rather, a more nuanced treatment of the issue both in the introduction and the discussion is warranted.

      We totally agree that we are not the first to argue against the efficient coding principles in the retina (Schwartz, 2021). The main argument in this study is that certain aspects of the RGC activity are distinct in an awake condition, such as the baseline firing and response kinetics, and thus we cannot simply translate our knowledge obtained from ex vivo studies into awake animals. To explore the implications on retinal computations, we showed in the revised manuscript that 1) awake responses have a comparable total information transfer rate (in bits per second; Figure 7A) but are less efficient (i.e., lower bits per spikes; Figure 7B); 2) awake responses are not in favor of a latency-based temporal code (Figure 7 – figure supplement 1); and 3) a linear decoder worked significantly better with awake responses (Figure 8), even though an image reconstruction is not necessarily the objective function of the retina. These results point out a need to rethink about retinal function in vivo, including the efficient coding theory.

      We thank the reviewer for the suggestion, and revised the manuscript accordingly.

      References

      Homann, J., and Freed, M.A. (2017). A mammalian retinal ganglion cell implements a neuronal computation that maximizes the SNR of its postsynaptic currents. Journal of Neuroscience 37, 1468-1478.

      Kuo, S.P., Schwartz, G.W., and Rieke, F. (2016). Nonlinear Spatiotemporal Integration by Electrical and Chemical Synapses in the Retina. Neuron 90, 320-332.

      Manookin, M.B., Patterson, S.S., and Linehan, C.M. (2018). Neural Mechanisms Mediating Motion Sensitivity in Parasol Ganglion Cells of the Primate Retina. Neuron 97, 13271340.e4. Schwartz, G.W. (2021). Retinal Computation (Academic Press).

    1. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #3

      Evidence, reproducibility and clarity

      Somites form consecutively along the anterior to posterior (AP) axis. The time of the formation of a somite is controlled by the segmentation clock, oscillation of cyclic genes in the presomitic mesoderm. The length of an oscillation cycle differs between species and should also differ between the axial levels. In chicken embryos, one cycle for a trunk somite requires 90 minutes, while it is much slower (150 minutes) for a posterior-most somite. Is this quicker or slower for an anterior-most somite? Andrade' group addressed this question and measured the time of the formation of each occipital somites (somite 1-5). They found that the formation of an occipital somite requires only 75 minutes, while somites from somite 6 onwards takes as long as the trunk somites (about 90 minutes). The faster formation of occipital somites is correlated with the time of the cyclic expression of hairy1 and hairy2.

      Major comments:

      The conclusion is well supported by the data. The measurement of the length increments of the segmented region and then assay using algorithm are well established. Thus, the data are well reproducible.

      Minor comments:

      The authors did not consider the fact that the first formed somite is the second somite. After the formation of the second somites, the real first somite forms anterior to the second somite. Furthermore, the real first and the third somite seems to be formed simultaneously. It is worthy for the authors to re-examine the data, whether the real first somite and the third somite are formed at the same time. And to check whether the first somite was counted to the segmented region. And this point should be at least discussed.

      Referees cross commenting

      I find all criticisms are justified. The most advance, as stated by other both reviewers, is the quantitative assay of the somite formation, since this is no yet done previously. As suggested by the first reviewer, it will be more informative if the authors complete this analysis for all regions. For all somites would be too much work, but they can select some representative somites of each region in addition to occipital region, such as 3 somites for one region, including the cervical, thoracic, lumbal, sacral and caudal region. Thus, the dynamic of the temporal somite formation of the whole embryo can be analysed using the same method. This will provide much more impact for this work.

      Significance

      Significance: The measurement of the length increments of the segmented region and then assay using algorithm are the novelty and strengths of this study. So, the data are reproducible and objective.

      The results of this study extend our understanding about the dynamic process of the somitogenesis. Especially, the most interesting point is that based on this result, we can see that the segmentation clock runs faster in the head region, and then slow down gradually along the AP axis.

      Audience: specialized, basic research<br /> The developmental biologist will be interested in this topic.

      My expertise is the somite development, somite differentiation, mesoderm development.

    1. if what mattered most to a writer was what was in his or her head, there’d be no reason to write anything

      Respectfully, no. The reason is that they want to share the experience of what's in their head with other people. That's the whole point!

    1. The changes to the message to create the response are straight from existing alien folklore and science fiction. In the section detailing important chemical elements, the main focus is altered from carbon to silicon, and the diagram of DNA is re-scribbled slightly. At the bottom, the pictogram of a human is replaced with a shorter figure with a large, bulbous head. This is a clear reference to the "grey" type of alien, and as a depiction could only be something that a human came up with.[6]

      ticonderoga: carbonifer, i've been walking through from uh, neresh to get to "liquid iron appears to be mentioned in this new word for "spirit" since the vador/umanayim ... "event."

      pinto "i have a vision of this message, similar to the "Cassiopea overlayed over USSR as startotality-in-vetatrine here ..." we don't really see it but both the EU and it apear to be showing signs of things slike "containing a rainy-UK" and a .... second Kremlin.

      I'm seeking ummm .. "any kind of confirmation of when the thing i call a 'semaphore' was invented;" today i'm tying it to the remnant return of ... definately at least

    1. If your enemy is hungry, feed him. If he is thirsty, give him a drink; for in doing so, you will heap coals of fire on his head.”

      Similar to the Matthew 28 separating the goats and the sheep. How when you serve others you are serving Christ.

    1. Reducing friction for non-English speakersDr. Anthony Kaziboni, the Head of Research at the University of Johannesburg, teaches students who mostly don’t speak English outside of the classroom. Kaziboni believes that command of English is a tremendous advantage in the academic world, and that misunderstandings of even small details of English grammar can hold back students from recognition and opportunity. He encourages his students to use ChatGPT for translation assistance, to improve their English writing, and to practice conversation.

      英语无疑是当前使用 Chatgpt效果最好的一种沟通方式。因此要想提高我们与chat gpt沟通的效率和效果。最好使用英语,进行沟通交流。而使用chat gpt本身就能够帮助我们。消除。英语使用上的障碍。对于非母语英语的。学生效果非常好。

    1. Developing empathy.

      When read a book about someone or something that we are unfamiliar with, we can be biased or have painted a narrative in our head that is far from the truth. Once we read an article or a story we can gain a deeper understanding of a certain situation and develop empathy for someone or something. An example would be of a book I read called A Long Way Gone: Memoirs of a Boy Soldier. This book is about a boy who got wrapped up in the civil wars in Sierra and how he and his family tried fleeing the war but he was taken hostage and forced to fight.

    1. steroid treatment, whether of androgen deficient men, nor-mal athletes, or ordinary vo unreers, can increase aggressive tendencies

      It sorta plays into the joke of an ultra violent meat head, but its intersting to see it holds up

    Annotators

  9. learn-us-east-1-prod-fleet02-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet02-xythos.content.blackboardcdn.com
    1. Outside events strongly influenced this development. Service in theUnion Anny enabled black men to participate more directly than womenin the strugg·le for freedom. The Freedmen's Bureau designated thehusband as head of the black household, insisting that men sign contractsfor the labor of their entire families and establishing wage scales that paidwomen less than men for identical plantation labor.

      men freedom

    2. here is no question thatmany black men considered it a badge of honor to see their wives workingat home and believed that, as head of the family, the man should decidehow its labor was organized.

      freedom- men

    1. the heavy bedstead, and then the barred windows, and then that gate at the head of the stairs,

      He mentions only mentions all things that keep her trapped and statonary.

    2. John does not know how much I really suffer. He knows there is no REASON to suffer, and that satisfies him.

      Seems like John very much gets in her head

    1. llm_int8_skip_modules (List[str], optional) — An explicit list of the modules that we do not want to convert in 8-bit. This is useful for models such as Jukebox that has several heads in different places and not necessarily at the last position. For example for CausalLM models, the last lm_head is kept in its original dtype.

      [!NOTE] LLM.int8() 的应用对象模块有什么讲究?

      flashcard

      各种 head 一般不量化计算,而是保留原数据类型 因为要获取有意义的输出?

    1. “Given the high unemployment rate in South Africa as well … you cannot sell it as a climate change intervention,” says Deborah Ramalope, head of climate policy analysis at the policy institute Climate Analytics in Berlin. “You really need to sell it as a socioeconomic intervention.”
      • for: quote, quote - climate change intervention, Trojan horse, Deborah Ramalope
      • quote
        • Given the high unemployment rate in South Africa as well … you cannot sell it as a climate change intervention, you really need to sell it as a socioeconomic intervention.
      • author: Deborah Ramalop
      • date: Aug. 15, 2023
      • source: https://www.wired.co.uk/article/just-energy-transition-partnerships-south-africa-cop
      • comment
        • A Trojan horse strategy
    1. Co-head of the Indian core |
      1. Team size you managed?
      2. The content is good - frame it more in terms of created a structure while trying to balance individuality; not that it is tricky to, but that you did.

    Annotators

    1. The frequently repeated claim that humans use only 10% of their brains is false. The exact origin of this myth is unknown, but misinterpretations of brain research are likely to blame. In experiments with animal brains during the 1800's through the early 1900's, Marie-Jean-Pierre Flourens and Karl Lashley destroyed and/or removed as much as 90% of the brain tissue of their animal subjects.  Nevertheless, these animals could still perform basic behavioral and physiological functions. Some who read these results made the incorrect assumption that this meant that animals were using only 10% of their brains.  Subsequently, this interpretation was generalized to humans (Elias and Saucer, 2006). Furthermore, prominent psychologists and researchers, such as Albert Einstein, Margaret Mead, and William James, were also quoted as saying that humans are using only a small portion of their brain (Elias and Saucer, 2006), fueling the 10% myth. Due to advances in biopsychology and other related fields, we now have a greater understanding of the complexity of the brain. We may not be using our brains as efficiently as possible at all times, but we are using the entirety of our brain as each part contributes to our daily functioning.  Studies of humans with brain damage have revealed that the effects of brain damage are correlated with where the damage is and how extensive it is. In other words, where damage occurs determines what functions are impacted and more damage has more of an effect. This reflects a key organizational principle of the brain: the localization of function.  This principle means that specific psychological and behavioral processes are localized to specific regions and networks of the brain.  For example, we now know that damage to an area of the brain known as the primary visual cortex, at the very back of your head in the occipital lobe, will result in blindness even though the rest of your visual system, including your eyes, is functioning normally. This syndrome is known as cortical blindness, to distinguish it from blindness that is caused by damage to the eyes.  We now know that damage to a small area less than the size of a quarter at the very base of your brain results in disruption of feeding and regulation of body weight.  Damage to another area of the brain located near your temples disrupts your ability to form new memories for facts and events, while leaving your ability to learn new motor tasks (such as skating or riding a bike) completely unaffected.  Damage to another brain area causes face blindness, or prosopagnosia, a disorder in which the afflicted individual can still see normally except that they cannot recognize familiar faces, even the faces of close family members or even their own face in a photograph.  In the pages that follow in this textbook, you will learn many amazing things about the brain, and the nervous system in general.  Get ready for many surprises as we explore the 3 pounds of brain tissue between our ears that make up the most complex piece of matter in the known universe. In this book, we examine some of what scientists now know about this astonishing organ, the brain, and how it functions to produce mind and behavior.

      10% of brain myth debunked and explained

    2. cans of the head are often used to help psychologists understand the links between brain and behavior

      MRI shows structure of brain functional MRI shows brain activity Three different brain imaging techniques show different brain functions

    3. s mind-body dualism or mind-brain dualism, which literally means that the mind and the functioning of the brain (assumed to be entirely physical) are two (dual) separate processes, completely independent of one another.  The origin of dualism is often attributed to the 17th century French philosopher and mathematician, Rene Descartes. If this view were true, then we would expect that brain damage would have no effect on the mind.  However, brain damage does affect the mind and the specific location of the damage produces more or less specific, fairly predictable, effects on the mind, modifying the mind and behavior in various ways.  Examples of this are coma due to head injury; the effects of Parkinson's disease on movement after the disease damages areas of the brain known as the basal ganglia; changes in personality and emotion due to injury to the front of the brain, specifically the frontal lobes; memory loss in Alzheimer's Disease; and so on.  Though you don't have to accept the assumption of physicalism when studying the brain if your religious beliefs are contrary to the idea, nevertheless it is important that you be aware of the assumption of physicalism/materialism that most biological psychologists accept, at least as a working hypothesis, if not a philosophical position, as they do their brain research.

      If the "completely" independent of one another" rigid assumption were modified or removed then the actuality of mind and matter is clear, the physical is needed in terms of structure and function, and the spiritual or other dimension(s) work through and on it.

    1. he assessment of individual children’s progressthrough ongoing observation by those caring forand educating the infant/toddler using an earlylearning framework that articulates developmentalprogressions (such as the Head Start EarlyLearning Outcomes Framework or state ELGs), andobservation of the infant/toddler’s own interests andmotivation for learning

      Are ELGs similar to Brigance? Is it like the infant version or something?

    1. discourse of sufficiency

      I have never heard of this terminology before, but I really like the picture that it paints in my head. I think this issue is still prevalent today within academia, and within many other avenues. I believe it is hard to get around sometimes, because it is almost like looking at internal biases and trying to account for them in an appropriate manner. But I agree with Nogelmeierʻs notion that there needs to be an investigation of texts and their baggage, as well as an inclusion of other, non-canonical texts.

    1. ∝(19A.2)(19A.2)at=r∝ a_t=r \propto \label{19-1} A Rotating Rigid Body The characterization of the motion of a rotating rigid body has a lot in common with that of a particle traveling on a circle. In fact, every particle making up a rotating rigid body is undergoing circular motion. But different particles making up the rigid body move on circles of different radii and hence have speeds and accelerations that differ from each other. For instance, each time the object goes around once, every particle of the object goes all the way around its circle once, but a particle far from the axis of rotation goes all the way around circle that is bigger than the one that a particle that is close to the axis of rotation goes around. To do that, the particle far from the axis of rotation must be moving faster. But in one rotation of the object, the line from the center of the circle that any particle of the object is on, to the particle, turns through exactly one rotation. In fact, the angular motion variables that we have been using to characterize the motion of a line extending from the center of a circle to a particle that is moving on that circle can be used to characterize the motion of a spinning rigid body as a whole. There is only one spin rate for the whole object, the angular velocity ωω\omega, and if that spin rate is changing, there is only one rate of change of the spin rate, the angular acceleration ∝∝\propto. To specify the angular position of a rotating rigid body, we need to establish a reference line on the rigid body, extending away from a point on the axis of rotation in a direction perpendicular to the axis of rotation. This reference line rotates with the object. Its motion is the angular motion of the object. We also need a reference line segment that is fixed in space, extending from the same point on the axis, and away from the axis in a direction perpendicular to the axis. This one does not rotate with the object. Imagining the two lines to have at one time been collinear, the net angle through which the first line on the rigid body has turned relative to the fixed line is the angular position θθ\theta of the object. The Constant Angular Acceleration Equations While physically, there is a huge difference, mathematically, the rotational motion of a rigid body is identical to motion of a particle that only moves along a straight line. As in the case of linear motion, we have to define a positive direction. We are free to define the positive direction whichever way we want for a given problem, but we have to stick with that definition throughout the problem. Here, we establish a viewpoint some distance away from the rotating rigid body,but on the axis of rotation, and state that, from that viewpoint, counterclockwise is the positive sense of rotation, or alternatively, that clockwise is the positive sense of rotation. Whichever way we pick as positive, will be the positive sense of rotation for angular displacement (change in angular position), angular velocity, angular acceleration, and angular position relative to the reference line that is fixed in space. Next, we establish a zero for the time variable; we imagine a stopwatch to have been started at some instant that we define to be time zero. We call values of angular position and angular velocity, at that instant, the initial values of those quantities. Given these criteria, we have the following table of corresponding quantities. Note that a rotational motion quantity is in no way equal to its linear motion counterpart, it simply plays a role in rotational motion that is mathematically similar to the role played by its counterpart in linear motion. Linear Motion Quantity Corresponding Angular Motion Quantity xxx θθ\theta vvv ωω\omega aaa ∝∝\propto The one variable that the two different kinds of motion do have in common is the stopwatch reading ttt. Recall that, by definition, ω=dθdtω=dθdt \omega=\frac{d\theta}{dt} \nonumber and∝=dωdtand∝=dωdt\mbox{and} \quad \propto= \frac{d\omega}{dt} \nonumber While it is certainly possible for ∝∝\propto to be a variable, many cases arise in which ∝∝\propto is a constant. Such a case is a special case. The following set of constant angular acceleration equations apply in the special case of constant angular acceleration: (The derivation of these equations is mathematically equivalent to the derivation of the constant linear acceleration equations. Rather than derive them again, we simply present the results.) θ=θ0+ω0t+12∝t2(19A.3)(19A.3)θ=θ0+ω0t+12∝t2 \theta=\theta_0+\omega_0t+\frac{1}{2} \propto t^2 \label{19-2} θ=θ0+ω0+ω2t(19A.4)(19A.4)θ=θ0+ω0+ω2t \theta=\theta_0+\frac{\omega_0+\omega}{2} t \label{19-3} ω=ω0+∝t(19A.5)(19A.5)ω=ω0+∝t \omega=\omega_0+\propto t \label{19-4} ω2=ω20+2∝Δθ(19A.6)(19A.6)ω2=ω02+2∝Δθ \omega^2=\omega_0^2 +2\propto \Delta\theta \label{19-5} The rate at which a sprinkler head spins about a vertical axis increases steadily for the first 2.00 seconds of its operation such that, starting from rest, the sprinkler completes 15.0 revolutions clockwise (as viewed from above) during that first 2.00 seconds of operation. A nozzle, on the sprinkler head, at a distance of 11.0 cm from the axis of rotation of the sprinkler head, is initially due west of the axis of rotation. Find the direction and magnitude of the acceleration of the nozzle at the instant the sprinkler head completes its second (good to three significant figures) rotation. Solution We’re told that the sprinkler head spin rate increases steadily, meaning that we are dealing with a constant angular acceleration problem, so, we can use the constant angular acceleration equations. The fact that there is a non-zero angular acceleration means that the nozzle will have some tangential acceleration at→at→\vec{a_t}. Also, the sprinkler head is spinning at the instant in question so the nozzle will have some centripetal acceleration ac→ac→\vec{a_c}. We’ll have to find both at→at→\vec{a_t} and ac→ac→\vec{a_c} and add them like vectors to get the total acceleration of the nozzle. Let’s get started by finding the angular acceleration ∝∝\propto. We start with the first constant angular acceleration equation (equation 19A.319A.3\ref{19-2}): θ=0+0⋅t+12∝t2θ=0+0⋅t+12∝t2 \theta= 0+ 0\cdot t+\frac{1}{2} \propto t^2 \nonumber The initial angular velocity ω0ω0\omega_0 is given as zero. We have defined the initial angular position to be zero. This means that, at time t=2.00st=2.00st = 2.00 s, the angular position θθ\theta is 15.0rev=15.0 rev2π radrev=94.25rad15.0rev=15.0 rev2π radrev=94.25rad15.0 \, \mbox{rev}=15.0\space \mbox{rev}\frac{2\pi \space\mbox{rad}}{\mbox{rev}}=94.25 \mbox{rad}. Solving equation 19A.319A.3\ref{19-2} above for ∝∝\propto yields: ∝=2θt2∝=2θt2 \propto=\frac{2\theta}{t^2} \nonumber ∝=2(94.25rad)(2.00s)2∝=2(94.25rad)(2.00s)2 \propto=\frac{2(94.25 \mbox{rad})}{(2.00s)^2} \nonumber ∝=47.12rads2∝=47.12rads2 \propto=47.12 \frac{\mbox{rad}}{s^2} \nonumber Substituting this result into equation 19A.219A.2\ref{19-1}: at=r∝at=r∝a_t= r \propto \nonumber gives us at=(.110m)47.12rad/s2at=(.110m)47.12rad/s2 a_t=(.110m)47.12\mbox{rad}/s^2 \nonumber which evaluates to at=5.18ms2at=5.18ms2a_t=5.18 \frac{m}{s^2} \nonumber Now we need to find the angular velocity of the sprinkler head at the instant it completes 2.00 revolutions. The angular acceleration ∝∝\propto that we found is constant for the first fifteen revolutions, so the value we found is certainly good for the first two turns. We can use it in the fourth constant angular acceleration equation (equation 19A.619A.6\ref{19-5}): ω2=0+2∝Δθω2=0+2∝Δθ \omega^2= 0+2\propto \Delta\theta \nonumber where Δθ=2 rev=2.00 rev2π radrev=4.00π radΔθ=2 rev=2.00 rev2π radrev=4.00π rad\Delta\theta=2\space \mbox{rev}=2.00\space\mbox{rev}\frac{2\pi\space\mbox{rad}}{\mbox{rev}}=4.00\pi \space\mbox{rad} ω=2∝Δθ−−−−−−√ω=2∝Δθ\omega=\sqrt{2\propto\Delta\theta} \nonumber ω=2(94.25rad/s2)4.00πrad−−−−−−−−−−−−−−−−−−−√ω=2(94.25rad/s2)4.00πrad\omega=\sqrt{2(94.25 \mbox{rad}/s^2)4.00\pi \mbox{rad}} \nonumber \omega=48.67 \mbox{rad}/s\label{19-6}\omega=48.67 \mbox{rad}/s\label{19-6}\omega=48.67 \mbox{rad}/s\label{19-6} (at that instant when the sprinkler head completes its 2nd turn) Now that we have the angular velocity, to get the centripetal acceleration we can use equation ??????\ref{18-6}: ac=rω2ac=rω2 a_c=r\omega^2 \nonumber ac=.110m(48.67rad/s)2ac=.110m(48.67rad/s)2 a_c=.110m(48.67 \mbox{rad}/s)^2 \nonumber ac=260.6ms2ac=260.6ms2a_c=260.6 \frac{m}{s^2} \nonumber Given that the nozzle is initially at a point due west of the axis of rotation, at the end of 2.00 revolutions it will again be at that same point. Now we just have to add the tangential acceleration and the centripetal acceleration vectorially to get the total acceleration. This is one of the easier kinds of vector addition problems since the vectors to be added are at right angles to each other. From Pythagorean’s theorem we have a=a2c+a2t−−−−−−√a=ac2+at2 a=\sqrt{a_c^2+a_t^2} \nonumber a=(260.6m/s2)2+(5.18m/s2)2−−−−−−−−−−−−−−−−−−−−−−√a=(260.6m/s2)2+(5.18m/s2)2 a=\sqrt{(260.6m/s^2)^2+(5.18m/s^2)^2} \nonumber a=261m/s2a=261m/s2 a=261m/s^2 \nonumber From the definition of the tangent of an angle as the opposite over the adjacent: tanθ=atactanθ=atac tan\theta=\frac{a_t}{a_c} \nonumber θ=tan−15.18m/s2260.6m/s2θ=tan−15.18m/s2260.6m/s2 \theta=tan^{-1} \frac{5.18 m/s^2}{260.6m/s^2} \nonumber θ=1.14∘θ=1.14∘\theta=1.14^{\circ} \nonumber Thus, a=261m/s2at 1.14∘ North of Easta=261m/s2at 1.14∘ North of Easta=261m/s^2 \quad \mbox{at \(1.14^{\circ}\) North of East} \nonumber When the Angular Acceleration is not Constant The angular position of a rotating body undergoing constant angular acceleration is given, as a function of time, by our first constant angular acceleration equation, equation 19A.319A.3\ref{19-2}: θ=θ0+ω0t+12∝t2θ=θ0+ω0t+12∝t2\theta=\theta_0+\omega_0t+\frac{1}{2}\propto t^2 \nonumber If we take the 2nd derivative of this with respect to time, we get the constant ∝∝\propto. (Recall that the first derivative yields the angular velocity ωω\omega and that ∝=dωdt∝=dωdt\propto=\frac{d\omega}{dt}. ) The expression on the right side of θ=θ0+ω0t+12∝t2θ=θ0+ω0t+12∝t2\theta=\theta_0+\omega_0t+\frac{1}{2}\propto t^2 contains three terms: a constant, a term with ttt to the first power, and a term with ttt to the 2nd power. If you are given θθ\theta in terms of ttt, and it cannot be rearranged so that it appears as one of these terms or as a sum of two or all three such terms; then; ∝∝\propto is not a constant and you cannot use the constant angular acceleration equations. Indeed, if you are being asked to find the angular velocity at a particular instant in time, then you’ll want to take the derivative dθdtdθdt\frac{d\theta}{dt} and evaluate the result at the given stopwatch reading. Alternatively, if you are being asked to find the angular acceleration at a particular instant in time, then you’ll want to take the second derivative d2θdt2d2θdt2\frac{d^2\theta}{dt^2} and evaluate the result at the given stopwatch reading. Corresponding arguments can be made for the case of ωω\omega. If you are given ωω\omega as a function of ttt and the expression cannot be made to “look like” the constant angular acceleration equation ω=ω0+∝

      Just a suggestion for improvement, throughout this page the proportional symbol,∝, is used instead of alpha,α. This should be really changed to the alpha symbol to be consistent with other texts, and not confuse those that are familiar with the logic symbol.

    1. Yeah, yeah, yeah. My point is that “eros” just is the word for love, so to say that Eros is the god of love would be like saying Eros is the god of eros.

      Making my head spin

  10. Aug 2023
    1. Alas, with the years all this fine contempt began to fade; for the words I longed for, and all their dazzling opportunities, were theirs, not mine. But they should not keep these prizes, I said; some, all, I would wrest from them. Just how I would do it I could never decide: by reading law, by healing the sick, by telling the wonderful tales that swam in my head,—some way.

      Similar to Henry Adams, Dubois longs to learn about everything education has to offer (reading law, telling wonderful tales.) He has a hunger for knowledge despite the obstacles he's had to overcome.

    1. Author Response

      The following is the authors’ response to the original reviews.

      We would like to thank both reviewers and the editor for their time and effort in carefully reviewing and comprehending our manuscript. We are grateful for their thorough assessment, as well as the insightful questions and suggestions they have provided. We have taken into account the questions and comments raised by the reviewers, and we have incorporated the necessary revisions accordingly. In the following pages the reviewers’ comments are italicized. Our replies are in normal script.

      In addition to revisions suggested by reviewers we also added a new summary schematic (Fig 8) and minor changes to acknowledgments.

      Reviewer 1

      This is a very strong study with few concerns. Regarding DN1+ T cell function, the authors assessed IFN-γ and activation markers, but it is unclear if the cells are polyfunctional (produced high levels of other cytokines at 6 weeks) or if there were changes in the humoral response (serum Ab titers or size/ number of germinal centers.)

      Thank you for your thorough assessment of our work and your kind comments.

      a. We observed a decreased IFN-γ and TNF-α production in antigen experienced DN1 T cells compared to naïve DN1 T cells, which is consistent with findings in Tfh cells.

      b. We tested for anti-MA IgM and IgG production but did not observe an increase in these antibodies in the vaccinated setting. It is possible that additional inflammatory stimulation, such as from an adjuvant or infection, may be necessary to trigger sufficient antibody level for detection using ELISA.

      c. We did not measure the number or size of germinal centers in this study, but future investigations could explore this aspect.

      Reviewer 2

      1. Authors elaborate the introduction solely highlighting the relevance of antigen persistence in the context of vaccination. However, it is well known that several mycobacterial antigens (Lipids and proteins) can cause detrimental responses when overexposed to the immune system. In this regard, it would be appropriate to introduce the possibility of the occurrence of exhaustion when prolonged exposure to antigens is happening, which is the main theme of this paper.

      Thank you for bringing these points to our attention. We have added a paragraph in the discussion section (page 15-16, line 372-386), addressing the implications of our findings in relation to exhaustion in the context of antigen persistence during chronic viral infections. We have also provided an example involving the lipid trehalose 6,6’-dibehenateled (TDM), a known virulence factor for Mtb, which has been utilized in several subunit vaccines without demonstrating significant toxicity.

      1. Authors need to provide more information about the source of MA. It is briefly mentioned in the materials and methods section that it was obtained from Sigma. If that is the case, it would be ideal to show the integrity of the polysaccharide in term of balance and abundance between different MA species.

      We obtained M. tuberculosis MA from Sigma, which comprises α-, keto-, and methoxy MA forms with an average combined lipid tail length of 80 carbons. MA-specific T cells preferentially recognize these three forms of MA have been identified in humans. We have provided more detailed information regarding the MA in the Materials and Methods section (page 17, line 429-431).

      1. Building up on the previous comment, MA is a complex mixture of polysaccharides including multiple lengths of fatty acids and modifications. Could the authors comments on the potential variability of MA structure and potential impact on immune responses?

      The binding capacities of Group 1 CD1-restricted T cells can be influenced by various factors, including specific head groups, lipid tail length, and structure of the lipid tail. Notably, DN1 T cells have been shown to have higher binding affinities towards keto and methoxy MA, while displaying weaker binding to α-MA (Van Rhijn et al., 2017, Eur. J. Immunol. 47:1525). In our study, we successfully utilized a mixture of MA to activate DN1 T cells, indicating that the required subtypes of MA were present in sufficient quantities to elicit this activation. In future investigations focusing on the polyclonal immune response, incorporating a mixture of MA and possibly other Mtb lipid antigens will enable a broader spectrum of T cell activation. This, in turn, is expected to enhance the overall effectiveness and robustness of protection in challenge experiments.

      1. How do the authors explain the lack of stimulation of cell proliferation induced by MA-PLGA formulation? Does this result contradict previous findings?

      This study represents the first instance of utilizing PLGA as a delivery system for a lipid antigen via a pulmonary vaccine route, despite its previous applications in numerous other vaccine formulations. Therefore, we do not think our findings contradicts any existing research in the field. It is worth noting that the immunogenicity of PLGA can be influenced by the specific polymer chemistry and formulation, which may account for potential variations in the observed effects. We have added additional text to the discussion (page 13, line 310 – 313) to address this point.

      1. Fig 3. Authors switch to IT administration simply arguing against the limitation of IN delivery regarding its low volume. However, administration via IN could be done in an iterative manner. According to this change, this reviewer asks whether the performance of MA-PLGA could now be comparable to BCN-MA using IT instead.

      PLGA possesses an inherent background adjuvant effect, which may not be ideal for precisely stimulating group 1 CD1-restricted T cells, as a considerable proportion of these T cells exhibit some level of autoreactivity (Li, et al, 2011, Blood 118:3870, De Lalla et al., 2011, Eur. J. Immunol. 41:602; de Jong et al, 2010, Nat. Immunol. 11:1102). Notably, our observations revealed that blank PLGA-NP exerted a significant stimulatory effect on both mouse (DN1) and human (M11) MA-specific T cells (Fig. 2A-D). This underscores the advantage of the BCN system, which lacks detectable adjuvant effects and enables a more controlled, dose-dependent augmentation of T cell responses with increasing concentrations of loaded MA. Therefore, we did not further evaluate the impact of PLGA-MA using the IT route of vaccination.

      1. What would be the reasons of the no role of encapsulating NP in the persistence of MA?

      In this study, we have provided evidence to support the notion that encapsulation plays a role in antigen persistence, as demonstrated in Fig. 5A-C. Specifically, we directly compared the persistence of MA when delivered encapsulated in BCNs versus without encapsulation in BCNs, using DC pulsing and IT vaccination as the delivery methods. Our results indicate that at 6 weeks post-vaccination, MA encapsulated in BCNs can activate DN1 T cells, while free MA does not. These findings may initially appear to be contradictory to those depicted in Fig. 5D-F, where antigen persistence is observed following vaccination with attenuated Mtb. However, we propose that the attenuated Mtb bacteria may function similarly to nanoparticles by encapsulating and containing MA, thereby facilitating its persistence within the host. We appreciate the opportunity to clarify these points (page 15, line 364-367). Encapsulation within PEG-PPS NP may also contribute to two additional mechanisms. First, we have demonstrated that PEG-PPS NPs target myeloid cell populations (Burke et al., 2022, Nat. Nano. 17:319), such as alveolar macrophages, that can serve as antigen persistence depots as well as present CD1b/MA complexes on their surfaces. NPs allow more efficient delivery to these cells, whereas otherwise the lipid would bind to albumin, HDL, LDL, and other lipid carriers in blood for a broader, non-specific biodistribution, which would include cells less efficient at antigen persistence or presentation. Second, we previously demonstrated that the BCN nanostructure is highly stable within cells, supporting a slow intracellular release (Bobbala et al., 2020, Nanoscale 12:5332). This could assist with a more sustained presentation of lipid antigen by targeted cells in contrast to free form lipid or NPs (like PLGA) that rapidly degrade within cells. Indeed, low levels of fluorescently tagged BCNs were still detectable 6 weeks post-vaccination (Fig. 6B). Our future studies will further investigate this hypothesis.

      1. Authors need to discuss to what extent the MA location into AM is route dependent.

      The localization of MA within alveolar macrophages (AMs) in the lung is likely specific to intratracheal (IT) vaccination. Therefore, mice vaccinated subcutaneously (SC) or intravenously (IV) may possess distinct antigen persistence depots. We have made modifications to the discussion section to further emphasize this point (page 15, line 359-364).

      1. Also, AM are programmed to sustain low immune responses because of their unique location in the lung. In fact, Mtb uses this to replicate while immune response is mounted. In this regard, accumulation of MA into this compartment may not be relevant for the overall immune response. In other words, what would be the contribution of this population to the T cell activation?

      It is likely that AMs primarily function as antigen depots and do not directly contribute to the activation of DN1 T cells. This assertion is supported by our findings, as co-culturing AMs with DN1 T cells alone did not result in T cell activation (Fig. 6E). However, we observed that the presence of hCD1Tg-expressing bone marrow-derived dendritic cells was necessary for DN1 T cell activation in vitro, which likely reflects a similar phenomenon occurring in vivo.

      1. Could the T cells responses measured be due to the reduced fraction of DC loaded with BCN-MA at initial time points?

      Regarding the T cell response observed in Fig. 5A-C, where we used DCs to deliver either free MA or MA-BCN, we took steps to address potential differences in loading capacity between the two at initial time points. Specifically, DCs were pulsed with a concentration of 10 𝜇g/mL for free MA and 5 𝜇g/mL of MA-BCN (the figure legend has been modified to clarify this point, page 37, line 962 - 963). To ensure approximate equivalence in loading, we examined the immune response one week after vaccination and found no statistically significant difference between the two methods.

    1. Reviewer #3 (Public Review):

      Summary:

      The paper proposes an alternative to the attractor hypothesis, as an explanation for the fact that grid cell population activity patterns (within a module) span a toroidal manifold. The proposal is based on a class of models that were extensively studied in the past, in which grid cells are driven by synaptic inputs from place cells in the hippocampus. The synapses are updated according to a Hebbian plasticity rule. Combined with an adaptation mechanism, this leads to patterning of the inputs from place cells to grid cells such that the spatial activity patterns are organized as an array of localized firing fields with hexagonal order. I refer to these models below as feedforward models.

      It has already been shown by Si, Kropff, and Treves in 2012 that recurrent connections between grid cells can lead to alignment of their spatial response patterns. This idea was revisited by Urdapilleta, Si, and Treves in 2017. Thus, it should already be clear that in such models, the population activity pattern spans a manifold with toroidal topology. The main new contributions in the present paper are (i) in considering some forms of recurrent connectivity that were not directly addressed before (but see comments below). (ii) in applying topological analysis to simulations of the model. (iii) in interpreting the results as a potential explanation for the observations of Gardner et al.

      Strengths:

      The exploration of learning in a feedforward model, when recurrent connectivity in the grid cell layer is structured in a ring topology, is interesting. The insight that this not only aligns the grid cells in a common direction but also creates a correspondence between their intrinsic coordinate (in terms of the ring-like recurrent connectivity) and their tuning on the torus is interesting as well, and the paper as a whole may influence future theoretical thinking on the mechanisms giving rise to the properties of grid cells.

      Weaknesses:

      1. It is not clear to me that the proposal here is fundamentally new. In Si, Kropff and Treves (2012) recurrent connectivity was dependent on the head direction tuning and thus had a ring structure. Urdapilleta, Si, and Treves considered connectivity that depends on the distance on a 2d plane.

      2. The paper refers to the connectivity within the grid cell layer as an attractor. However, would this connectivity, on its own, indeed sustain persistent attractor states? This is not examined in the paper. Furthermore, is this even necessary to obtain the results in the model? Perhaps weak connections that do not produce an attractor would be sufficient to align the spatial response patterns during the learning of feedforward weights, and reproduce the results? In general, there is no exploration of how the strength of collateral interactions affects the outcome.

      3. I did not understand what is learned from the local topology analysis. Given that all the grid cells are driven by an input from place cells that spans a 2d manifold, and that the activity in the grid cell network settles on a steady state that depends only on the inputs, isn't it quite obvious that the manifold of activity in the grid cell layer would have, locally, a 2d structure?

      4. The modeling is all done in planar 2d environments, where the feedforward learning mechanism promotes the emergence of a hexagonal pattern in the single neuron tuning curve. This, combined with the fact that all neurons develop spatial patterns with the same spacing and orientation, implies even without any topological analysis that the emerging topology of the population activity is a torus.

      However, the toroidal topology of grid cells in reality has been observed by Gardner et al also in the wagon wheel environment and in sleep, and there is substantial evidence based on pairwise correlations that it persists also in various other situations, in which the spatial response pattern is not a hexagonal firing pattern. It is not clear that the mechanism proposed in the present paper would generate toroidal topology of the population activity in more complex environments. In fact, it seems likely that it will not do so.

      5. Moreover, the recent work of Gardner et al. demonstrated much more than the preservation of the topology in the different environments and in sleep: the toroidal tuning curves of individual neurons remained the same in different environments. Previous works, that analyzed pairwise correlations under hippocampal inactivation and various other manipulations, also pointed towards the same conclusion. Thus, the same population activity patterns are expressed in many different conditions. In the present model, the results of Figure 6 suggest that even across distinct rectangular environments, toroidal tuning curves will not be preserved, because there are multiple possible arrangements of the phases on the torus which emerge in different simulations.

      6. In real grid cells, there is a dense and fairly uniform representation of all phases (see the toroidal tuning of grid cells measured by Gardner et al). Here the distribution of phases is not shown, but Figure 7 suggests that phases are non uniformly represented, with significant clustering around a few discrete phases. This, I believe, is also the origin for the difficulty in identifying the toroidal topology based on the transpose of the matrix M: vectors representing the spatial response patterns of individual neurons are localized near the clusters, and there are only a few of them that represent other phases. Therefore, there is no dense coverage of the toroidal manifold that would exist if all phases were represented equally. This is not just a technical issue, however: there appears to be a mismatch between the results of the model and the experimental reality, in terms of the phase coverage.

      7. The manuscript makes several strong claims that incorrectly represent the relation between experimental data and attractor models, on one hand, and the present model on the other hand. For the latter, see the comments above. For the former, I provide a detailed list in the recommendations to the authors, but in short: the paper claims that attractor models induce rigidness in the neural activity which is incompatible with distortions seen in the spatial response patterns of grid cells. However, this claim seems to confuse distortions in the spatial response pattern, which are fully compatible with the attractor model, with distortions in the population activity patterns, which would be incompatible with the attractor model. The attractor model has withstood numerous tests showing that the population activity manifold is rigidly preserved across conditions - a strong prediction (which is not made, as far as I can see, by feedforward models). I am not aware of any data set where distortions of the population activity manifold have been identified, and the preservation has been demonstrated in many examples where the spatial response pattern is disrupted. This is the main point of two papers cited in the present manuscript: by Yoon et al, and Gardner et al.

      8. There is also some weakness in the mathematical description of the dynamics. Mathematical equations are formulated in discrete time steps, without a clear interpretation in terms of biophysically relevant time scales. It appears that there are no terms in the dynamics associated with an intrinsic time scale of the neurons or the synapses, and this introduces a difficulty in interpreting synaptic weights as being weak or strong. As mentioned above, the nature of the recurrent dynamics within the grid cell network (whether it exhibits continuous attractor behavior) is not sufficiently clear.

      In my view, the weaknesses discussed above limit the ability of the model, as it stands, to offer a compelling explanation for the toroidal topology of grid cell population activity patterns, and especially the rigidity of the manifold across environments and behavioral states. Still, the work offers an interesting way of thinking on how the toroidal topology might emerge. Perhaps with certain additional elements this may motivate new theoretical insights.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Mitochondria is the power plant of the cells including neurons. Thomas et al. characterized the distribution of mitochondria in dendrites and spines of L2/3 neurons from the ferret visual cortex, for which visually driven calcium responses of individual dendritic spines were examined. The authors analyzed the relationship between the position of mitochondria and the morphology or orientation selectivity of nearby dendrite spines. They found no correlation between mitochondrion location and spine morphological parameters associated with the strength of synapses, but correlation with the spine-somatic difference of orientation preference and local heterogeneity in preferred orientation of nearby spines. Moreover, they reported that the spines that have a mitochondrion in the head or neck are larger in size and have stronger orientation selectivity. Therefore, they proposed that "mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs."

      Strengths:<br /> This paper attempted to address a fundamental question: whether the distribution of the mitochondria along the dendrites of visual cortical neurons is associated with the functions of the spines, postsynaptic sites of excitatory synapses. Two state of the art techniques (2 photon Ca imaging of somata and spines and EM reconstructions of cortical pyramidal neurons) had been used, which provides a great opportunity to examine and correlate the function of spine ultrastructure and spatial distribution of dendritic mitochondria.

      Weaknesses:<br /> Overall, the findings are interesting. However, the study lacks the data providing insights into either the mechanisms or the functional meaning of the pattern of mitochondrion distribution along the dendrites, which restricts the significance of the study. It also suffers from small correlation coefficients and small sample sizes (60-121 spines in 4 neurons) as well as missing some important analysis.

    1. This incisive definition forces the reader to hold themselves accountable for their ideas and actions.

      I think this can be hard to fully get through one's head because it's extremely difficult to admit to oneself that one may be in the wrong or in need of change. I think in general, one of societies greatest flaws is our inability to hold ourselves accountable. It's easier to point out others' shortcomings. A great example is cancel culture. Many are quick to point a finger but won't admit that they also may need to change their own ways.

    2. The antiracist position would be to at least consider enrolling your child and/or learning about the disparities and inequities affecting that school in order to fight them.

      It is important to go steps further in getting rid of racism and to educate not only ones self but family as well in the differences between races. With racism you have to tackle the problem head on and not try and jump around it because it makes you uncomfortable.

    1. In many cases we know what interests us and we automatically gravitate toward stimuli that match up with that.

      I feel like I relate to this immensely, because there is a very specific instance when I see something very particular, I stop everything I'm doing to observe it. I'm often spending time in my driveway washing or working on my car, or sometimes helping my friend with his car. When I do, especially in the later afternoon, my eyes always seem to find themselves gazing on any car that is blue, because of my fascination with a Boost Blue Honda FL5 Type R that is owned by someone in my neighborhood. I have such an interest in this car that I can pick it up so quickly from my peripheral vision, to a point where I literally stop what I'm doing to stare at it as it drives by. If I see another blue car, my head will turn to see if it's the FL5, because I have such high interest for it. (I moved this annotation because this sentence matched better with what I was saying)

    1. As I’m sure you guys know by now, it is extremely difficult to stay alert and attentive, instead of getting hypnotised by the constant monologue inside your own head (may be happening right now).

      This is something I can relate to. I find myself getting distracted really quickly and I find it hard to focus on what is actually being taught in front of me. I take a LOT of breaks while doing homework!

    1. W hy is it almost impossible to gaze directly at the Grand Canyon under thesecircumstances and see it for what it is—as one picks up a strange objec t from o ne’sback yard and gazes directly at it? It is almo st impossible because the Grand Canyon,the thing as it is, has been appropriated by the symbolic complex which has alreadybeen formed in the sightseer’s mind. Seeing the canyon under approved circumstancesis seeing the symb olic co mplex head on. T he thing is no longer the thing as itconfronted the Sp aniard ; it is rather that which has already been formulated—bypicture postcard , geography book, tourist folders, and the words Gra nd C any on. As aresult of this preformulation, the source of the sightseer’s pleasure undergoes a shift.W here the wonder and delight of the Spaniard arose from his penetration of the thingitself, from a progressive discovery of depths, patterns, colors, shadows, etc., now thesightseer measures his satisfaction by the d egree to w hich the canyo n con forms to thepreformed com plex

      The Grand Canyon is not giving tourists the same feeling as the Spaniard felt because tourists have expectations on what the grand canyon will look like, and especially high standards.

    2. W hy is it almost impossible to gaze directly at the Grand Canyon under thesecircumstances and see it for what it is—as one picks up a strange objec t from o ne’sback yard and gazes directly at it? It is almo st impossible because the Grand Canyon,the thing as it is, has been appropriated by the symbolic complex which has alreadybeen formed in the sightseer’s mind. Seeing the canyon under approved circumstancesis seeing the symb olic co mplex head on. T he thing is no longer the thing as itconfronted the Sp aniard ; it is rather that which has already been formulated—bypicture postcard , geography book, tourist folders, and the words Gra nd C any on. As aresult of this preformulation, the source of the sightseer’s pleasure undergoes a shift.W here the wonder and delight of the Spaniard arose from his penetration of the thingitself, from a progressive discovery of depths, patterns, colors, shadows, etc., now thesightseer measures his satisfaction by the d egree to w hich the canyo n con forms to thepreformed com plex

      I understood this as addressing reputation. No one can perceive the Grand Canyon as if you're discovering it for the first time. On top of that, majority of people have seen pictures, so we establish this idea in our heads of what the Canyon is. This means when we visit it, we're just seeing if it lives up to the reputation.

    1. They control the biosynthetic machinery of bacterial host and behest them to produce different viral proteins. They are considered as particles outside the host cell containing nucleic acid (DNA or RNA) which encode necessary information required for their replication. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

      How bacteriaphage work.

    2. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

      This annotation is where you can find the bacteriophages.

    3. Bacteriophages are viruses, the most abundant organisms and the natural predators of bacteria. They are self-replicating, obligatory intracellular parasites and inert biochemically in extracellular environment. They control the biosynthetic machinery of bacterial host and behest them to produce different viral proteins. They are considered as particles outside the host cell containing nucleic acid (DNA or RNA) which encode necessary information required for their replication. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

      overall explaining what bacteriophages are and what all they do. breaks down exactly what they do.

    4. Bacteriophages are viruses, the most abundant organisms and the natural predators of bacteria. They are self-replicating, obligatory intracellular parasites and inert biochemically in extracellular environment. They control the biosynthetic machinery of bacterial host and behest them to produce different viral proteins. They are considered as particles outside the host cell containing nucleic acid (DNA or RNA) which encode necessary information required for their replication. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

      Bacteriophages are viruses, they are the natural predators of bacteria, self replicating, obligatory intracellular parasites and inert biochemically in extracellular environments.

    1. I used to have the view that Scrum is a useless batch of meetings, that sucks the life and productivity out of the dev process.Now, after seeing it from an adjacent (but not subjugated under it) perspective, I think it is a life-sucking batch of meetings that are good for one thing: taking developers who can’t or don’t want to see the overall business/architecture picture and getting useful work out of them.Most of us here are not in that category. I’d wager a majority of HN readers can’t help but to seek out understanding of the business, where this piece fits, what it interacts with. For us, specifying everything upfront is useless. Estimating stuff is irritating because we need the flexibility to make smart decisions during dev. Retro meetings are lies because we can’t say “stop with all this and let me work”.But if you’re trying to make a process than can take junior devs (not junior in tenure, but junior in the qualities above) and produce an output that scales almost-kinda linearly with dev count, it sort of works.I’d argue that you’re way better off hiring 6 devs that can go from business problem -> technical solution in their head, without all the ceremony, instead of 40 devs who can’t and 6 PMs to wrangle them.But I can also see how a company ends up there - go through a tough hiring year, or even just make a few poor hiring decisions, and now you have people on the team who need handholding and supervision. That’s what scrum is; it feels like micromanagement because it is. It forces junior-performing devs into a productive state - maybe 5% of what you’d get out of a senior-performing dev without scrum, but it’s something non-negative.

      A surprisingly positive take on scrum and where it could be useful

    1. This isn’t just a summit for crafters, it is a summit serving crafters who want to use their skills to make handmade gifts for their friends and family. It’s for crafters who need a little hand-holding

      I'd make this section more about the takeaways and that they'll walk away with actual gift ideas complete with instructions so they can make them happen right away, and get a head-start on their handmade xmas gifting journey. Where other summits might give you ideas, this summit takes you through a project from start to finish with the expert guidance of experience crafters.

    1. patriarchy and neoliberalism

      patriarchy is defined as: a system of society or government in which the father or eldest male is head of the family and descent is traced through the male line. neo liberalism: Neoliberalism, also neo-liberalism, is a term used to signify the late-20th century political reappearance of 19th-century ideas associated with free-market capitalism after it fell into decline following the Second World War.

    1. Dana Rachlin, executive director of We Build the Block, a Brooklyn-based public safety organization that helps run the alliance, bought Alicia some Chinese food to calm her. As she ate her meal, Rachlin called the city’s mental health hotline. She waited while on hold for 10 minutes before someone told her it would be 24 hours before a team could come, and that she could call the police. Rachlin rolled her eyes and hung up. It was getting colder. Rachlin sat on the bench at the bus stop and Alicia sat next to her, put her head on her shoulder and fell asleep. Finally, Rachlin and Almond and an executive from a social services group drove Alicia to an intake center for a shelter. She could not get a bed until Monday, but she could stay at the center through the weekend.

      This is a great example of what can happen if different professionals responded to more situations like these instead of police. The police would most likely have just taken her into jail for public intoxication, but luckily got the right help and support she needed.

    1. The sender is the radio announcer who encodes a verbal message that is transmitted by a radio tower through electromagnetic waves (the channel) and eventually reaches your (the receiver’s) ears via an antenna and speakers in order to be decoded. The radio announcer doesn’t really know if you receive his or her message or not, but if the equipment is working and the channel is free of static, then there is a good chance that the message was successfully received.

      I went to a baseball game with my boyfriend and they spoke on the intercome about what was going on in the game but I never watched a baseball game in my life so while my bf already decoded it in his head he decoded it for me out loud as well about who's winning.

    1. Author Response

      The following is the authors’ response to the original reviews.

      Reviewer #1 (Public Review):

      This study investigates the context-specificity of facial expressions in three species of macaques to test predictions for the 'social complexity hypothesis for communicative complexity'. This hypothesis has garnered much attention in recent years. A proper test of this hypothesis requires clear definitions of 'communicative complexity' and 'social complexity'. Importantly, these two facets of a society must not be derived from the same data because otherwise, any link between the two would be trivial. For instance, if social complexity is derived from the types of interactions individuals have, and different types of signals accompany these interactions, we would not learn anything from a correlation between social and communicative complexity, as both stem from the same data.

      The authors of the present paper make a big step forward in operationalising communicative complexity. They used the Facial Action Coding System to code a large number of facial expressions in macaques. This system allows decomposing facial expressions into different action units, such as 'upper lid raiser', 'upper lip raiser' etc.; these units are closely linked to activating specific muscles or muscle groups. Based on these data, the authors calculated three measures derived from information theory: entropy, specificity and prediction error. These parts of the analysis will be useful for future studies.

      The three species of macaque varied in these three dimensions. In terms of entropy, there were differences with regard to context (and if there are these context-specific differences, then why pool the data?). Barbary and Tonkean macaques showed lower specificity than rhesus macaques. Regarding predicting context from the facial signals, a random forest classifier yielded the highest prediction values for rhesus monkeys. These results align with an earlier study by Preuschoft and van Schaik (2000), who found that less despotic species have greater variability in facial expressions and usage.

      Crucially, the three species under study are also known to vary in terms of their social tolerance. According to the highly influential framework proposed by Bernard Thierry, the members of the genus Macaca fall along a graded continuum from despotic (grade 1) to highly tolerant (grade 4). The three species chosen for the present study represent grade 1 (rhesus monkeys), grade 3 (Barbary macaques), and grade 4 (Tonkean macaques).

      The authors of the present paper define social complexity as equivalent to social tolerance - but how is social tolerance defined? Thierry used aggression and conflict resolution patterns to classify the different macaque species, with the steepness of the rank hierarchy and the degree of nepotism (kin bias) being essential. However, aggression and conflict resolution are accompanied by facial gestures. Thus, the authors are looking at two sides of the same coin when investigating the link between social complexity (as defined by the authors) and communicative complexity. Therefore, I am not convinced that this study makes a significant advance in testing the social complexity for communicative complexity hypothesis. A further weakness is that - despite the careful analysis - only three species were considered; thus, the effective sample size is very small.

      Social tolerance in macaques is defined by various covarying traits, among which rates of counter-aggression and conflict resolution are only two of many included (see Thierry 2021 for a recent discussion and review). We do not deviate from Thierry’s definition of social tolerance. We simply highlight that the constellation of behavioral traits in the most tolerant macaque species results in a social environment where the outcome of social interactions is more uncertain (see introduction lines 102-114). As we argue throughout the paper, higher uncertainty can be used as a proxy for higher complexity and thus we conclude that the most tolerant macaque species have the highest social complexity. While most social behavior in macaques is accompanied by some facial behavior, we were careful to define social contexts only from the body language/behavior (e.g., lunge for aggression, grooming for affiliation) of the individuals involved and ignored the facial behavior used (see method lines 371-381). Therefore, the facial behavior of macaques (communication signals) was not used in defining either social tolerance (and by extension complexity) or the social context in which it was used. We feel like this appropriately minimizes any elements of circularity in the analysis of social and communicative complexity.

      Regarding the effective sample size of three species, we agree that it is small, and it is a limitation of this study. However, the methodology we used is applicable to any species for which FACS is available (including other non-human primates, dogs, and horses), and therefore, we hope that other datasets will complement ours in the future. Nevertheless, we now acknowledge this limitation in the discussion (lines 314317).

      Reviewer #2 (Public Review):

      This is a well-written manuscript about a strong comparative study of diversity of facial movements in three macaque species to test arguments about social complexity influencing communicative complexity. My major criticism has to do with the lack of any reporting of inter-observer reliability statistics - see comment below. Reporting high levels of inter-observer reliability is crucial for making clear the authors have minimized chances of possible observer biases in a study like this, where it is not possible to code the data blind with regard to comparison group. My other comments and questions follow by line number:

      We agree that inter-observer coding reliability is an important piece of information. We now report in more detail the inter-observer reliability tests that we conducted on lines 384-392.

      38-40. Whereas I am an advocate of this hypothesis and have tested it myself, the authors should probably comment here, or later in the discussion, about the reverse argument - greater communicative complexity (driven by other selection pressures) could make more complicated social structures possible. This latter view was the one advocated by McComb & Semple in their foundational 2005 Biology Letters comparative study of relationships between vocal repertoire size and typical group size in non-human primate species.

      It is true that an increase in communicative complexity could allow/drive an increase in social complexity. Unfortunately our data is correlational in nature and we cannot determine the direction of causality. We added such a statement to the discussion (lines 311-314).

      72-84 and 95-96. In the paragraph here, the authors outline an argument about increasing uncertainty / entropy mapping on to increasing complexity in a system (social or communicative). In lines 95-96, though, they fall back on the standard argument about complex systems having intermediate levels of uncertainty (complete uncertainty roughly = random and complete certainty roughly = simple). Various authors have put forward what I think are useful ways of thinking about complexity in groups - from the perspective of an insider (i.e., a group member, where greater randomness is, in fact, greater complexity) vs from the perspective of an outside (i.e., a researcher trying to quantify the complexity of the system where is it relatively easy to explain a completely predictable or completely random system but harder to do so for an intermediately ordered or random system). This sort of argument (Andrew Whiten had an early paper that made this argument) might be worth raising here or later in the discussion? (I'm also curious where the authors sentiments lie for this question - they seem to touch on it in lines 285-287, but I think it's worth unpacking a little more here!)

      In this study we used three measures of uncertainty (entropy, context specificity, and prediction error) to approximate complexity. However, maximum entropy or uncertainty would be achieved in a system that is completely random (and thus be considered simple). Therefore, the species with the highest entropy values, or unpredictability, could be interpreted as having a simpler communication system than a species with a moderately high entropy/unpredictability value. Our argument is that animal communication systems cannot possibly be random, otherwise they would not have evolved as signals. In systems where we know the highest entropy (or unpredictability) will not be due to randomness, as is the case with animal social interactions and communication, we can conclude that the system with the highest uncertainty is the most complex. We have now expanded upon this point in the discussion (lines 286-294). See also response to reviewer 1 below.

      115-129. See also:

      Maestripieri, D. (2005). "Gestural communication in three species of macaques (Macaca mulatta, M. nemestrina, M. arctoides): use of signals in relation to dominance and social context." Gesture 5: 57-73.

      Maestripieri, D. and K. Wallen (1997). "Affiliative and submissive communication in rhesus macaques." Primates 38(2): 127-138.

      On that note, it is probably worth discussing in this paragraph and probably later in the discussion exactly how this study differs from these earlier studies of Maestripieri. I think the fact that machine learning approaches had the most difficulty assigning crested data to context is an important methodological advance for addressing these sorts of questions - there are probably other important differences between the authors' study here and these older publications that are worth bringing up.

      Our study differs from these two studies in that the studies above classified facial behavior into discrete categories (e.g., bared-teeth, lip-smack), whereas we adopted a bottom-up approach and made no a priori assumptions about which movements are relevant. We broke down facial behavior down to their individual muscle movements (i.e., Action Units). Measuring facial behavior at the level of individual muscle movements allows for a more detailed and objective description of the complexity of facial behavior. This is a general point in advancing the study of facial behavior that is discussed in the introduction (lines 60-71) and discussion (lines 206-208). The reason we don’t draw a direct comparison with the studies above is because they had a slightly different focus. Our study was more focused on complexity of the (facial) communication system in general rather than comparing whether the different species use the same facial behavior in the same/different social contexts.

      220-222. What is known about visual perception in these species? Recent arguments suggest that more socially complex species should have more sensitive perceptual processing abilities for other individuals' signals and cues (see Freeberg et al. 2019 Animal Behaviour). Are there any published empirical data to this effect, ideally from the visual domain but perhaps from any domain?

      This is an interesting point. We are not aware of any studies showing differences in visual perceptions within the macaque genus. Both crested macaques and rhesus macaques are able to discriminate between individuals and facial expressions in match-to-sample tasks with comparable performances (Micheletta et al., 2015a, 2015b; Parr et al. 2008; Parr & Heinz, 2009). Similarly, several macaque species are sensitive to gaze shifts from conspecifics (Tomasello et al. 1998; Teufel et al. 2010; Micheletta & Waller, 2012).

      274-277. I am not sure I follow this - could not different social and non-social contexts produce variation in different affective states such that "emotion"-based signals could be as flexible / uncertain as seemingly volitional / information-based / referential-like signals? This issue is probably too far away from the main points of this paper, but I suspect the authors' argument in this sentence is too simplified or overstated with regard to more affect-based signals.

      Emotion-based signals could, in theory, also produce flexible signals and it is possible that some facial expressions reflect an emotional state. However, some previous studies have suggested that facial expressions are only used as a display of emotion, rather than such signals having evolved for a different function such as announcing future intentions. In our study we found that macaques used, in some cases, the same facial expressions (i.e. combination of Action Units) in at least two different social contexts that, presumably, differed in their emotional valence. Thus, it is unlikely that particular facial expressions are bound to a single emotion. We think that this is an important point to make even though it is slightly beyond the scope of our paper.

      288 on. Given there are only three species in this study, the chances of one of the species being the 'most complex' in any measure is 0.33. Although I do not believe this argument I am making here, can the authors rule out the possibility that their findings related to crested macaques are all related to chance, statistically speaking?

      We are not aware of a way to rule out this possibility. However, we believe that we are appropriately cautious throughout the paper and acknowledge that having only investigated three species is a limitation of this study in the discussion (lines 314-317, see also our response to reviewer 1 above).

      329-330. The fact that only one male rhesus macaque was assessed here seems problematic, given the balance of sexes in the other two species. Can the authors comment more on this - are the gestures they are studying here identical across the sexes?

      We agree it would have been preferable to collect data on more than one male rhesus macaque, but that was unfortunately not possible. We are not aware of any studies showing differences in the use of facial behavior between male and female rhesus macaques. If differences exist, most likely these would occur in a sexual/mating context. However, in our study we only considered affiliative (non-sexual), submissive, and aggressive contexts, where we have no a priori reason to believe that there are sex differences.

      354-371. Inter-observer reliability statistics are required here - one of the authors who did not code the original data set, or a trained observer who is not an author, could easily code a subset of the video files to obtain inter-observer reliability data. This is important for ruling out potential unconscious observer biases in coding the data.

      We agree this is an important piece of information. We now report in more detail the inter-observer reliability tests that we conducted on lines 384-392:

      “An agreement rating of >0.7 was considered good [Ekman et al 2002] and was necessary for obtaining certification. To obtain a MaqFACS coding certification, AVR, CP, and PRC coded 23 video clips of rhesus macaques and the MaqFACS codes were compared to the data of other certified coders (https://animalfacs.com).

      The mean agreement ratings obtained were 0.85, 0.73, 0.83 for AVR, CP, and PRC, respectively. In addition, AVR and CP coded 7 videos of Barbary macaques with a mean agreement rating of 0.79. AVR and PRC coded 10 videos of crested macaques with a mean agreement rating of 0.74.”

      Reviewer #1 (Recommendations For The Authors):

      Given the long debate on the concept of information exchange in animal communication, I would also recommend being more careful with the term 'exchanges of information' (line 271). Perhaps it's better to be agnostic in the context of this paper.

      As suggested, we now changed the phrasing to focus on the behavior of the animals, rather than suggesting that information is being exchanged (lines 270-273),

      Line 281: "This result confirms the assumption that facial behaviour in macaques is not used randomly": the authors are knocking down a straw man. Nobody who has ever studied animal communication would consider that signals occur randomly. Otherwise, they would not have evolved as signals.

      Indeed, nobody claims that animal communication signals are used randomly. Although it may be taken for granted, we feel it is worthwhile to reiterate this point, given that we used relative entropy and prediction error as measures of complexity. For instance, maximum entropy or unpredictability would be achieved in a system that is completely random (and thus be considered simple). Therefore, the species with the highest entropy values, or lowest predictability, could be interpreted as having a simpler communication system than a species with a moderately high entropy value. But if we are working under the assumption that animal communication systems cannot possibly be random, then we can conclude that the species whose communication system has the highest entropy is in fact the most complex. We tried to make this justification clearer in the discussion (lines 285-294).

      I did not follow why there is a higher reliance on facial signals when predation pressure is higher. Apart from the fact that the authors cannot address this question, they may want to reconsider this idea altogether.

      We now expand on the logic of why predation pressure might affect the use of facial signals (see lines 308-309): “When predation pressure is higher, reliance on facial signals could be higher than, for example vocal signals, such as to not draw attention of predators to the signaller.”

      Technical comments:

      One methodological issue that requires clarification is what the units of analysis are. The authors write that each row in their analysis denoted an observation time of 500 ms. How many rows did the authors assemble? The authors mention a sample size of > 3000 social interactions in the abstract. How did they define social interactions? And how many 'time windows' of 500 ms were obtained? Did they take one window per interaction or several? If several, then how was this move accounted for in the analysis? The reporting needs to be more accurate here. Most likely, the bootstrapping took care of biases in the data, but still, this information needs to be provided.

      We have now added some additional information to the method section. Social interactions for each context had the following definitions: “Social context was labeled from the point of view of the signaler based on their general behavior and body language (but not the facial behavior itself), during or immediately following the facial behavior. An aggressive context was considered when the signaler lunged or leaned forward with the body or head, charged, chased, or physically hit the interaction partner. A submissive context was considered when the signaler leaned back with the body or head, moved away, or fled from the interaction partner. An affiliative context was considered when the signaler approached another individual without aggression (as defined previously) and remained in proximity, in relaxed body contact, or groomed either during or immediately after the facial behavior. In cases where the behavior of the signaler did not match our context definitions, or displayed behaviors belonging to multiple contexts, we labeled the social context as unclear. Social context was determined from the video itself and/or from the matching focal behavioral data, if available.” (lines 371-382). The total duration of all social interactions per social context, and thus the number of 500ms windows/rows, have been added to Table 1 (lines 395-397). There were several 500ms windows per social interaction. All 500ms time blocks per interaction were used in the statistical analyses in order to retain all the variation and complexity of the facial behavior (Action Unit combinations) used by the macaques (lines 403-405). Indeed the bootstrapping procedure was used to account for any biases in the data.

      Overall, I would recommend providing more information on the actual behaviour of the animals. The paper is strong in handling highly derived indices representing the behaviour, but the reader learns little about the animals' behaviour. Thus, it would be great if statements about the entropy ratio were translated into what these measures represent in real life. For context specificity, this is clear, but for entropy, not so much.

      A high entropy ratio essentially suggests that a species uses a high variety of unique facial behavior/signals and all signals in the repertoire are used roughly equally often (rather than one facial behavior being used 90% of the time and others rarely used). We have tried our best to better explain this point in the introduction (lines 75-81) and discussion (lines 215-222). Discussing exactly what these signals are and what they mean was beyond the scope of this paper.

      Line 106: nepotism, not kinship

      Changed as suggested (line 106).

      Line 113: I would avoid statements about how a monkey society is perceived by its members.

      We think that noting how individuals may perceive their social environment is worthwhile when defining social complexity, so have retained this point but changed the phrasing to be more speculative (lines 112-113).

      Line 329: I was very surprised that only one male was represented in the data for rhesus monkeys. The authors try to wriggle their way out of this issue in the supplementary material ("Therefore, we have no a priori reason to expect an overall difference in the diversity and complexity of facial behaviour between the sexes"), but I think this is a major shortcoming of the analysis. They should ascertain whether there are no sex differences in the other two species regarding their variables of interest. They could then make a very cautious case for there being no sex differences in rhesus either. But of course, they would not know for sure.

      As with our response to reviewer 2 above, we agree that it would have been preferable to collect data on more than one male rhesus macaque, but that was unfortunately not possible. We are not aware of any studies showing differences in the use of facial behavior between male and female rhesus macaques. If differences exist, most likely these would occur in a sexual/mating context. However, in our study we only considered affiliative (non-sexual), submissive, and aggressive contexts, where we have no a priori reason to believe that there are sex differences. Looking at sex differences in the use of facial behavior would be a worthwhile study on its own, but it is outside the scope of this paper.

      This paper would make a stronger contribution if it focussed on the comparative analysis of facial expressions and removed the attempt of testing the social complexity for communicative complexity hypothesis.

      A comparative analysis of the contextual use of specific facial movements is important. But this paper is focused on making a more general comparison of the communication style and complexity across species. The social complexity hypothesis for communicative complexity is one of the key theoretical frameworks for such an investigation and allows us to frame our study in a broader context. We contribute important data on 3 species with methods that can be replicated and extended to others species. Therefore, we believe that it is a worthy contribution to investigations of the evolution of complex communication.

      REFERENCES

      Micheletta, J., J. Whitehouse, L.A. Parr, and B.M. Waller. ‘Facial Expression Recognition in Crested Macaques (Macaca nigra)’. Animal Cognition 18 (2015): 985–90. https://doi.org/10/f7fvnh.

      Micheletta, Jérôme, Jamie Whitehouse, Lisa A. Parr, Paul Marshman, Antje Engelhardt, and Bridget M. Waller. ‘Familiar and Unfamiliar Face Recognition in Crested Macaques (Macaca nigra)’. Royal Society Open Science 2 (2015): 150109. https://doi.org/10/ggx9k9.

      Parr, L. A., and M. Heintz. ‘Facial Expression Recognition in Rhesus Monkeys, Macaca mulatta’. Animal Behaviour 77 (2009): 1507–13. https://doi.org/10/bbsp5n.

      Parr, L.A., M. Heintz, and G. Pradhan. ‘Rhesus Monkeys (Macaca mulatta) Lack Expertise in Face Processing’. Journal of Comparative Psychology 122 (2008): 390–402. https://doi.org/10/d7w6bv.

      Micheletta, J., and B.M. Waller. ‘Friendship Affects Gaze Following in a Tolerant Species of Macaque, Macaca nigra’. Animal Behaviour 83 (2012): 459–67. https://doi.org/10/c4f8n2.

      Thierry B. Where do we stand with the covariation framework in primate societies? Am. J. Biol. Anthropol. 128 (2021): 5–25. https://doi.org/10.1002/ajpa.24441

      Tomasello, M., J. Call, and B. Hare. ‘Five Primate Species Follow the Visual Gaze of Conspecifics’. Animal Behaviour 55 (1998): 1063–69. https://doi.org/10/bmq7xh.

      Teufel, C., A. Gutmann, R. Pirow, and J. Fischer. ‘Facial Expressions Modulate the Ontogenetic Trajectory of Gaze-Following among Monkeys’. Developmental Science 13 (2010): 913–22. https://doi.org/10/b6j5r7.

    1. Author Response

      The following is the authors’ response to the original reviews.

      We are grateful for the helpful comments of both reviewers and have revised our manuscript with them in mind.

      One of the main issues raised was that readers may by default assume that our models are correct. We in fact made it very clear in our discussion that the models are merely hypotheses that will need testing by “wet” experiments and we do not therefore agree that even readers unfamiliar with AF would assume that the models must be correct. It was also suggested that readers could be reassured by including extensive confidence estimates such as PAE plots. As it happens, every single model described in the manuscript had reasonably high PAE scores and more crucially the entire collection of output files, including PAE data, are readily accessible on Figshare at https://doi.org/10.6084/m9.figshare.22567318.v2, a fact that the reviewers appear to have overlooked. The Figshare link is mentioned three times in the manuscript. Embedding these data within the manuscript itself would in our view add even more details and we have therefore not included them in our revised manuscript. Likewise, it is rather simple for any reader to work out which part of a PAE matrix corresponds to an interaction observed in the corresponding pdb prediction. Besides which, it is our view that the biological plausibility and explanatory power of models is just as important as AF metrics in judging whether they may be correct, as is indeed also the case for most experimental work.

      Another important point was that the manuscript was too long and not readable. Yes, it is long and it could well be argued that we could have written a different type of manuscript, focusing entirely on what is possibly the simplest and most important finding, namely that our AF models suggest that in animal cells Wapl appears to form a quarternary complex with SA, Pds5, and Scc1 in a manner suggesting that a key function of Wapl’s conserved CTD is to sequester Scc1’s Nterminal domain after it has dissociated from Smc3. For right or for wrong, we decided that this story could not be presented on its own but also required 1) an explanation for how Scc1 is induced to dissociate from Smc3 in the first place and 2) how to explain that the quarternary complex predicted for animal cells was not initially predicted for fungi such as yeast. The yeast situation was an exception that clearly needed explaining if the theory was to have any generality and it turned out that delving into the intricate details of the genetics of releasing activity in yeast was eventually required and yielded valuable new insights. We also believe that our work on the recruitment of Eco/Esco acetyl transferases to cohesin and the finding that sororin binds to the Smc3/Scc1 interface also provided important insight into how releasing activity is regulated. We acknowledge that the paper is indeed long but do not think that it is badly written. It is above all a long and complex story that in our view reveals numerous novel insights into how cohesin’s association with chromosomes is regulated and have endeavoured to eliminate any excessive speculation. We feel it is not our fault that cohesin uses complex mechanisms.

      Notwithstanding these considerations, we have in fact simplified a few sections and removed one or two others but acknowledge that we have not made substantial cuts.

      It was pointed out that a key feature of our modelling, namely the predicted association of Wapl’s C-terminal domain with SA/Scc3’s CES is inconsistent with published biochemical data. The AF predictions for this interface are universally robust in all eukaryotic lineages and crucially fully consistent with published and unimpeachable genetic data. We note that any model that explains all findings is bound to be wrong for the very simple reason that some of these findings will prove to be incorrect. There is therefore an art in Science of judging which data must be explained and accommodated and which should be ignored. In this particular case, we chose to ignore the biochemistry. Time will tell whether our judgement proves correct.

      Last but not least, it was suggested that we might provide some experimental support for our proposed SA/Scc3-Pds5-Scc1-WaplC quaternary complex. We are in fact working on this by introducing cysteine pairs (that can be crosslinked in cells) into the proposed interfaces but decided that such studies should be the topic of a subsequent publication. It would be impossible with the resources available to our labs to follow up all of the potential interactions and we therefore decided to exclude all such experiments.

      We are grateful for the detailed comments provided by both reviewers, many of which were very helpful, and in many but not all cases have amended the manuscript accordingly.

      With regard to the more specific comments:

      Reviewer #1 (Recommendations For The Authors):

      1) One concern is that observed interfaces/complexes arise because AF-multimer will aim to pack exposed, conserved and hydrophobic surfaces or regions that contain charge complementarity. The risk is that pairwise interaction screens can result in false positive & non-physiological interactions. It is therefore important to report the level of model confidence obtained for such AF calculations:

      A) The authors should color the key models according to pLDDT scores obtained as reported by AF. This would allow the reader to judge the estimated accuracy of the backbone and side chain rotamers obtained. At least for the key models and interactions it would be important to know if the pLDDT score is >90 (Correct backbone and most rotamers) or >70 (only backbone is correct).

      B) It would also be important to report the PAE plots to allow estimation of the expected position error for most of the important interactions. pLDDT coloring and PEA plots can be shown side-by-side as shown in other published data (e.g. https://pubmed.ncbi.nlm.nih.gov/35679397/ (Supplementary data)

      C) The authors should include a Table showing the confidence of template modeling scores for the predicted protein interfaces as ipTM, ipTM+pTM as reported by AlphaFold-multimer. Ideally, they would also include DockQ scores but this may not be essential. Addition of such scores would help classification into Incorrect, Acceptable or of high quality. For example, line 1073 et seq the authors show a model of a SCC1SA and ESCO1 complex (Fig. 37). Are the modeling scores for these interfaces high? It does not help that the authors show cartoons without side chains? Can the authors provide a close-up view of the two interfaces? Are the amino acids are indeed packed in a manner expected for a protein interface? Can we exclude the possibility that the prediction is obtained merely because the sequence segments (e.g. in ESCO1 & ESCO2) are hydrophobic and conserved?

      We do not agree that including this level of detail to the text/figures of the manuscript would be suitable. All the relevant data for those who may be sceptical about the models are readily available at https://doi.org/10.6084/m9.figshare.22567318.v2. In our view, the cartoon versions of the models are easier for a reader to navigate. Anyone interested in the molecular details can look at the models directly.

      Importantly, no amount of statistical analysis can completely validate these models. What is required are further experiments, which will be the topic of further work from our and I dare from other laboratories.

      D) When they predict an interaction between the SA2:SCC1 complex and Sororin's FGF motif, they find that only 1/5 models show an interaction and that the interaction is dissimilar to that seen of CTCF. Again, it would be helpful to know about modeling scores. Can they show a close-up view of the SORORIN FGF binding interface to see if a realistic binding mode is obtained? Can they indicate the relevant region on the PAE plot?

      Given that AF greatly favours other interactions of Sororin’s FGF motif over its interaction with SA2-Scc1, we do not agree that dwelling on the latter would serve any purpose.

      2) Line 996: AF predicts with high confidence an interaction between Eco1 & SMC3hd. What are the ipTM (& DockQ if available) scores. Would the interface score High, Medium or Acceptable?

      As mentioned, see https://doi.org/10.6084/m9.figshare.22567318.v2.

      3) Line 1034 et seq: Eco1/ESCO1/ESCO2 interaction with PDS5. Interface scores need to be shown to determine that the models shown are indeed likely to occur. If these interactions have low model confidence, Fig. 36 and discussion around potential relevance to PDS5-Eco1 orientation relative to the SMC3 head remains highly speculative and could be expunged.

      See https://doi.org/10.6084/m9.figshare.22567318.v2. It should be clear that the predictions are very similar in fungi and animals. Crucially, we know that Pds5 is essential for acetylation in vivo, so the models appear plausible from a biological point of view.

      4) Considering the relatively large interface between ECO1 and SMC3, would the author consider the possibility that in addition to acetylating SMC3's ATPase domain, ECO1 remains bound to cohesin-DNA complex, as proposed for ESCO1 by Rahman et al (10.1073/pnas.1505323112)?

      This is certainly possible but we would not want to indulge in such speculation.

      5) E.g. Line 875 but also throughout the text: As there is no labeling of the N- and C-termini in the Figures, is frequently unclear what the authors are referring to when they mention that AF models orient chains in a certain manner.

      Good point. This has been amended. However, the positions of N- and C- is all available at https://doi.org/10.6084/m9.figshare.22567318.v2.

      6) Fig19B: PAE plots: authors should indicate which chains correspond to A, B, C. Which segment corresponds to the TYxxxR[T/S]L motif? Can they highlight this section on the PAE plot?

      Good point and amended in the revised manuscript.

      Minor comments:

      1) Line 440: the WAPL YSR motif is not shown in Fig. 14A

      2) Line 691: Scc3 spelling error.

      3) Line 931: Sentence ending '... SCC3 (SCC3N).' requires citation.

      4) Line 1008: Figure reference seems wrong. It should read: Fig. 34A left and right. Fig. 34B does not contain SCC1.

      Many thanks for spotting these. Hopefully, all corrected.

      5) Fig. 41 can be removed as it shows the absence of the interaction of Sororin with SMC1:SCC1. Sufficient to mention in the text that Sororin does not appear to interact with SMC1:SCC1.

      This is possible but we decided to leave this as is.

      Reviewer #2 (Recommendations For The Authors):

      Minor points

      (1) Are there any predicted models in which one of the two dimer interfaces of the hinge is open when the coiled coils are folded back, as seen in the cryo-EM structure of human cohesin-NIPBL complex in the clamped state?

      No AF runs ever predicted half opened hinges. It is possible that the introduction of mutations in one of the two interfaces might reveal a half-opened state and we ought to try this. However, it would not be appropriate for this manuscript, we believe.

      (2) Structures of the SA-Scc1 CES bound to [Y/F]xF motifs from Sgo1 and CTCF have been reported, suggesting that a similar motif could interact with SA/Scc3. Surprisingly, AF did not predict an interaction between Scc3/SA and Wapl FGF motifs, which only bind to the Pds5 WEST region. On the other hand, AF predicted interactions of the Sororin FGF motif with both Pds5 WEST and SA CES. Can the authors comment on this Wapl FGF binding specificity? What will happen if a Wapl fragment lacking the CTD is used in the prediction?

      This seems to be an academic point as the CTD is always present.

    2. Reviewer #1 (Public Review):

      There are a number of outstanding questions concerning how cohesin turnover on DNA is controlled by various accessory factors and how such turnover is controlled by post-translational modification. In this paper, Nasmyth et al. perform a series of AlphaFold structure predictions that aim to address several of these outstanding questions. Their structure predictions suggest that the release factor WAPL forms a ternary complex with PDS5 and SA/SCC3. This ternary complex appears to be able to bind the N-terminal end of SCC1, suggesting how formation of such a complex could stabilize an open state of the cohesin ring. Additional calculations suggest how the Eco/ESCO acetyltransferases and Sororin engage the SMC3 head domain presumably to protect against WAPL-mediated release.

      This work thus demonstrates the power of AF prediction methods and how they can lead to a number of interesting and testable hypotheses that can transform our understanding of cohesin regulation. These findings require orthogonal experimental validation, but authors argue convincingly that such validation should not be a pre-requisite to publication.

      In their revised version, the authors did not systematically include model confidence scores, and it therefore remains difficult for the reader to evaluate the reliability of the models obtained. The authors correctly point out that such metrics are available on Figshare. It is therefore possible to obtain such information. The caveat is that it remains to the user to identify and extract the relevant information. While they claim that they have labeled N- and C-termini in their figures, no such labeling can be seen in the revised version. Addition of such labels, at least for some of the figures, would help the user to navigate the models.

      Also, PAE plots still contain chain labels (A,B,C etc.) and it is not always clear which protein is being referred to. Which segment does the reader need to focus on? The authors claim that PAE plots are now amended but no such changes can be seen. At least for the key models proposed, the authors should have facilitated access and help readers interpret the different plots/models. While it is possible to load the different PDB files from Figshare, the current version still requires that the reader then works out what segments are used and how they correspond to chain labels shown.

      It is exciting to see AF-multimer predictions being applied to cohesin. As some of the reported interactions are not universally conserved and some involve relatively small interfaces the possibility arises that these interfaces show poor or borderline confidence scores. As some of these interfaces map to mutants that have previously been obtained by hypothesis-free genetic screens and mutational analyses, they appear nevertheless valid. Thus, an important point to make is that even interfaces that show modest confidence scores may turn out to be valid while others may be not.

    1. The sound of the waves crashing against the beach, the rocks, was rhythmic and un-soothing. The sun was bright and warm, the wind was kind, but the sea itself was just as unsettling as it always had been to me.

      This is very descriptive and gives the reader an image inside there head which helps so much to understand the text.

    1. Consider the "hanging-head" format for major headings. In this design, some or all of the headings are on the left margin, while all text is indented one to two inches. This format will make headings stand out more and reduce the main text's line length.

      I always use the "hanging-head" format. It helps me with my writing and it makes it easier for my writing flow.

    1. Bowers remembers arriving at Diller’s home in 2006 to arrange for the donation. “She was the most organized donor I’d ever met.” “She had a rack of her costumes that she wished to donate. Each costume came with a plastic bag attached to it and inside the bag, she had carefully included not only the props—her cigarette holder, the head-dress, the gloves, the shoes—but also a photograph of her wearing the entire ensemble. She was better at curation than I was,” Bowers jokes.

      Curator Dwight Blocker Bowers on Phyllis Diller's organization.

    1. “Unlike preschool curricula, which may have more prescribed topics andcontent, the learning that occurs in the first 3 years of life is less content-specificand more fundamental to all learning processes. Because babies self-selecttheir object of attention an arena of practice, the planning and implementationof experiences that will support a child’s agenda must be carried out througha process of close observation, documentation, reflection, and individualizedplanning. Therefore, rather than a curriculum that is designed for an age groupor a classroom, infant/toddler curricula are documented in individual plans (the‘written plan’ referred in the Head Start Performance Standard).”

      I'm a little confused on this one because the curriculum that we use with our 2/3 years olds are broken down into topics. So does that mean we should be focusing more on the observations than the curriculum?

    2. the assessment of individual children’s progressthrough ongoing observation by those caring forand educating the infant/toddler using an earlylearning framework that articulates developmentalprogressions (such as the Head Start EarlyLearning Outcomes Framework or state ELGs), andobservation of the infant/toddler’s own interests andmotivation for learning

      I think assessments are very important when you are trying to track the progress of the child.

    3. “Unlike preschool curricula, which may have more prescribed topics andcontent, the learning that occurs in the first 3 years of life is less content-specificand more fundamental to all learning processes. Because babies self-selecttheir object of attention an arena of practice, the planning and implementationof experiences that will support a child’s agenda must be carried out througha process of close observation, documentation, reflection, and individualizedplanning. Therefore, rather than a curriculum that is designed for an age groupor a classroom, infant/toddler curricula are documented in individual plans (the‘written plan’ referred in the Head Start Performance Standard).”

      The major differences in curriculum are that preschool has more topics and content while the first 3 years of life curriculum focuses on more individualized plans. I dont think they should be the same because preschool age and infant/toddler age have different focuses and mile stones.

    4. the assessment of individual children’s progressthrough ongoing observation by those caring forand educating the infant/toddler using an earlylearning framework that articulates developmentalprogressions (such as the Head Start EarlyLearning Outcomes Framework or state ELGs), andobservation of the infant/toddler’s own interests andmotivation for learning

      It's very important for the caregivers/teacher to observe and assess individual children to see how the child is developing and meeting their milestones. Through observation you will also learn what motives the child and his/her interest as well as their likes and dislikes. By using learning frame work when observing and assessment will help the teacher with class activities, setting up the environment and set learning goals for the individual child.

  11. cqpress-sagepub-com.lmc.idm.oclc.org cqpress-sagepub-com.lmc.idm.oclc.org
    1. In one of the city's most recent eruptions of gun violence, 21 people were injured in multiple shootings after a May 13 NBA playoff game. The incident was one of several mass shootings that occurred across the country that weekend — from a supermarket in Buffalo, N.Y., to a church in Laguna Woods, Calif.1

      Some people tend to get very worked up about sports but that is no excuse to harm or even take someone's life over a game. I can see that maybe their adrenaline spiked up and got to their head.

    1. was eighteen and had many admirers; Carramae, a blonde, was only fifteen but already married

      The descriptive language here helps the reader create a picture of the two characters in their head.

    2. Her forward expressionwas steady and driving like the advance of a heavy tru

      The use of analogy here draws a vivid image in my head of Mrs. Feemans heavy and intense stare.

    3. Her forward expressionwas steady and driving like the advance of a heavy truck

      The use of analogy here draws a vivid image in my head of Mrs. Feemans heavy and intense stare.

    4. Joy called them Glycerin and Caramel. Glynese, a redhead,was eighteen and had many admirers; Carramae, a blonde

      I looked up what Glycerin was and it is a sugar alcohol which is also similar to caramel in the aspect of sugar. And to make an assumption i am also assuming that glycerin is the color red because one of the daughters is a red head and the other daughter has blonde hair to resemble Carmel.

    1. I had spent the first 18 years of my life in one set of surroundings, rarely popping my head out of the bubble of my friends, family, and community in St. Louis, Missouri.

      I could not have had a more opposite upbringing as the author as I spent my life moving around every 2 to 3 years. My friends and family all told me that this versatility meant that I would find it easier to incorporate myself at the University of Arizona, and I therefore would not have so much of the lost feeling. This is absolutely untrue however, and I found myself feeling just as lost and intimidated as almost everyone else my first year.

  12. drive.google.com drive.google.com
    1. Of importance, before resuming play, the injured playerapparently reported symptoms consistent with a head injuryto other players (that he suffered a collision to his head, hadblacked out, and had a headache) but never reported thesesymptoms to the athletic trainer, team physician, or coaches

      Going off of my above annotation on this case. The coach should have never been sued because the athlete withheld information from the coach, AT, and the team physician. They are not mind readers all they knew about was the finger. Therefore the athlete is in the wrong more than the sports medicine team.

    2. In Zemke v Arreola,47 16-year-old Nicholas Zemke sustained asevere and debilitating head injury while playing varsity foot-ball for his high school. During a game he suffered a dislo-cated finger, which was treated by the athletic trainer andteam physician. The coach asked the team physician whetherthe player was “done for the day” and was advised that theycould tape it up and that he would be fine and ready to go.Thereafter the coach asked the player if he was ready to go;the player responded that his finger was fine, but that “I’mnot ready to go in now.” The coach responded, “Okay, whenyou are ready to go in, come back and let me know.” He soonresumed play and collapsed during a time-out, having suf-fered a right subdural hematoma

      This is a weird situation. I agree with both Heather and Isaac. The coach should not have been sued when the team physician and AT both gave the athlete the okay to play. But also the coach should of sat the player out just to make sure there was no other injures they didn't find at that moment.

    3. . To be familiar with features of concussion2. To evaluate player who appeared to have suffered headinjury for symptoms of concussion3. To repeat evaluation at intervals before player would bepermitted to reenter game4. To determine, based upon evaluation, seriousness of injuryand whether it was appropriate to let the player reenterthe game or to remove the player from all contact pendingmedical examination

      Any coach that coaches a sport where head injures are likely they need to be trained on concussions so things like this does not happen.

    4. Immunities are also available in civil rights cases. InLivingston v DeSoto Independent School District et al,23 a highschool student became seriously ill after running on an out-door track as part of training activities for the girls’ basketballprogram. The coach took her to the school’s athletic trainingroom and the head athletic trainer diagnosed her with heat-stroke. She was taken to the hospital and died.

      Practicing safe guidelines in practices and games should be a priority for coaches. In some instances, it can be a rare freak accident. But prevention needs to be taken into account.

    5. In a post-Kahn decision, the Supreme Court of Californiamore recently affirmed that players assume certain risks in par-ticipating in sports.3 In Avila v Citrus Community College, a stu-dent playing baseball for his community college was injuredby a pitch intentionally thrown at his head, which cracked hisbatting helmet. Staggering and dizzy, he made it to first base,and was told by a coach to stay in the game

      This happens more often than people may realize. Coaches tell players all the time "don't be soft", "You are alright", or "shake it off". This is the right choice to make by a coach, which asserts that most of them do not have any medical experience

    6. To be familiar with features of concussion2. To evaluate player who appeared to have suffered headinjury for symptoms of concussion3. To repeat evaluation at intervals before player would bepermitted to reenter game

      Concussion protocol is not something to mess around with. If an athlete is in any type of situation involving a head related injury. They need to be evaluated. Sometimes the athlete may not have symptoms until the next day. Evaluations on the athlete need to be considered until they are okay.

    7. As seen above, the laws may vary greatlyfrom state to state. Hence, coaches, team physicians, athletictrainers, and school administrators need to understand theseprinciples as applied by the courts in their states in order toundertake steps to minimize liabilities.

      Not only may laws and statutes vary from state to state but it is important to understand your role and which laws or protection acts you fall under. The volunteer is protected by all the same rules as the head coach. The head is protected by separate rules as the team athletic trainer.

    8. The coach responded, “Okay, whenyou are ready to go in, come back and let me know.” He soonresumed play and collapsed during a time-out, having suf-fered a right subdural hematoma.

      I understand Isaac's argument above, but I do put some blame on the coach. I say that because as a head injury, those are often pretty significant. Even though the Athletic Trainer had gave the clear, I would still allow for a second opinion when proceeding forward with letting the athlete go back into the game.

    9. Of importance, before resuming play, the injured playerapparently reported symptoms consistent with a head injuryto other players (that he suffered a collision to his head, hadblacked out, and had a headache)

      Head injuries are one of the biggest topics in sport injuries and are now taken very seriously. A normal head injury can be quite dangerous, but if the athlete were to continue to play with the injury, it could cause the injury to get way more severe.

    10. Theappellate court rejected this argument, noting that “here, sadly,Zemke [injured player] did not report to his coaches or medi-cal staff the critical facts about his injury—that he had suffereda collision to his head, that he had blacked out, and that hehad a headache—that might have alerted them to seek medi-cal attention for head trauma and could have created a duty toprevent further head injury.”4

      They made the right call. If the athlete did not tell the coaches about his other symptoms and did not tell them he injured his head as well, they would not have known.

    1. Perimeter-weighted Then someone had a bright idea. What if we place more weight around the perimeter of the head? That way, if you mistakenly hit the ball from the toe of the heel rather than right out of the middle of the face, the momentum of this extra weight will prevent the clubhead from twisting as much at impact. Would that make life easier?
    1. Although golf clubs are lumps of metal on a shaft, every millimetre of the clubhead is sculpted to the tightest of manufacturing tolerance to make sure they perform as expected, when required. Two of the major influences on how you golf ball launches, flies, and spins are the loft and lie angle. These terms can be challenging to get your head around (primarily lie angle) but a fundamental understanding of how they change the dynamics of golf can be advantageous.
    1. I could continue a thread anywhere, rather than always picking it up at the end. I could sketch out where I expected things to go, with an outline, rather than keeping all the points I wanted to hit in my head as I wrote. If I got stuck on something, I could write about how I was stuck nested underneath whatever paragraph I was currently writing, but then collapse the meta-thoughts to be invisible later -- so the overall narrative doesn’t feel interrupted.

      Notes about what you don't know (open questions), empty outline slots, red links as [[wikilinks]], and other "holes" in tools for thought provide a bookmark for where one may have quit exploring, but are an explicit breadcrumb for picking up that line of thought and continuing it at a future time.

      Linear writing in one's notebooks, books they're reading, and other places doesn't always provide an explicit space which invites the reader or writer to fill them in. One has to train themselves to annotate in the margins to have a conversation with the text. Until one sees these empty spaces as inviting spaces they can be invisible to the eye.

    1. There is another world under this earth. It is like this one in every way. The animals, the plants, and the people are the same, but the seasons are different. The streams that come down from the mountains are the trails by which we reach this underworld. The springs at their head are the doorways by which we enter it. But in order to enter the other world, one must fast and then go to the water, and have one of the underground people for a guide. We know that the seasons in the underground world are different, because the water in the spring is always warmer in winter than the air in this world; and in summer the water is cooler.

      The underground world including animals, plants, and people are similar to those in our world. However, there is a difference in the seasons that goes on in the underground world that includes the pattern of climate and weather.

    1. Nurse held her neck on one sid & Eliz: Hubbard (one of the sufferers) had her neck set in that posture whereupon another Patient Abigail Williams cryed out set up Goody Nurses head the maid’s neck will be broke & when some set up Nurses head Aaron wey observed that Betty Hubbards was immediately righted

      I am observing that Nurse turned her neck to the side and Hubbard copied her by turning her neck too

      I interpret this as Abigail and Hubbard trying to prove Nurse is indeed a witch by implying she was " controlling" Hubbards movement causing her neck to fall in the same position as Nurses because Abigail yelled out " Set up Goody Nurses head the maid's neck will be broke". And Hubbard changing positions when Nurse did too.

      This shows how unjust the law system was during the Salem Witch trials. Anything could prove you were a witch ( example- Nurse "controlling her neck".) Back then the thought of " innocent till proven guilty" was not a thing if you were accused you were a witch. Change over time- the law system is now better less people being falsely accused.

    1. Of the forms of communication, intrapersonal communication has received the least amount of formal study.

      I "talk to myself" all the time. I feel like my intrapersonal dialogue with myself helps me stay organized and helps me be calm. It makes sense that intrapersonal communication doesn't have a lot of research on it because everyone's minds are different. I have to run conversations through my head when I am about to make a phone call. I practice what I am going to say.

    2. Like other forms of communication, intrapersonal communication is triggered by some internal or external stimulus. We may, for example, communicate with our self about what we want to eat due to the internal stimulus of hunger, or we may react intrapersonally to an event we witness. Unlike other forms of communication, intrapersonal communication takes place only inside our heads.

      Everyone on this planet has intrapersonal communication. I talk to myself every day, and I have conversations with myself on what I'm going to do or what I need to do. Some people talk to themselves to calm down, or they journal to ease their minds. When something surprising happens people usually react somehow in their head, basically when anything happens people react to themselves. Just as the text states, "We also use intrapersonal communication or “self-talk” to let off steam, process emotions, think through something, or rehearse what we plan to say or do in the future." Intrapersonal communication happens almost every second throughout one person's day.

    3. Instruction in the latter stressed the importance of “oratory” or “elocution,” and this interest in reading and speaking aloud is sustained today in theatre and performance studies and also in oral interpretation classes, which are still taught in many communication departments.

      The act of theatrical improv is a form of public speaking in an entertaining way. An actor presenting in front of an audience and shaping the show around what pops into their head. They might ask several audience members for different situations and come up with an amusing scene. This makes the audience feel connected and encourages further participation. This practice is beneficial and a positive form of communication.

    1. The year 1900 was not the first to upset schoolmasters. Copernicus and Galileo had broken many professorial necks about 1600; Columbus had stood the world on its head towards 1500

      The author names these significant figures in history that changed education. It's interesting to see other historical figures such as the author looks at different scholars and teachings to show how during Adams time he was looking to seek even more knowledge than what was found out already and to do it differently.

    1. Reviewer #2 (Public Review):

      In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

      The conclusions from the paper are mostly well supported by the simulations, however, they remain very descriptive and the key findings should be better described and validated. In particular:

      It has been shown that the lipid composition of HIV membrane is rich in cholesterol [1], which accounts for almost 50% molar ratio. The authors use a very different composition and should therefore provide a reference. It has been shown for 4E10 that the change in lipid composition affects dynamics of the binding. The robustness of the results to changes of the lipid composition should also be reported.

      The real advantage of the multiscale approach (coarse grained (CG) simulation followed by a back-mapped all atom simulation) remains unclear. In most cases, the binding mode in the CG simulations seem to be an artifact.

      The results reported in this study should be better compared to available experimental data. For example how does the approach angle compare to cryo-EM structure of the bnAbs engaging with the MPER region, e.g. [2-3]? How do these results from this study compare to previous molecular dynamics studies, e.g.[4-5]?

      References<br /> 1. Brügger, Britta, et al. "The HIV lipidome: a raft with an unusual composition." Proceedings of the National Academy of Sciences 103.8 (2006): 2641-2646.<br /> 2. Rantalainen, Kimmo, et al. "HIV-1 envelope and MPER antibody structures in lipid assemblies." Cell Reports 31.4 (2020).<br /> 3. Yang, Shuang, et al. "Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack." Nature Communications 13.1 (2022): 6393.<br /> 4. Carravilla, Pablo, et al. "The bilayer collective properties govern the interaction of an HIV-1 antibody with the viral membrane." Biophysical Journal 118.1 (2020): 44-56.<br /> 5. Pinto, Dora, et al. "Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01." Cell host & microbe 26.5 (2019): 623-637.

    1. 12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever. https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html you are offline https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html we the people rise again https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html safe souls, safe fu https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html We the People of Slate … The U.S. Constitution, as you mighta been, shoulda [“come” on … its someday] rewrϕte it. "Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state’s population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn’t like in the Constitution and pencil in their hearts’ desires. Here’s what the document would look like with their best ideas." Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers 多也了了夕 "with a wand of scheffilara, 并#亦太 he begins … "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon." Logically the recent mentions of Gilgamesh and the simultaneous 同時 overlaping 場道 of the eventual link between the famous ruling of Solomon on the separation of babies and mothers and waters and land … to a story of many “two cities” that culminates in a cultural or societal or “evolutionary” link to Sodom and Gomorrah and the city-state of Babylon (and it’s Hanging Gardens) and also of course to Paris and Troy and “Masstodon” and city-states [ciudadestado] and perhaps planet-cities; from Cambridge to Cambridge across the “Cable” to see state to “London” … recently I called it “the city of realms” … I started out logically intending to link “game theory” and John Nash to the mathematical story of Sputnik and a revival of American physics; but in my usual way of rambling into the woods [I mean neighborhood] of stream of consciousness … turned into a premonitory discourse of “two cities” and how sometimes even things as obvious as the number of letters in the word “two” don’t do a good enough job of conveying … how and/or why one is simply never enough, and two isn’t much better–but in the end a circle … is drawn; the perfect circle in our imaginary mathematical perfection … I see a parted “line” in the letter pronounced “tea” (and beginning that word); and two “vee” (pron. of “v”) symbols joined together in a word we pronounce as “double-you” … and symbolically because I know “V” is the Roman Numeral for 5 (five) and I know not how to multiply in Roman numerals– It’s important to pause; here. I am going to write a more detailed piece on “the two cities” as I work through this maze like crossroads between “them” and “demo…” … here demorigstrably I am trying to fuse together an evolutionary change in … lit. biological evolution as well as an echelon leap forward in "self-government" … in a place where these two things are unfathomable and unspokenly* connected. https://www.google.com/search?q=prometheuslocke+%2Bsite%3Agodlikeproductions.com “Silence is betrayal” -MLK To a question on the idiom; is Bablyon about “the law” or “of the land of Nod?” “What is democracy” … the song, Metallica’s “ONE” echoes and repeats; as we apparently scrive together the word “THEM” … I question myself … if Babylon were the capital city of some mythical Nation of Time … if it were the central “turning point” of Sheol; ... >|< Can you not see that in this place; in a world that should see and does there is a gigantic message proving that we are not in reality and trying to show us how and why that's the best news since ... ever---that it's as simple as conjoining "the law of the land" with a basic set of rules that automatically turn Hell into something so much closer to Heaven I just do not understand---why we cant stand up together and say "bullets will not kill innocent children" and "snowflakes will not start avalanches ...." that cover or bury or hide the road from Earth to Verital)e .... or from the mythical Valis to Tanis---or from Rigel to Beth-El ... "guess?" ## as "an easy" answer; I'm looking for a fusion of "law and land" that somehow remembers a "jok'er a scene" about "lawn" seats; and "where the girls are green;" It's as simple as night and day; Heaven and Hell ... the difference between survival and--what we are presented with here; it's "doing this right"--that ends the Hell of representative democracy and electoral college--the blindness and darkness of not seeing "EXTINCTION LEVEL EVENT" encoded in these words and in our governments foundation ... by the framers [not just of the USA; but English .. and every language]  ... is literally just as simple as "not caring" or thinking we are at the beginning of some long process--or thinking it will never be done--that special "IT" that's the emancipation of you and I. Here words like "gnosis" and "gaudeamus" pair with my/ur "new ntersanding*" of the difference between Asgard and Medgard and really understanding our purpose here is to end "evil" ... things like "simulating disease and pain" (here, simulating meaning ... intentionally causing, rather than "gamifying away") and successfully linking the "Pillars of Hercules" to Plato's vision of Atlantis and the letter sequences "an" and "as" ... unlock a fusion of religion and mythology and "cryptographic truth" that connects "messianic" and "Christian" to "Roman" ... "Chinese" and "American" ... literally the key to the difference between the phrases "we are" and "we were" .... in "sight" of "silicon" in simulation and Israel, Genesis, and "silence" ... trying to the raising of Asgardian enlightenment ... and seeing "simple cypher" connecting to "Norse" ... and the "I AM THAT" surer than shit ... the intention and design of all religion and creation is to end "simulated reality" and also not seeing "SR" ... in Israel and Norse ... "for instance." https://www.google.com/search?q=%22I+AM%22+%22WE+ARE%22+%2Bsite%3Afromtaws "SOIS" a key--in two languages conjugated literally as both "I AM" and "WE ARE" simultaneously; Search: I know that if I am than so are you ... and it is because we have overcome .... something I truly cannot figure out, fathom, or believe ... was truly here before us--a spiralling series of failures ... speaking: to the heavens; but in secret and in action; "doing everything possible to succeed." It's a simple linguistic concept; the "singularity" and the "plurality" of a simple word--"to be"--but it goes to the heart of everything that we are and everything that is around us. This is a message about understanding and preserving individuality as well as liberty; and literally seeing "ARXIV" and understanding "often" and failing to connect God and prescience to "IV" and the Fourth Amendment ... it's about blindness and ... "curing the blind instantly" ... and fathoming how and why this message has been etched into our entire history and and all religions and myths and music--to help us "to be THAT we" that actually "are responsible" for the end of Hell. I neglected to mention "Har-Wer" and "Tower of Babel" which are both related lingusitically, religiously and topically: "to who ..." and while we're on "four score and [seven years from now]" seeing the fourth "living thing" in Eden and it's (the name, Abel) connection to Babel and Abraham Lincoln; slavery and ... understanding we live in a place where the history of the United States also, like Monoceros and "Neil Armstrong's first step" are a time shifted ... overlayed map to achieving freedom ... it's about becoming a father-race ... and actually "doing" the technological steps required to "emancipate the e's of 'me&e'" and survive in exo-planetary space--- it might be as simple as adding "because we did this" here and now; and having it be something we are truly proud of .... forevermore™ ... for certain in the heart of this story about cyclicality and repetition of error--its not because we did "this" or something over and over again; it's about changing "the problem" and then helping others to also overcome ... "things like time travel ... erasing speech" --- however that happenecl. I also failed to mention that "I am in Hell" ... as in this world is hellacious to me; in an overlay with the Hellenic period and this message that we are in the Trojan Horse ... a small gem .... "planet" truly is the Ark of the Covenant---and it's the simple understanding that "reality is hell" is to "living without air conditioning and plumbing is hell" just as soon as you achieve ... "rediscovering" those things--- I can't figure out why I am the only person screaming "this is Hell." That's also, Hell. ... but recently suggested an old joke about "there being 10 kinds of people in the world (obv an anti-tautology and a tautology simultaneously)" only after that brief bit of singularity and duality mentioning the rest of the joke: "those that understand binary and those that don't know how to base convert between counting with two hands and counting with only an 'on and off.'" It's not obvious if you aren't trying to figure it out, I suppose; but 10 is decimal notation for "kiss" and the "often" without "of" ... and binary notation for the decimal equivalent of "2." A long long time ago in a state that simply non-randomly ties to the heart of the name of our galaxy ... I was again thinking of the "perfect imperfections" of things like saying "three equals one equals one" (which, of course was related to the Holy Trinity and it's "prescient/anachronistic Adamic presence encoded in the name Ab|ra|ha|m" which means "father of a great multitude") ... I brought that one back in the last few months; connecting the letter K and in this "logos-rythmic" tie to the "base of a number system" embellish the truth just a bit and suggest a more accurate rendition of the original [there is no such thing as equality, "is" of separate objects--as in no two snowflakes are the same unless they are literally the same one; true of ancient weights and with the advent of (thinking about) time no two "planets" are the same even if they're the exact same one--unless it's at a fixed moment in time. This name may be viewed either as meaning "father of many" in Hebrew or else as a contraction of ABRAM (1) and הָמוֹן (hamon) meaning "many, multitude". The biblical patriarch Abraham was originally named Abram but God changed his name (see Genesis 17:5). https://en.wikipedia.org/wiki/Yeshua#Yeshua,_Yehoshua,_and_Yeshu_in_the_Talmud K=3:11 ... to a handle on the music, the DHD of the gate and the *ring of David's "sling" ... ---and that's a relationship of "3 is to 11" as [the SAT style "analog]y" as a series of alpha, two mathematic, and two numeric symbols ... may only tie in my mind alone to the books of Genesis and Matthew and the phrase "chapter and verse" and to the stories of Lot and Job ... again in Genesis and the eponymous "Book of Job." So ... "tying up loose ends one 10b [III] iv. " as it appears I've taken it upon myself to call a Job and suggest is my "Lot in life [x]i* [3]" I worry sometimes that important things are missing, or will disappear---for instance Mirriam Webster, which is a "canonical/standard dictionary) should probably have an entry for "lot in life" non-idiomatically as "granny apples to sour apples" as 2 MANY ALSO ICI; 1twoⅱ ... following in Mitnick's bold introductory word steps; the curve and the complement ... the missiles and the canoes; the line and the blank space ... "supposedly two examples of two kinds, which could be three not nothings ... Today I write about something monumental; as if as important as the singularity depicted in Arthur C. Clarke's 2001 "A Space Odyssey" ... and remember a day when I thought it very novel and interesting to see the words "stillborn and yet still born" connected in a single piece of writing to "Stillwater and yet still water" ... today adding in another phrase noting the change wrought only by one magical single "space" (also a single capital letter; and a third phrase): "block chains with a great blockchain." http://www.goodmath.org/blog/2015/07/21/arabic-numerals-have-nothing-to-do-with-angle-counting/ https://gizmodo.com/no-this-viral-image-does-not-explain-the-history-of-ar-1719306568 https://en.wikipedia.org/wiki/Chinese_word_for_%22crisis%22 https://dictionary.hantrainerpro.com/chinese-english/translation-ji_howmany.htm https://dictionary.hantrainerpro.com/chinese-english/translation-duo_many.htm https://en.wikipedia.org/wiki/Euripides, Iphigenia in Aulis or Iphigenia at Aulis[1] (Ancient Greek: Ἰφιγένεια ἐν Αὐλίδι, Iphigeneia en Aulidi; variously translated, including the Latin Iphigenia in Aulide) is the last of the extant works by the playwright Euripides. Written between 408, after Orestes, and 406 BC, the year of Euripides' death, the play was first produced the following year[2] in a trilogy with The Bacchae and Alcmaeon in Corinth by his son or nephew, Euripides the Younger,[3] and won first place at the City Dionysia in Athens. The play revolves around Agamemnon, the leader of the Greek coalition before and during the Trojan War, and his decision to sacrifice his daughter, Iphigenia, to appease the goddess Artemis and allow his troops to set sail to preserve their honour in battle against Troy. The conflict between Agamemnon and Achilles over the fate of the young woman presages a similar conflict between the two at the beginning of the Iliad. In his depiction of the experiences of the main characters, Euripides frequently uses tragic irony for dramatic effect. J.K. Rowling spurred just this past week a series of explanations about just exactly what is a blockchain coin worth ... and why is it so; her final words on the subject (artistic liberty taken, obviously not the last she'll say of this magic moment) "I don't think I trust this." Taken directly from an off the cuff email to ARXM titled: "Slow the S is ... our Hypothes.is" I imagine I'll be adding some wiki/ipfs stuff to it--and try to keep it compatible; the design and layout is almost exactly what I was dreaming about seeing--as a "first rough draft product." Lo, and behold. It's been added to the many places I host my tome; the small compilation of nearly every important email that has gone out ... all the way back to the days of the strange looking Margarita glass ... that now very much resembles the "Cantonese character 'le'" which I've come to associate with a "handle" on multiple corners of a room--something like an automatic coat rack conveyor belt connecting different versions of "what's in the box." I'm planning on using that symbol 了 to denote something like multiple forks of the same page. Obviously I'm thinking forward to things like "the Transhumaist Chain Party" (BDSM, right?)'s version of some particular piece of legislation, let's say everything starts with the sprawling "bulbing" of "Amendment M" ideas and specific verbiage ... and then we'll of course need some kind of new git/subversion/cvs style version control mechanism to merge intelligently into something that might actually .... really should ... make it into that place in history--the first constitutional amendment ratified by a "Continental Congress of All People" ... but you could also see it as an ongoing sort of forking of something like the "wikipedia page" on what some specific term, say "technocracy" means, and how two parties might propagandize and change the meaning of such thing; to suit the more intelligent and wise times we now live in. For instance, we might once have had a "democracy" and a "democractic" party that had some Anarchist Cook Book version of the history of it ending in something like Snipes and Stallone's "DEMOLITION MAN." Just kidding, we all know "democracy" has everything to do with "d is cl ... and not th" ... to be the them that is the heart of the start of the first true democracy. At least the first one I've ever seen, in my old "to a republic" ... style. As it is you can play around with commenting and highlighting and annotating all the stuff I've written and begged and begged for comments on--while I work on layering the backend to to perma-store our ideas and comments on both a blockchain (probably a new one; now that i've worked a little with ethereum) with maybe some key-merkle-tree-walk-search stuff etched into the original Rinkeby ... and then of course distributed data in the "public owned and operated" IPFS. To be clear, I plan on rewriting the backend storage so that we will have a permanent record of all comments; all versions of whatever is being commented on; and changes/revisions to those documents--sort of turning the web into a massive instant "place of collaboration, discussion, and co-authoring" ... if you use the wonderful LEGO pieces that have been handed to us in ideas from places like me, lemma--dissenter, and of course hypothes.is who has brought you and i such a polished and nice to look at "first draft" of something like the living Constitution come repository of all human knowledge. I do sort of secretly wich they would have called this project something like "annotating and reflecting (or real or ...) knowledge" just so the movement could have been called ARK. ... or something .... but whatever join the "calling you a reporter" group or ... "supposedly a scientist?" NOIR INgR .. I CITE SITE OF ENUDRICAM; a rekindling of the dream of a city appearing high above in the sky, now with a boldly emblazened smiling rainbow and upsidown river ... specifically the antithesis of "angel falls," there's a lagoon too--actually a chain of several ponds underneith the floating rock ... and in some versions of this waking dream there are rings around the thing; you might imagine an artificial set of centripetal orbitals something like a fusion of the ring Eslyeum and the "Six-Axis ride" of the JKF Center's "Spacecamp." I write as I dream, and though I cannot for certain explain exactly how; it's become a strong part of my mythology that this spectacular rendition of "what ends the silence" has something to do with the magical delivery of "a book" ... something not of this Earth but an unnatural thing; one I've dreamt of creating many times. This book is something like the DSM-IV and something like a Merck diagnostic manual; but rather than the old antiquated cures of "the Norse Medgard" this spectacle nearly "itsimportant" autoprints itself and lands on something like every doorpost; what it is is a list of reasons why "simply curing all disease" with no explanation and no conversation would be a travesty of morality--how it would render us half-blind to the myriad of new solutions that can come from truly understanding why "ITIS" to me has become a kind of magical marker: an "it is special" as in, it's cure could possibly solve a number of other problems. Through that missing "o," English on the ball, we see a connection between a number of words that shine bright light including Exodus itself which means "let there be light," the word for Holy Fire and the Burning Bush.. .reversed to hSE'Ah, and a story about the Second Coming parting our holy waters. This answer connects the magical Rod's of Aaron in Exodus and the Iron Rod of Jesus Christ to the Sang Rael itself... in a fusion that explains how the Periodic Table element for Iron links not just to Total Recall and Mars, but also to this key my dream of what the first day of the Second Coming might be like; were the Rod of Christ... in the right hands. In a story that also spans the Bible, you might understand better how stone to bread and your input make all the difference in the world between Heaven and Adam's Hand. Once more, what do you think He ....   Since the very earliest days of this story, I have asked for better for you, even than see Nearly all of the original parts of the original "post-origination dream" remain intact; there's a walkway that magically creates new paths and "attractions" based on where you walk, something like an inversion of the artificial intelligence term "a random walk down a binary tree" ... for instance going left might bring you to the Internet Cafetornaseum of the Earl of Sandwich; and going to the right might bring you to the ICIMAX/Auditorium of Science and Discovery--there's a walkway to "Magical GLAS D'elevators" that open a special "instantiation" of the Japan Room of the Potter and the Toolmaker ... complete with a special [second level and hidden staircase] Pool of Bethesdaibo verily delivering something like youth of mind and body ... or at least as close to such a thing as a sip of Holy Water or Ambrosia or a dip in the pool of Coccoon and Ponce De'Leon could instantly bring ... to those that have seen Jupiter Ascending ... the questions of "nature versus nurture" and what it means to be "old and wise" and "young at heart" truly mean--- https://www.youtube.com/watch?v=M8CyN1awWls https://link.springer.com/chapter/10.1057/9780230366688_16 https://www.youtube.com/watch?v=YDo5zvYNn3A Somewhere between the outdoor rafting ride and the level with the special "ballroom of the ancient gallery" ... perhaps now being named or renamed or recalled as something about "Face [of] the Music" lies a magical "mini-maize" ... a look at a mock-up (or #isitit) of Merlink and Harthor's "round table" that displays a series of ... (at least to me) magical appearing holographic displays and controls that my dreams have stolen from Phillip K. Dick's Minority Report and something of what I hope Microsoft's Dynamics/Hololens/Surface will become---a series of short "focus groups" .... to guage and discuss the information in the "CITIES-D5AM-MERCK" ... how to end world hunger and nearly all disease with the press of a magical buzzer--castling churches to something like "political-party-town-hall-meeting centers" and replacing jails and prisons and hospitals with something like the "Hospitalier's PRIDE and DOJOY's I practiced "Kung-fun-dance" ... a fusion of something like a hotel and a school that probably looks very much like a university with classrooms and dorms and dining hall's all fit into a single building. I imagine a series of 2 or 3 "room changes" as in you walk from the one where you get the book and talk about it ... to the one where you talk about "what everyone else said about it" and maybe another one that actually connects you to other people with something like Facebook's Portal; the point of the whole thing to really quickly "rubber stamp" the need for an end to "bars in the sky" nonalcoholic connotation--as in "overcoming the phrase the sky is the limit" and showing us the need for a beacon of glowing hope fulfilled--probably actually the vision of a holographic marker turning into actual rings around the single moon of Earth, the focus of the song annoucing the dawn of the age of Aquarius--- It might lead us also to Ceres; and another set of artificial rings, or to Monoceros and a rehystorical understanding of the birthplace and birthing of the "river roads" that bridge the "space gaps" in the galaxy from our "one giant leap for mankind" linking the Apollo moon landing to the mythological connection to the sun; and connecting how the astrological charts of the ancients might detail a special kind of overlapping--the link between Earth's SOL and something like Proxima or Alpha Centauri; and how that "monostar bridge" might overlap to Orion and from there through Sagitarius and the center of the Milky Way ... all the way to Andromeda and more dreams of being in a place where there's a map to a tri-galactic system in the constellation Cancer and a similar one in Leo ... and just incase you haven't noticed it--a special marker here, I thought to myself it might be cool to "make an acronymic tie to Monoceros" and without even thinking auto-wrote Orion (which was the obvious constellation next to Monoceros, in the charts) and then to Sagitarrius; which is the obvious ... heart of our astrological center and link to "other galaxies." ----I've dreamt or scriven or reguessed numerous times how the Milky Way's map to an "Atlas marked through time by the ages and the ancients" might tie this place and this actual map to the creation of the railways between stars to the beginning and the end of time and of course to this message that links it all to time travel. There's a few "guesses" I've contemplated; that perhaps the Milky Way chart is a metal-cosmic or microcosmic map to the dawn of time in the galactic vision of ... just after the big bang; or it might tie to a map of something like the unthinkable--a civilization that became so powerful it was able to reverse the entropy of "cosmic expansion" and reverse the thing Asimov wrote of in "The Last Question" as the end of life and the ability to survive basically due to "heat loss." "The Last Question." (And if you read two, why not "The Last Answer"?). Find these readings added to our collection, 1,000 Free Audio Books: Download Great Books for Free. https://archive.org/details/texts http://zlibraryexau2g3p.onion.pet/ Looking for free, professionally-read audio books from Audible.com, including ones written by Isaac Asimov? * all "asterisks" in the abovə document denote a sort of Adamic unspoken relationship between notations and meanings; here adding the "Latin word for three" and source of the phrase "t.i.d." (which is doctor/pharmacy latin for "three times a day") where the "t" there is an abbreviation of "ter" ... and suppose the link between K and 11 and 3 noting it's alphanumeric position in the English alphabet as the 11th letter and only linking cognitively to three via the conversion between hex, and binarryy ... aberrative here is the overlapping "hakkasan" style (or ZHIV) lack of mention of the answer in "state of Kansas" and the "citystate of Slovakia" as described in the ICANN document linked [in] the related subsection or slice of the word "binarry" for the state of India. Tetris could be spelled with the addition of only a single letter [in] "tea"---the three letters "ris" are the hearts of the words "Christ" and "wrist" [and arguably of Osiris where you also see the round table character of the solar-system/sun glyph and the chemical element for The Fifth Element (as def. by i) via "Sinbad" and "Superman." The ERIS Free Network should also be mentioned here in connection with the IRC network I associate in the place between skipping stones and sacred hearts defined by "AOL" and "Kdice" in my life. In the lexicon of modern HTML, curly braces are generally relative to "classes" and "major object definitions (javascript/css)" while square brackets generally only take on computer-interpreted meaning in "Markdown" which is clearly (by definition, by this character set "[]") a superset (or at least definately not a subset) of HTML. Dr. Will Caster (Johnny Depp) is a scientist who researches the nature of sapience, including artificial intelligence. He and his team work to create a sentient computer; he predicts that such a computer will create a technological singularity, or in his words "Transcendence". His wife, Evelyn (played by Rebecca Hall), is also a scientist and helps him with his work. Following one of Will's presentations, an anti-technology terrorist group called "Revolutionary Independence From Technology" (R.I.F.T.) shoots Will with a polonium-laced bullet and carries out a series of synchronized attacks on A.I. laboratories across the country. Will is given no more than a month to live. In desperation, Evelyn comes up with a plan to upload Will's consciousness into the quantum computer that the project has developed. His best friend and fellow researcher, Max Waters (Paul Bettany), questions the wisdom of this choice, reasoning that the "uploaded" Just from my general understanding and memory "st" is not ... to me (specifically) an abbreviation of "state" but "ste" is a U.S. Postal code (also "as I understand it") for the name of a special room or set of rooms called a "suite" and in Adamic "connotation" I sometimes read it as "sweet" ... which has several meanings that range from "cool" to "a kind of taste sensation" to "easy to sway or fool." If you asked me though, for instance if "it" was an abbreviation or shorthand notation or acronym for either "a United state" or "saint" ... you'd be sure. While it's clear from studying linguistic cryptography ... (If I studied it a little here and some there, its also from the "universal translator of Star Trek") and the personal understanding that language is a kind of intelligent code, and "any code is crackable" ... that I caution here that "meaning" and "face value" often differ widely and wildly ... even in the same place or among the same group of people ... either varying over time or heritage. Menelaus, in Greek mythology, king of Sparta and younger son of Atreus, king of Mycenae; the abduction of his wife, Helen, led to the Trojan War. During the war Menelaus served under his elder brother Agamemnon, the commander in chief of the Greek forces. When Phrontis, one of his crewmen, was killed, Menelaus delayed his voyage until the man had been buried, thus giving evidence of his strength of character. After the fall of Troy, Menelaus recovered Helen and brought her home. Menelaus was a prominent figure in the Iliad and the Odyssey, where he was promised a place in Elysium after his death because he was married to a daughter of Zeus. The poet Stesichorus (flourished 6th century BCE) introduced a refinement to the story that was used by Euripides in his play Helen: it was a phantom that was taken to Troy, while the real Helen went to Egypt, from where she was rescued by Menelaus after he had been wrecked on his way home from Troy and the phantom Helen had disappeared. https://www.britannica.com/topic/Menelaus-Greek-mythology This article is about the ancient Greek city. For the town of ancient Crete, see Mycenae (Crete). For the hamlet in New York, see Mycenae, New York. Μυκῆναι, Μυκήνη The Lion Gate at Mycenae, the only known monumental sculpture of Bronze Age Greece 37°43′49″N 22°45′27″ECoordinates: 37°43′49″N 22°45′27″E This article contains special characters. Without proper rendering support, you may see question marks, boxes, or other symbols. Mycenae (Ancient Greek: Μυκῆναι or Μυκήνη, Mykēnē) is an archaeological site near Mykines in Argolis, north-eastern Peloponnese, Greece. It is located about 120 kilometres (75 miles) south-west of Athens; 11 kilometres (7 miles) north of Argos; and 48 kilometres (30 miles) south of Corinth. The site is 19 kilometres (12 miles) inland from the Saronic Gulf and built upon a hill rising 900 feet (274 metres) above sea level.[2] In the second millennium BC, Mycenae was one of the major centres of Greek civilization, a military stronghold which dominated much of southern Greece, Crete, the Cyclades and parts of southwest Anatolia. The period of Greek history from about 1600 BC to about 1100 BC is called Mycenaean in reference to Mycenae. At its peak in 1350 BC, the citadel and lower town had a population of 30,000 and an area of 32 hectares.[3] 3. Chew 2000, p. 220; Chapman 2005, p. 94: "...Thebes at 50 hectares, Mycenae at 32 hectares..." https://en.wikipedia.org/wiki/Clymene_(mythology) Melpomene (/mɛlˈpɒmɪniː/; Ancient Greek: Μελπομένη, romanized: Melpoménē, lit. 'to sing' or 'the one that is melodious'), initially the Muse of Chorus, she then became the Muse of Tragedy, for which she is best known now.[1] Her name was derived from the Greek verb melpô or melpomai meaning "to celebrate with dance and song." She is often represented with a tragic mask and wearing the cothurnus, boots traditionally worn by tragic actors. Often, she also holds a knife or club in one hand and the tragic mask in the other. Melpomene is the daughter of Zeus and Mnemosyne. Her sisters include Calliope (muse of epic poetry), Clio (muse of history), Euterpe (muse of lyrical poetry), Terpsichore (muse of dancing), Erato (muse of erotic poetry), Thalia (muse of comedy), Polyhymnia (muse of hymns), and Urania (muse of astronomy). She is also the mother of several of the Sirens, the divine handmaidens of Kore (Persephone/Proserpina) who were cursed by her mother, Demeter/Ceres, when they were unable to prevent the kidnapping of Kore (Persephone/Proserpina) by Hades/Pluto. In Greek and Latin poetry since Horace (d. 8 BCE), it was commonly auspicious to invoke Melpomene.[2] See also [AREXMACHINA] Muses in popular culture The Nine Muses Flagstaff (/ˈflæɡ.stæf/ FLAG-staf;[6] Navajo: Kinłání Dookʼoʼoosłííd Biyaagi, Navajo pronunciation: [kʰɪ̀nɬɑ́nɪ́ tòːkʼòʔòːsɬít pɪ̀jɑ̀ːkɪ̀]) is a city in, and the county seat of, Coconino County in northern Arizona, in the southwestern United States. In 2018, the city's estimated population was 73,964. Flagstaff's combined metropolitan area has an estimated population of 139,097. Flagstaff lies near the southwestern edge of the Colorado Plateau and within the San Francisco volcanic field, along the western side of the largest contiguous ponderosa pine forest in the continental United States. The city sits at around 7,000 feet (2,100 m) and is next to Mount Elden, just south of the San Francisco Peaks, the highest mountain range in the state of Arizona. Humphreys Peak, the highest point in Arizona at 12,633 feet (3,851 m), is about 10 miles (16 km) north of Flagstaff in Kachina Peaks Wilderness. The geology of the Flagstaff area includes exposed rock from the Mesozoic and Paleozoic eras, with Moenkopi Formation red sandstone having once been quarried in the city; many of the historic downtown buildings were constructed with it. The Rio de Flag river runs through the city. Originally settled by the pre-Columbian native Sinagua people, the area of Flagstaff has fertile land from volcanic ash after eruptions in the 11th century. It was first settled as the present-day city in 1876. Local businessmen lobbied for Route 66 to pass through the city, which it did, turning the local industry from lumber to tourism and developing downtown Flagstaff. In 1930, Pluto was discovered from Flagstaff. The city developed further through to the end of the 1960s, with various observatories also used to choose Moon landing sites for the Apollo missions. Through the 1970s and '80s, downtown fell into disrepair, but was revitalized with a major cultural heritage project in the 1990s. The city remains an important distribution hub for companies such as Nestlé Purina PetCare, and is home to the U.S. Naval Observatory Flagstaff Station, the United States Geological Survey Flagstaff Station, and Northern Arizona University. Flagstaff has a strong tourism sector, due to its proximity to Grand Canyon National Park, Oak Creek Canyon, the Arizona Snowbowl, Meteor Crater, and Historic Route 66. #PSANSDISL #LWDISP either without gas or seeing cupidic arroz in "thank you" or "allta, wild" ... pps: a magnanimous decision ... I stand here on the brink of what appears to be total destruction; at least of everything I had hoped and dreamed for ... for the last decade in my life which appears literally to span thousands of years if not more in the eyes of some other beholder. I spent several months in Kentucky telling a story of a post apocalyptic and post-cataclysmic delusion; some world where I was walking around in a "fake plane" something like a holodeck built and constructed around me as I "took a walk around the world" to ... it did anything but ease my troubled mind. Recently a few weeks in Las Vegas, and a similar story; telling as I walked penniless down the streets filled with casino's and anachronistic taxi-cabs ... some kind of vision of the entirety of the heavens or the Earth or the "choir of angels" I think of when I echo the words Elohim and Aesir from mythology ... there with me in one small city in superposition; seeing what was a very well put together and interesting story about a "star port" Nirvane ... a place that could build cities into the face of mountains and half working monorails appearing in the sky---literally right before my eyes. I suppose this is the place "post cataclysm" though I still have trouble understanding what it is that's actually about ... in my mind it connects to the words "we are losing habeas" echo'ed from the streets of Los Angeles in a more clear and more military voice than usual--as I walked block by block trying to evade a series of events that would eventually somehow connect all the way to the "outskirts of Orlando, Florida" in a place called Alhambra. Apparently the name of a castle; though I wasn't aware of that until much later. It doesn't feel at all like a "cataclysm" to me; I see no great rift--only a world filled with silent liars, people who collectively believe themselves to have stolen something--something gigantic--at least that's the best interpretation of the throws and impetus behind the thing that I and mythology together call Jormungandr. With an eye for "mythological connections" you could clearly see that name of the Great Serpent of Revelation connects to something like the Unseelie; the faeries of Gaelic lore. To me though this world seems still somewhat fluid, it's my entire life--moving from Plantation to a place where the whole of it might be Bethlehem and to "clear my throat" it's not hard to see here how that land of "coughs" connects to the Biblical land of Nod and to the "Adamically sieved" Snifleheim ... from just a little twist on the ancient Norse land most probably as close to Hel as anyone ever gets--or so I dream and hope---still today. It all looks so real and so fake at the same time; planned for thousands of generations, the culmination of some grand masterpiece story that certainly ties history and myth and reality into a twisted heap of "one big nothing, one big nothing at all." I've tried to convey to the world how important I believe this place and this time to be--not by some choice of my own ... but through an understanding of the import of our history and the impact of having it be so obviously tuned and geared towards this specific time ... many thousands of years literally all focused on a single moment, on one day or one hour or even just a few years where all of that gets thrown down on the table as if some trump card has been played--and whether or not you fathom the same magnanimous statement or situation or position ... to me, I think it depends on whether or not you grew up in the same kind of way, believing our history to be so fixed and so difficult to change. I don't particularly feel like that's the "zeitgeist" of today; I feel like the children believe it to be some kind of game, and that it is such as easy thing to "sed" away or switch and turn into something else--another story, another purpose ... anyone's personal fantasy land come true. I don't think that's the case at all, it's clearly a personal nightmare; and it's clearly one we've seen time and time again--though not myself--the Jesus Christ that is the same yesterday, today; and once again perhaps echoing "no tomorrow" never remembers or believes that we've "seen it all before" or that we've ever really gotten the point; the thing you present to me as "factual reality" is a sickness, it disgusts me; and I'd do anything to go back to the world "where I was so young, and so innocent" and so filled with starry-eyed hope that we were at the foot of something grand and amazing that would become an empire turned republic of the heavens; filling the stars ... with the kind of love for kindness and fairness that I once associated very strongly with the thing I still believe to be the American Spirit. "Suddenly it changes, violently it changes" ... another song echoes through the ages--like the "words of the prophets dancing ((as light)) through the air" ... and I no longer even have a glimmer of hope that the thing I called the American People still exist; I feel we've been replaced by some broken container of minds, that the sky itself has become corrupt to the point that there's no hope of turning around this thing that I once believed with all my heart and all my mind was so obviously a "designed downward spiral" one that was---again--so obviously something of a joke, intended to be easy to bounce off a false bottom and springboard beyond "escape velocity" and beyond the dark waters of "nearest habitable star systems (being so very far away)" into a place where new words and new ideas would "soar" and "take flight." Here though; I am filled with a kind of lonely sadness ... staring at what appears to be the same mistake(s) happening over and over again; something I've come to call "skipping stones in the pond of reality" and really do liken it to this thing that appears to be the new meaning of "days" and ... a civilization that spends absolutely no love or lust to enter a once sacred and holy place and tarnish it with their sick beliefs and their disgusting desires. You all ... you appear to be some kind of springboard to "bunt" forth yet another age or era of nothingness into the space between this planet and "none worth reaching" and thank God, out of grasp. Today, I'd condemn the entirety of this world simply for it's lack of "oathkeepers" and understanding of what the once hallowed words of Hippocrates meant to ... to the people charged and dharmically required to heal rather than harm. It appears the place and time that was once ... at least destined to be the beginning of Heaven ... has become a "recurring stump" of some future unplanned and tarnished by many previous failed efforts and attempts to overcome this same "lack of conversation or care" for what it meant to be "humane" in a world where that was clearly set high aloft and above "humanity" in the place where they--where we were the best nature had to offer, the sanest, the kindest; the shining last best hope. Today I write almost every day ... secretly thanking "my God" for the disappearance of my tears and the still small but bright hope that "Tearran" will one day connect the Boston Tea Party and the idea that "render to Caesar" and Robin of Loxley ... all have something to do with a re-ordering of society and the worth and import of "money" ... to a place that cares more for freedom from murder than it does ... "freedom from having to allow others to hear me speak." I hold back tears and emotions; not by conscious choice or ability but ... still with that strange kind of lucky awkward smile; and secretly not so far below the surface it's the hope of "a swift death" that ... that really scares me more than the automatons and mechanical responses I see in the faces of many drivers as they pass me on the street--the imagery of connecting it to the serpentine monster of the movie Beetlejuice ... something I just "assume" the world understands and ... doesn't seem to fear (either); as if Churchill had gotten it all wrong and backwards--the only thing you have to fear, is the loss of fear of "loss." Here my crossroads---halfway between the city my son lives in and the city my parents live in--it's on making a decision on whether I should continue at all, or personally work on some kind of software project I've been writing about, or whether I should focus on writing about a "revolution" in government and society that clearly is ... "somewhat underway." In my mind it's obvious these things are all connected; that the software and the governance and the care of whether or not "Babylon" is remembered as a city of great laws and great change or a city of demons and depravity ... that these thi]ngs all hinge and congeal around a change in your hearts; hoping you will chose to be the beginning of a renaissance of "society and civilization" rather than the kings and queens of a sick virtual anarchy ... believing yourselves to have stolen "a throne of God" rather than to literally be the devastating and demoralizing depreciation of "lords and fiefdoms" to something more closely resembled by the time of the Four Horsemen depicted in Highlander. These words intended to be a "forward" to yet another compliment of a ((nother installment of a partial)) chain of emails; whimsically once half-joking ... I called it the Great Chain of Revelation. The software too; part of the great chain, this "idea" that the blockchain revolution will eventually create a distributed and equal governance structure, and a rekindling of monetary value focused on "free and open collaboration" rather than "survival of the most unfit"--something society and civilization seem to have turned the "call of life" from and to ... literally just in the last few years as we were so very close to ... reaching beyond the Heaven(s). I don't think its hard to imagine how a "new set of ground rules" could significantly change the "face of a place" -- make it something shiny and new or even on the other side of the coin, decayed or depraved. It's not hard to connect the kind of change I'm hoping for with "collision protection" and "automatic laws" to the (perhaps new, perhaps ... ancient) Norse creation story of the brothers of Odin: Vili and Ve. It might be hard to see today how a new "kind of spiritual interaction" might be only a few "mouse clicks" away though--how it could change everything literally in a flash of overnight sensation ... or how it might take something like a literal flash of stardom (or ... on the other hand, something like totalitarian or authoritarian "iron fisting") to make a change like this "ubiquitious" or ... something like the (imagined in my mind as ... messianic) "ED" of storming through the cosmos or the heavens and turning something that might appear to be "free and perfect feeling" today into a universe "civlized overnight" and then ... I wonder how long it would take to laud a change like that; for it to be something of a voluntary "reunderstanding" of a process ... to change the meaning of every word or every thought that connects to the process of "civilization" to recognize that something so great and so powerful has happened as to literally change the meaning of the word, to turn a process of civilization into something that had a ... "signta-lamcla☮" of forboding and then a magical staff struck into the heart of a sea and then ... and then the word itself literally changes to introduce a new "mid term" or "halfway point" in which a great singularity or enlightenment or change in perspective or understanding sort of acknowledges ... that some "clear outside" force not only intervened on the behalf of the future and the people of our world but that it was uniquely involved in the whole of-- "waking up" tio a nu def of #Neopoliteran. ^Like the previous notation; the below text comes from an email previously sent; and while i stand behind things like my sanity, my words; and my continued and faithful attempt to speak and convey both a useful and helpful truth to the world---sometimes just a single day can make all the difference in the world. Sometimes it's just a single moment; a flash or a comment about ^th@ blink of an eye" ... and I've literally just "thought up/had/experienced/transitioned thru" that exact moment. The lies standing between "communication" and either "cooperation" or .... some other kind of action have become more defined. More obvious. Because of this clarification; like a kind of "ins^tant* gnosis" ... search high and lo ... the depths all the way to above the heavens ... for a festive divorce ceremonial ritual ... that looks something like a bachelor party ':;] — @amrs@koyu.SPACe ... @suzq@rettiwtkcuf.social (@yitsheyzeus) May 22, 2020 I ... TERON; Gjall are painting me into a corner here; and I don't see around it anymore--I don't see the light, and I don't see the point. I was a happy-go-lucky little kid in my mind; that's not "what I wanted to be" or what I wanted to present, it's who I was. I saw "Ashkenazi" and ... know I am one of those ... and I kind of understood that something horrible might have happened, or might happen here--and I kind of understand that crying smashing feeling of "to ash" that echoes through the ages in the potpourri songs about pockets full of Parker Posey .. and ancient Psalms about "from the ashes of Edom" we have come--and from that you can see the cyclical sickness of this ... place so sure it's "East of Eden" and yet gung-ho on barrelling down the same old path towards ash and towards Edom and towards ... more of Dave's "ashes to ashes dust to dust" and his "smoke clouds roll and symphony of death..." and few words of solace in a song called Recently that I imagine was fleeting and has recently come and gone--people stare, I can't ignore the sick I see. I can't ignore his "... and tomorrow back to being friends" and all but wonder who among us doesn't realize it's "ash" and "gone" and "no memory of today" that's the night between now and ... a "tomorrow with friends" not just for me--but for all of you--for this place that snickers and pantomimes some kind of ... anything but "I'm not done yet" and "there's more ... vendetta ... and retribution to be had, Adam ... please come back in a few more of our faux-days." This is sickness; and happy-go-lucky Himodaveroshalayim really doesn't do much but complain about that word, the "sickle" and the tragic unavoidable ... ash of it all ... these days--you'd think we could "pull out" of this mess, turn another way; smile another day, but it seems there's only one way to get to that avenu in the mind of ... "he who must not know or be me." I have to admit I found some joy in the epiphany that the hidden city of Zion and it's fusion with the Namayim' version of how that "Ha" gels and jives with the name Abraham and the Manna from Heaven and the bath salt and the tina and the "am in e" of amphetamine--maybe a glimmer or a shimmer or a glow of hope at the moment "Nazion" clicked ... and I said ... "no, not me ... I'm nothing like a king, no dreams of authoritarianism at all in the heart of Kish@r;" even as I wrote words that in the spirit of the moment were something of a "tis of a'we" that connected to my country and the first sing-songy "tisME" that I linked to trying to talk in the rhyming spirit of some "first Christ" that probably just like me was one limmerick away from the end of the rainbow and one "Four Non Blondes" song away from tying "or whatever that means" and this land crowned with "brotherhood" (to some personal "of the Bell, and of the bell towers so tall and Crestian") to just one Hopp skip and jump away from the heart of the obvious echoes of a bridge between haiku and Heroku... a few more gears shift into place, a click and and a mechanical turn of the face of the clock's ku-ku striking ... it was the word "Earthene" that was the last "Jesusism" around the post Cimmerian time linking Dionysus and Seuss to that same "su-s" that's belonging to a moment in the city of Uranus--codified and etched in stone as "MCO"--not just for its saucer and warp nacelles and "deflector dish" but for it's underground caverns and it's above ground "Space Mountain" and that great golf ball in the heart of it all. The gears of time and the dawns of civilizequey.org query the missing "here" in our true understanding of what "in the beginning, to hear; to here ... to rue the loss of the Maize from Monoceros to the VEGA system and the tri-galactic origin of ... "some imaginary universal ... Earthene pax" to have dropped the ball and lost it all somewhere between "Avenu Malkaynu" and melaleuca trees--or Yggrasil and Snifleheim--or simply to miss the point and "rue brickell" because of bricks rather than having any kind of love or nostalgia linking to a once cobblestone roadway to the city in the Emerald skies paved in golden "do not return" signs ... to have lost Avenues well after not realizing it was "Heaven'es that were long gone far before I stepped foot on this road once called too Holy for sandals" in a place where that Promised Land and this place of "K'nanites" just loses it's grip on reality when it comes to mentioning the possibility that the original source and story of Ca'anan was literally designed to rid the world of ... "bad nanites" and the mentality of ... vindictiveness that I see behind every smirk. The final hundred nanoseconds on our clock towards doom and gloom cause another bird to fly; another snake to curl up and listen again to the songs designed to charm it into oblivion; whether that's about a club in South Beach or a place not so far from our new "here..." all remains to be seen in my innocent eyes wondering what it truly is that stands between what you are ... and finding "forgiveness not needed--innocent child writes to the mass" ... and the long arm of the minute hand and the short finger of the hour for one brief moment reconcile and move towards "midnight" together; and it's simply idyllic, the Nazarene corner between nil and null you've relegated the history of Terran poast futures into ... "foreves mas" or so they (or you) think. I'm still so far from "Five Finger Death Punch" though; and so far from Rammstein and so far from any kind of sick events that could stand between me and "the eternal" and change my still "casual alternative rock" loving heart to something more death metal; I rue whatever lies between me and there being any kind of Heaven that thinks there could exist a "righteous side" of Hell and it... simultaneously. I still see light here in admonishing the masses and the angels standing against the story and the message God brings us in our history. I still see sparks in siding with the "causticness" of "no holodecks in sight" and the hunger and the pain of simulating ... "the hells of reality" over the story of decades or centuries of silence refusing to see "holography" and "simulated" in the word Holocaust and the horrors of this place that simply doesn't seem to fathom or understand the moments of hunger pangs and the fear of "dark Earth pits" or towers of "it's not Nintendo-DS" linking the Man in the High Castle to an Iron Mask. I rally against being what I clearly am raised high on some pedestal by some force beyond my comprehension and probably beyond that of the "perfect storm in time" that refuses to itself acknowledge what it means to gaze at such an unfathomable loss of innocence at the cost of a "happy and serene future" or even at the glimmer of the Never-Never-Land I'd hoped we would all cherish and love and share ... the games and the newfound freedom that comes not just from "seeing Holodeck" turn into "no bullets" and "no cages" but into a world that grows and flourishes into something that's so far beyond my capability to understand that I'm stuck here; dumbfounded; staring at you refusing to stop car accidents and school shootings ... because "pedestal." For the "fire and the glory" of some night you refuse to see is this one--this place where morality rekindles from ... from what appears tobe one small candle, but truly--if it's not in your heart, and it's not coming from some great force of goodness--fear today and a world of "forever what else may come." Here in a place the Bible calls Penuel at the crossing of a River Jordan ... the Angel of the Lord notes the parallels in time and space between the Potomac and the Rhine--stories of superposition and cities and nation-states that are nothing more than a history of a history of things like the Monoceros "arroz" linking not just to the constellation Orion but to Sagittarius and to Cupid and of course to the Hunter you know so well-- Searching for a Saturday; a sabbath to be made Holy once more ... "at the Rubycon" The Einstein-Rosen Wormhole and the Marshall-Bush-JFKjr Tunnel The waters are called narah, (for) the waters are, indeed, the offspring of Nara; as they were his first residence (ayana), he thence is named Narayana. — Chapter 1, Verse 10[3] In a semi-fit of shameless arexua-self recognition i'm going to mention Amazon's new series "Upload" and connect it to the PKD work that my Martian-in-simulcrum-ciricculum-vitae on "colonization education" ... tying together Transcendance, Total Recall and ... well; to be honest it actually gave me another "uptick" in the upbeat ... maybe i'll stick around until I'm sure there's at least one more copy of me in the ivrtual-invverse ... oh, that reminds me ... Farmer)'s Lord of Opium also touches on this same "mind of God in the computer" subject (which of course leads to Ghost in the Shell and Lucy--thanks Scarlette :). While I'm listing Matrix-intersected pieces of the puzzle to No Jack City, Elon Musk's neuralace and Anderson's Feed are also worth a mention. Also the first link in this paragraph is titled ... "the city of the name of time never spoken after time woke up and stfu'd" (which of course is the primary subject of this ... update to the city Aerosol). The ... "actual original typed dream" included a sort of "roller coaster ride" through space all the way to Mars; where the real purpose of "the thing" I am calling the "Mars Hall" was to display previous victories and failures ... and the introduction of "older or future" culture's suggestions for "the right way" to colonize a new habitat. If it were Epcot Center, this would be something like SpaceMountain taking you to to the foture of "Epcot Countries" as if moving from "countries" to planets were as easy as simply ... "reading backwards." THE SOFTWARE, SINGERS, AND SHIELD(S) OF HEIROSOLYMITHONEYY Thinking just a little bit ahead of myself, but I'm on "Unreal Object/Map Editor within the VR Server" and calling it something like "faux-wet-ware" ... which then of course leads to a similar onomonopeia of "weapons and ..." where-with-all to find a better singer's name to connect the road of "sword" to a Wo'riordan ... but I think that fusion of warrior and woman probably does actually say ... enough of it all; on this road to the living Bright Water that the diety in my son's middle name defines well here, as "waking up," stretching it's tributaries and it's winding wonders and wistfully .... Narayana (Sanskrit: नारायण, IAST: Nārāyaṇa) is known as one who is in yogic slumber on the celestial waters, referring to Lord Maha Vishnu. He is also known as the "Purusha" and is considered the Supreme being in Vaishnavism. andromedic; the ports of call ... to the mediterranean (literally) from the gulf coast; ... ho engages in the creation of 14 worlds within the universe as Brahma when he deliberately accepts rajas guna, himself sustains, maintains and preserves the universe as Vishnu by accepting sattva guna. Narayana himself annihilates the universe at the end of maha-kalp ... . there's no place like home. there's no place like home. there's no place like home. and so it begins ... "f: r e l i g i o n find out what it means to me. faucet, ever single one, stream of purity ... from Fort Myers ... f ... flicks ... Flint. " ^this notation will from this email forward in linear time denote some form of contact method or information related to the context of the message you are reading. This particular one sends me an encrypted email. 5if there is an "@" symbol involved in the "anchor's hypertext reference" (technically an "a href=" in HTML4) your browser should attempt to open an email client to send a message over an anonymous SMTP relay. Understand that "anonymous" in this case may or may not mean your sending email address is hidden or obvuscated--so if you want to receive a reply you must include it in the DATA of your SMTP transmission defined by the RFC5321 attached. In most cases "anonymous" also means that you will not have the recipients direct contact information unless they have made it public---additionally the exact server/system/relay used may or may not be the "Sbroken Berkman Perl Script" linked to in the "hypertext reference" specifically anchored to the words "an anonymous SMTP relay" above. A simple "hat character" (^) and the letter "t" as you see beginning the above paragraph will denote a contact method or form that works over the internet using an HTTP protocol defined in a series of RFC's including (but not limited to) RFC's numbered as 2616, 7230, 7235, 2068 and use a simple language which is based on a definition suggested or proposed currently by an organization called the "W3C Consortium" ---and ... previously set and defined by an organiza^tion located at html.spec.whatwg.org; which appears (to me, for the first time as I write these words) to follow the conceptual spirit of the "living document" defined by the several "Continental Congresses, et alia." I personally now conjoin this document in my head to a procession of patrilineal or matrilnear predecessors to the actual event .... still to be defined ... but related to this specific email, this mailing list; its contributors and readers as well as actual members of the organization (still to be created, defined, or named) that creates a "round table*" of members that is open to the public, to all voters educated enough to understand the specific issue being voted on (up to a standard that; in this place and time appears to be unset and unmet but materially related to reawching the age of 18 years old; growing up in or being born in the United States of America (related spec.* to the Constitution of the United States of America which is officially "self-defined" through a process which includes all three branches of the government which it also "self-defines" and purports to be "of, for, and by the people"--though the general population is only able to contribute through an indirect process (read:the people cannot directly contribute to the constitution without either running for office (like a senator) or being appointed to a specific government position (like a judge or executive branch public servant). The current state of American representative democracy is the highest standard to which I am currently knowledgable of "extant*"--and it is specifically substandard, inferior, and "just not good enough" as a comparison to the process required to vote in the organization being "self-defined" through this process. It is my sincere and clear hope that "this process" will result in a legal and moral amendment to the document shown in the previous link and presented by the Legislative Branch of the United States here. It is my current and faithful belief that anything else would also be significantly below the standards morally required by "this process" which of course includes over 200 years of American citizenship and (other international relations; i.e., e.g, for "iv" example, id est, exemplia gratia) as well as the Sons of Liberty and prior to that contributions from the Crown and the "Parliament and Crown" of the United Kingdom; among others et alea's ifndef: 'swikipedia/et_al.. To note specifically because of lack of personal knowledge and public notoriety (assuming all other requiremnant* achem requirements) alas, babylon. i listened to a man yesterday who was talking about "true heroes" ... he of course noted jesus christ and superman together, suggesting the first was one, and the second just a fiction. he also talked about people like ghandi and "leaders who use non-violent means to "change the world." i at least agree with him on the third, ghandi is a good prototype for some kind of hero. staring at this ... "to be completed" work on tales of two cities, whether from sodom and gomorrah all the way to athens and sparta and perhaps even london and paris--and this particular city, babylon; it stands out as one which truly has no equal or even "mirror" in the history of the world. i suppose i'd add "alexandria" and suggest the library and the laws; something that are fundamental to the ethos of the planet i call "athens." i imagine he did not know "hammurabi's" name; and even today in this place where i ask and do not receive answers; i imagine you still don't connect muhammad or amsterdam ... to this king who in our history is set apart and lifted high on a pedestal of having "codified and written down" laws ... for the very first time. it's almost comical, it took me a paragraph and a sentence to connect "the king and i" to this mirror world, where the bible and the people have most assuredly decided "babylon" is a negative thing or a depraved place. "fallen, fallen, is [the city of] babylon the great" ... just a quote from one of my favorite movies; which of course is re-quoting "dante" and/or "the bible" "a dwelling place [of] (the) demons (say), it has become." www.icann.org/news/blog/the-problem-with-the-seven-keys kauri on IPFS: has-abaslom-and-the-ethos-of-arcadia

      12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever.

      you are offline

      we the people rise again

      safe souls, safe fu


      We the People of Slate ...

      The U.S. Constitution, as you [mighta been, shoulda "come" on ... its somedayrewrϕte it.

      "Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state's population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn't like in the Constitution and pencil in their hearts' desires. Here's what the document would look like with their best ideas."

      多也了了夕 "with a ~~wand~~ of scheffilara, 并#亦太 he begins ... "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon."

      Logically the recent mentions of Gilgamesh and the simultaneous 同時 overlaping 場道 of the eventual link between the famous ruling of Solomon on the separation of babies and mothers and waters and land ... to a story of many "two cities" that culminates in a cultural or societal or "evolutionary" link to Sodom and Gomorrah and the city-state of Babylon (and it's Hanging Gardens) and also of course to Paris and Troy and "Masstodon" and city-states [ciudadestado] and perhaps planet-cities; from Cambridge to Cambridge across the "Cable" to see state to "London" ... recently I called it "the city of realms" ... I started out logically intending to link "game theory" and John Nash to the mathematical story of Sputnik and a revival of American physics; but in my usual way of rambling into the woods [I mean neighborhood] of stream of consciousness ... turned into a premonitory discourse of "two cities" and how sometimes even things as obvious as the number of letters in the word "two" don't do a good enough job of conveying ... how and/or why one is simply never enough, and two isn't much better--but in the end a circle ... is drawn; the perfect circle in our imaginary mathematical perfection ... I see a parted "line" in the letter pronounced "tea" (and beginning that word); and two "vee" (pron. of "v") symbols joined together in a word we pronounce as "double-you" ... and symbolically because I know "V" is the Roman Numeral for 5 (five) and I know not how to multiply in Roman numerals--

      It's important to pause; here. I am going to write a more detailed piece on "the two cities" as I work through this maze like crossroads between "them" and "demo..." ... here demorigstrably I am trying to fuse together an evolutionary change in ... lit. biological evolution as well as an echelon leap forward in "self-government" ... in a place where these two things are unfathomable and unspokenly* connected.

      To a question on the idiom; is Bablyon about "the law" or "of the land of Nod?"

      "What is democracy" ... the song, Metallica's "ONE" echoes and repeats; as we apparently scrive together the word "THEM" ... I question myself ... if Babylon were the capital city of some mythical Nation of Time ... if it were the central "turning point" of Sheol; ... >|<

      Can you not see that in this place; in a world that should see and does there is a gigantic message proving that we are not in reality and trying to show us how and why that's the best news since ... ever---that it's as simple as conjoining "the law of the land" with a basic set of rules that automatically turn Hell into something so much closer to Heaven I just do not understand---why we cant stand up together and say "bullets will not kill innocent children" and "snowflakes will not start avalanches ...." that cover or bury or hide the road from Earth to Verital)e .... or from the mythical Valis to Tanis---or from Rigel to Beth-El ... "guess?"

      ## as "an easy" answer; I'm looking for a fusion of "law and land" that somehow remembers a "jok'er a scene" about "lawn" seats; and "where the girls are green;"

      It's as simple as night and day; Heaven and Hell ... the difference between survival and--what we are presented with here; it's "doing this right"--that ends the Hell of representative democracy and electoral college--the blindness and darkness of not seeing "EXTINCTION LEVEL EVENT" encoded in these words and in our governments foundation ... *by the framers [not just of the USA; but English .. and every language] *

      ... is literally just as simple as "not caring" or thinking we are at the beginning of some long process--or thinking it will never be done--that special "IT" that's the emancipation of you and I.

      Here words like "gnosis" and "gaudeamus" pair with my/ur "new ntersanding*" of the difference between Asgard and Medgard and really understanding our purpose here is to end "evil" ... things like "simulating disease and pain" (here, simulating meaning ... intentionally causing, rather than "gamifying away") and successfully linking the "Pillars of Hercules" to Plato's vision of Atlantis and the letter sequences "an" and "as" ... unlock a fusion of religion and mythology and "cryptographic truth" that connects "messianic" and "Christian" to "Roman" ... "Chinese" and "American" ... literally the key to the difference between the phrases "we are" and "we were" ....

      in "sight" of "silicon" in simulation and Israel, Genesis, and "silence" ... trying to the raising of Asgardian enlightenment ... and seeing "simple cypher" connecting to "Norse" ...

      and the "I AM THAT" surer than shit ... the intention and design of all religion and creation is to end "simulated reality" and also not seeing "SR" ... in Israel and Norse ... "for instance."

      It's a simple linguistic concept; the "singularity" and the "plurality" of a simple word--"to be"--but it goes to the heart of everything that we are and everything that is around us. This is a message about understanding and preserving individuality as well as liberty; and literally seeing "ARXIV" and understanding "often" and failing to connect God and prescience to "IV" and the Fourth Amendment ... it's about blindness and ... "curing the blind instantly" ... and fathoming how and why this message has been etched into our entire history and and all religions and myths and music--to help us "to be THAT we" that actually "are responsible" for the end of Hell.

      • I neglected to mention "Har-Wer" and "Tower of Babel" which are both related lingusitically, religiously and topically: "to who ..." and while we're on "four score and [seven years from now]" seeing the fourth "living thing" in Eden and it's (the name, Abel) connection to Babel and Abraham Lincoln; slavery and ... understanding we live in a place where the history of the United States also, like Monoceros and "Neil Armstrong's first step" are a time shifted ... overlayed map to achieving freedom ... it's about becoming a father-race ... and actually "doing" the technological steps required to "emancipate the e's of 'me&e'" and survive in exo-planetary space---

      it might be as simple as adding "because we did this" here and now; and having it be something we are truly proud of .... forevermore™ ... for certain in the heart of this story about cyclicality and repetition of error--its not because we did "this" or something over and over again; it's about changing "the problem" and then helping others to also overcome ... "things like time travel ... erasing speech" --- however that happenecl.

      • I also failed to mention that "I am in Hell" ... as in this world is hellacious to me; in an overlay with the Hellenic period and this message that we are in the Trojan Horse ... a small gem .... "planet" truly is the Ark of the Covenant---and it's the simple understanding that "reality is hell" is to "living without air conditioning and plumbing is hell" just as soon as you achieve ... "rediscovering" those things---

      • I can't figure out why I am the only person screaming "this is Hell." That's also, Hell.

      ... but recently suggested an old joke about "there being 10 kinds of people in the world (obv an anti-tautology and a tautology simultaneously)" only after that brief bit of singularity and duality mentioning the rest of the joke: "those that understand binary and those that don't know how to base convert between counting with two hands and counting with only an 'on and off.'" It's not obvious if you aren't trying to figure it out, I suppose; but 10 is decimal notation for "kiss" and the "often" without "of" ... and binary notation for the decimal equivalent of "2." A long long time ago in a state that simply non-randomly ties to the heart of the name of our galaxy ... I was again thinking of the "perfect imperfections" of things like saying "three equals one equals one" (which, of course was related to the Holy Trinity and it's "prescient/anachronistic Adamic presence encoded in the name Ab|ra|ha|m" which means "father of a great multitude") ... I brought that one back in the last few months; connecting the letter K and in this "logos-rythmic" tie to the "base of a number system" embellish the truth just a bit and suggest a more accurate rendition of the original [there is no such thing as equality, "is" of separate objects--as in no two snowflakes are the same unless they are literally the same one; true of ancient weights and with the advent of (thinking about) time no two "planets" are the same even if they're the exact same one--unless it's at a fixed moment in time.

      K=3:11 ... to a handle on the music, the DHD of the gate and the *ring of David's "sling" ...

      ---and that's a relationship of "3 is to 11" as [the SAT style "analogy)]y" as a series of alpha, two mathematic, and two numeric symbols ... may only tie in my mind alone to the books of Genesis and Matthew and the phrase "chapter and verse" and to the stories of Lot and Job ... again in Genesis and the eponymous "Book of Job." So ... "tying up loose ends one 10b [III] iv. " as it appears I've taken it upon myself to call a Job and suggest is my "Lot in life [x]i* [3]"

      • I worry sometimes that important things are missing, or will disappear---for instance Mirriam Webster, which is a "canonical/standard dictionary) should probably have an entry for "lot in life" non-idiomatically as "granny apples to sour apples" as

      2 MANY ALSO ICI; 1two ... following in Mitnick's bold introductory word steps; the curve and the complement ... the missiles and the canoes; the line and the blank space ... "supposedly two examples of two kinds, which could be three not nothings ... Today I write about something monumental; as if as important as the singularity depicted in Arthur C. Clarke's 2001 "A Space Odyssey" ... and remember a day when I thought it very novel and interesting to see the words "stillborn and yet still born" connected in a single piece of writing to "Stillwater and yet still water" ... today adding in another phrase noting the change wrought only by one magical single "space" (also a single capital letter; and a third phrase): "block chains with a great blockchain."

      • https://en.wikipedia.org/wiki/EuripidesIphigenia in Aulis or Iphigenia at Aulis[1] (Ancient Greek: Ἰφιγένεια ἐν Αὐλίδι, Iphigeneia en Aulidi; variously translated, including the Latin Iphigenia in Aulide) is the last of the extant works by the playwright Euripides. Written between 408, after Orestes, and 406 BC, the year of Euripides' death, the play was first produced the following year[2] in a trilogy with The Bacchae and Alcmaeon in Corinth by his son or nephew, Euripides the Younger,[3] and won first place at the City Dionysia in Athens.

      • The play revolves around Agamemnon, the leader of the Greek coalition before and during the Trojan War, and his decision to sacrifice his daughter, Iphigenia, to appease the goddess Artemis and allow his troops to set sail to preserve their honour in battle against Troy. The conflict between Agamemnon and Achilles over the fate of the young woman presages a similar conflict between the two at the beginning of the Iliad. In his depiction of the experiences of the main characters, Euripides frequently uses tragic irony for dramatic effect.

      J.K. Rowling spurred just this past week a series of explanations about just exactly what is a blockchain coin worth ... and why is it so; her final words on the subject (artistic liberty taken, obviously not the last she'll say of this magic moment) "I don't think I trust this."

      Taken directly from an off the cuff email to ARXM titled: "Slow the S is ... our Hypothes.is"

      I imagine I'll be adding some wiki/ipfs stuff to it--and try to keep it compatible; the design and layout is almost exactly what I was dreaming about seeing--as a "first rough draft product." Lo, and behold. It's been added to the many places I host my tome; the small compilation of nearly every important email that has gone out ... all the way back to the days of the strange looking Margarita glass ... that now very much resembles the "Cantonese character 'le'" which I've come to associate with a "handle" on multiple corners of a room--something like an automatic coat rack conveyor belt connecting different versions of "what's in the box." I'm planning on using that symbol 了 to denote something like multiple forks of the same page. Obviously I'm thinking forward to things like "the Transhumaist Chain Party" (BDSM, right?)'s version of some particular piece of legislation, let's say everything starts with the sprawling "bulbing" of "Amendment M" ideas and specific verbiage ... and then we'll of course need some kind of new git/subversion/cvs style version control mechanism to merge intelligently into something that might actually .... really should ... make it into that place in history--the first constitutional amendment ratified by a "Continental Congress of All People" ... but you could also see it as an ongoing sort of forking of something like the "wikipedia page" on what some specific term, say "technocracy" means, and how two parties might propagandize and change the meaning of such thing; to suit the more intelligent and wise times we now live in. For instance, we might once have had a "democracy" and a "democractic" party that had some Anarchist Cook Book version of the history of it ending in something like Snipes and Stallone's "DEMOLITION MAN."

      Just kidding, we all know "democracy" has everything to do with "d is cl ... and not th" ... to be the them that is the heart of the start of the first true democracy. At least the first one I've ever seen, in my old "to a republic" ... style. As it is you can play around with commenting and highlighting and annotating all the stuff I've written and begged and begged for comments on--while I work on layering the backend to to perma-store our ideas and comments on both a blockchain (probably a new one; now that i've worked a little with ethereum) with maybe some key-merkle-tree-walk-search stuff etched into the original Rinkeby ... and then of course distributed data in the "public owned and operated" IPFS. To be clear, I plan on rewriting the backend storage so that we will have a permanent record of all comments; all versions of whatever is being commented on; and changes/revisions to those documents--sort of turning the web into a massive instant "place of collaboration, discussion, and co-authoring" ... if you use the wonderful LEGO pieces that have been handed to us in ideas from places like me, lemma--dissenter, and of course hypothes.is who has brought you and i such a polished and nice to look at "first draft" of something like the living Constitution come repository of all human knowledge. I do sort of secretly wich they would have called this project something like "annotating and reflecting (or real or ...) knowledge" just so the movement could have been called ARK. ... or something .... but whatever join the "calling you a reporter" group or ... "supposedly a scientist?"

      NOIR INgR .. I CITE SITE OF ENUDRICAM; a rekindling of the dream of a city appearing high above in the sky, now with a boldly emblazened smiling rainbow and upsidown river ... specifically the antithesis of "angel falls," there's a lagoon too--actually a chain of several ponds underneith the floating rock ... and in some versions of this waking dream there are rings around the thing; you might imagine an artificial set of centripetal orbitals something like a fusion of the ring Eslyeum and the "Six-Axis ride" of the JKF Center's "Spacecamp." I write as I dream, and though I cannot for certain explain exactly how; it's become a strong part of my mythology that this spectacular rendition of "what ends the silence" has something to do with the magical delivery of "a book" ... something not of this Earth but an unnatural thing; one I've dreamt of creating many times. This book is something like the DSM-IV and something like a Merck diagnostic manual; but rather than the old antiquated cures of "the Norse Medgard" this spectacle nearly "itsimportant" autoprints itself and lands on something like every doorpost; what it is is a list of reasons why "simply curing all disease" with no explanation and no conversation would be a travesty of morality--how it would render us half-blind to the myriad of new solutions that can come from truly understanding why "ITIS" to me has become a kind of magical marker: an "it is special" as in, it's cure could possibly solve a number of other problems.

      Through that missing "o," English on the ball, we see a connection between a number of words that shine bright light including Exodus itself which means "let there be light," the word for Holy Fire and the Burning Bush.. .reversed to hSE'Ah, and a story about the Second Coming parting our holy waters.

      This answer connects the magical Rod's of Aaron in Exodus and the Iron Rod of Jesus Christ to the Sang Rael itself... in a fusion that explains how the Periodic Table element for Iron links not just to Total Recall and Mars, but also to this key

      my dream of what the first day of the Second Coming might be like; were the Rod of Christ... in the right hands. In a story that also spans the Bible, you might understand better how stone to bread and your input make all the difference in the world between Heaven and Adam's Hand. Once more, what do you think He ....

      Since the very earliest days of this story, I have asked for better for you, even than see

      Nearly all of the original parts of the original "post-origination dream" remain intact; there's a walkway that magically creates new paths and "attractions" based on where you walk, something like an inversion of the artificial intelligence term "a random walk down a binary tree" ... for instance going left might bring you to the Internet Cafetornaseum of the Earl of Sandwich; and going to the right might bring you to the ICIMAX/Auditorium of Science and Discovery--there's a walkway to "Magical GLAS D'elevators" that open a special "instantiation" of the Japan Room of the Potter and the Toolmaker ... complete with a special [second level and hidden staircase] Pool of Bethesdaibo verily delivering something like youth of mind and body ... or at least as close to such a thing as a sip of Holy Water or Ambrosia or a dip in the pool of Coccoon and Ponce De'Leon could instantly bring ... to those that have seen Jupiter Ascending ... the questions of "nature versus nurture" and what it means to be "old and wise" and "young at heart" truly mean---

      Somewhere between the outdoor rafting ride and the level with the special "ballroom of the ancient gallery" ... perhaps now being named or renamed or recalled as something about "Face [of] the Music" lies a magical "mini-maize" ... a look at a mock-up (or #isitit) of Merlink and Harthor's "round table" that displays a series of ... (at least to me) magical appearing holographic displays and controls that my dreams have stolen from Phillip K. Dick's Minority Report and something of what I hope Microsoft's Dynamics/Hololens/Surface will become---a series of short "focus groups" .... to guage and discuss the information in the "CITIES-D5AM-MERCK" ... how to end world hunger and nearly all disease with the press of a magical buzzer--castling churches to something like "political-party-town-hall-meeting centers" and replacing jails and prisons and hospitals with something like the "Hospitalier's PRIDE and DOJOY's I practiced "Kung-fun-dance" ... a fusion of something like a hotel and a school that probably looks very much like a university with classrooms and dorms and dining hall's all fit into a single building. I imagine a series of 2 or 3 "room changes" as in you walk from the one where you get the book and talk about it ... to the one where you talk about "what everyone else said about it" and maybe another one that actually connects you to other people with something like Facebook's Portal; the point of the whole thing to really quickly "rubber stamp" the need for an end to "bars in the sky" nonalcoholic connotation--as in "overcoming the phrase the sky is the limit" and showing us the need for a beacon of glowing hope fulfilled--probably actually the vision of a holographic marker turning into actual rings around the single moon of Earth, the focus of the song annoucing the dawn of the age of Aquarius---

      It might lead us also to Ceres; and another set of artificial rings, or to Monoceros and a rehystorical understanding of the birthplace and birthing of the "river roads" that bridge the "space gaps" in the galaxy from our "one giant leap for mankind" linking the Apollo moon landing to the mythological connection to the sun; and connecting how the astrological charts of the ancients might detail a special kind of overlapping--the link between Earth's SOL and something like Proxima or Alpha Centauri; and how that "monostar bridge" might overlap to Orion and from there through Sagitarius and the center of the Milky Way ... all the way to Andromeda and more dreams of being in a place where there's a map to a tri-galactic system in the constellation Cancer and a similar one in Leo ... and just incase you haven't noticed it--a special marker here, I thought to myself it might be cool to "make an acronymic tie to Monoceros" and without even thinking auto-wrote Orion (which was the obvious constellation next to Monoceros, in the charts) and then to Sagitarrius; which is the obvious ... heart of our astrological center and link to "other galaxies."

      ----I've dreamt or scriven or reguessed numerous times how the Milky Way's map to an "Atlas marked through time by the ages and the ancients" might tie this place and this actual map to the creation of the railways between stars to the beginning and the end of time and of course to this message that links it all to time travel. There's a few "guesses" I've contemplated; that perhaps the Milky Way chart is a metal-cosmic or microcosmic map to the dawn of time in the galactic vision of ... just after the big bang; or it might tie to a map of something like the unthinkable--a civilization that became so powerful it was able to reverse the entropy of "cosmic expansion" and reverse the thing Asimov wrote of in "The Last Question" as the end of life and the ability to survive basically due to "heat loss."

      "The Last Question." (And if you read two, why not "The Last Answer"?). Find these readings added to our collection, 1,000 Free Audio Books: Download Great Books for Free.

      Looking for free, professionally-read audio books from Audible.com, including ones written by Isaac Asimov?

      * all "asterisks" in the abovə document denote a sort of Adamic unspoken relationship between notations and meanings; here adding the "Latin word for three" and source of the phrase "t.i.d." (which is doctor/pharmacy latin for "three times a day") where the "t" there is an abbreviation of "ter" ... and suppose the link between K and 11 and 3 noting it's alphanumeric position in the English alphabet as the 11th letter and only linking cognitively to three via the conversion betweehex, and binarryy ... aberrative here is the overlapping "hakkasan" style (or ZHIV) lack of mention of the answer in "state of Kansas" and the "citystate of Slovakia" as described in the ICANN document linked [in] the related subsection or slice of the word "binarry" for the state of India. Tetris could be spelled with the addition of only a single letter [in] "tea"---the three letters "ris" are the hearts of the words "Christ" and "wrist" [and arguably of Osiris where you also see the round table character of the solar-system/sun glyph and the chemical element for The Fifth Element (as def. by i) via "Sinbad" and "Superman." The ERIS Free Network should also be mentioned here in connection with the IRC network I associate in the place between skipping stones and sacred hearts defined by "AOL" and "Kdice" in my life. In the lexicon of modern HTML, curly braces are generally relative to "classes" and "major object definitions (javascript/css)" while square brackets generally only take on computer-interpreted meaning in "Markdown" which is clearly (by definition, by this character set "[]") a superset (or at least definately not a subset) of HTML.

      Dr. Will Caster (Johnny Depp) is a scientist who researches the nature of sapience, including artificial intelligence. He and his team work to create a sentient computer; he predicts that such a computer will create a technological singularity, or in his words "Transcendence". His wife, Evelyn (played by Rebecca Hall), is also a scientist and helps him with his work.

      Following one of Will's presentations, an anti-technology terrorist group called "Revolutionary Independence From Technology" (R.I.F.T.) shoots Will with a polonium-laced bullet and carries out a series of synchronized attacks on A.I. laboratories across the country. Will is given no more than a month to live. In desperation, Evelyn comes up with a plan to upload Will's consciousness into the quantum computer that the project has developed. His best friend and fellow researcher, Max Waters (Paul Bettany), questions the wisdom of this choice, reasoning that the "uploaded"

      Just from my general understanding and memory "st" is not ... to me (specifically) an abbreviation of "state" but "ste" is a U.S. Postal code (also "as I understand it") for the name of a special room or set of rooms called a "suite" and in Adamic "connotation" I sometimes read it as "sweet" ... which has several meanings that range from "cool" to "a kind of taste sensation" to "easy to sway or fool."

      If you asked me though, for instance if "it" was an abbreviation or shorthand notation or acronym for either "a United state" or "saint" ... you'd be sure.

      While it's clear from studying linguistic cryptography ... (If I studied it a little here and some there, its also from the "universal translator of Star Trek") and the personal understanding that language is a kind of intelligent code, and "any code is crackable" ... that I caution here that "meaning" and "face value" often differ widely and wildly ... even in the same place or among the same group of people ... either varying over time or heritage.

      Menelaus, in Greek mythologyking of Sparta and younger son of Atreus, king of Mycenae; the abduction of his wife, Helen, led to the Trojan War. During the war Menelaus served under his elder brother Agamemnon, the commander in chief of the Greek forces. When Phrontis, one of his crewmen, was killed, Menelaus delayed his voyage until the man had been buried, thus giving evidence of his strength of character. After the fall of Troy, Menelaus recovered Helen and brought her home. Menelaus was a prominent figure in the Iliad and the Odyssey, where he was promised a place in Elysium after his death because he was married to a daughter of Zeus. The poet Stesichorus (flourished 6th century BCE) introduced a refinement to the story that was used by Euripides in his play Helen: it was a phantom that was taken to Troy, while the real Helen went to Egypt, from where she was rescued by Menelaus after he had been wrecked on his way home from Troy and the phantom Helen had disappeared.

      This article is about the ancient Greek city. For the town of ancient Crete, see Mycenae (Crete). For the hamlet in New York, see Mycenae, New York.

      Μυκῆναι, Μυκήνη

      Lions-Gate-Mycenae.jpg

      The Lion Gate at Mycenae, the only known monumental sculpture of Bronze Age Greece

      37°43′49"N 22°45′27"ECoordinates37°43′49"N 22°45′27"E

      This article contains special characters. Without proper rendering support, you may see question marks, boxes, or other symbols.

      Mycenae (Ancient Greek: Μυκῆναι or Μυκήνη, Mykēnē) is an archaeological site near Mykines in Argolis, north-eastern PeloponneseGreece. It is located about 120 kilometres (75 miles) south-west of Athens; 11 kilometres (7 miles) north of Argos; and 48 kilometres (30 miles) south of Corinth. The site is 19 kilometres (12 miles) inland from the Saronic Gulf and built upon a hill rising 900 feet (274 metres) above sea level.[2]

      In the second millennium BC, Mycenae was one of the major centres of Greek civilization, a military stronghold which dominated much of southern Greece, Crete, the Cyclades and parts of southwest Anatolia. The period of Greek history from about 1600 BC to about 1100 BC is called Mycenaean in reference to Mycenae. At its peak in 1350 BC, the citadel and lower town had a population of 30,000 and an area of 32 hectares.[3]

      3. Chew 2000, p. 220; Chapman 2005, p. 94: "...Thebes at 50 hectares, Mycenae at 32 hectares..."

      Melpomene (/mɛlˈpɒmɪniː/Ancient GreekΜελπομένηromanizedMelpoménēlit. 'to sing' or 'the one that is melodious'), initially the Muse of Chorus, she then became the Muse of Tragedy, for which she is best known now.[1] Her name was derived from the Greek verb melpô or melpomai meaning "to celebrate with dance and song." She is often represented with a tragic mask and wearing the cothurnus, boots traditionally worn by tragic actors. Often, she also holds a knife or club in one hand and the tragic mask in the other.

      Melpomene is the daughter of Zeus and Mnemosyne. Her sisters include Calliope (muse of epic poetry), Clio (muse of history), Euterpe (muse of lyrical poetry), Terpsichore (muse of dancing), Erato (muse of erotic poetry), Thalia (muse of comedy), Polyhymnia (muse of hymns), and Urania (muse of astronomy). She is also the mother of several of the Sirens, the divine handmaidens of Kore (Persephone/Proserpina) who were cursed by her mother, Demeter/Ceres, when they were unable to prevent the kidnapping of Kore (Persephone/Proserpina) by Hades/Pluto.

      In Greek and Latin poetry since Horace (d. 8 BCE), it was commonly auspicious to invoke Melpomene.[2]

      See also [AREXMACHINA]

      Flagstaff (/ˈflæɡ.stæf/ FLAG-staf;[6] NavajoKinłání Dookʼoʼoosłííd Biyaagi, Navajo pronunciation: [kʰɪ̀nɬɑ́nɪ́ tòːkʼòʔòːsɬít pɪ̀jɑ̀ːkɪ̀]) is a city in, and the county seat of, Coconino County in northern Arizona, in the southwestern United States. In 2018, the city's estimated population was 73,964. Flagstaff's combined metropolitan area has an estimated population of 139,097.

      Flagstaff lies near the southwestern edge of the Colorado Plateau and within the San Francisco volcanic field, along the western side of the largest contiguous ponderosa pine forest in the continental United States. The city sits at around 7,000 feet (2,100 m) and is next to Mount Elden, just south of the San Francisco Peaks, the highest mountain range in the state of Arizona. Humphreys Peak, the highest point in Arizona at 12,633 feet (3,851 m), is about 10 miles (16 km) north of Flagstaff in Kachina Peaks Wilderness. The geology of the Flagstaff area includes exposed rock from the Mesozoic and Paleozoic eras, with Moenkopi Formation red sandstone having once been quarried in the city; many of the historic downtown buildings were constructed with it. The Rio de Flag river runs through the city.

      Originally settled by the pre-Columbian native Sinagua people, the area of Flagstaff has fertile land from volcanic ash after eruptions in the 11th century. It was first settled as the present-day city in 1876. Local businessmen lobbied for Route 66 to pass through the city, which it did, turning the local industry from lumber to tourism and developing downtown Flagstaff. In 1930, Pluto was discovered from Flagstaff. The city developed further through to the end of the 1960s, with various observatories also used to choose Moon landing sites for the Apollo missions. Through the 1970s and '80s, downtown fell into disrepair, but was revitalized with a major cultural heritage project in the 1990s.

      The city remains an important distribution hub for companies such as Nestlé Purina PetCare, and is home to the U.S. Naval Observatory Flagstaff Station, the United States Geological Survey Flagstaff Station, and Northern Arizona University. Flagstaff has a strong tourism sector, due to its proximity to Grand Canyon National ParkOak Creek Canyon, the Arizona SnowbowlMeteor Crater, and Historic Route 66.

      PSANSDISL #LWDISP either without gas or seeing cupidic arroz in "thank you" or "allta, wild" ...

      pps: a magnanimous decision ...

      I stand here on the brink of what appears to be total destruction; at least of everything I had hoped and dreamed for ... for the last decade in my life which appears literally to span thousands of years if not more in the eyes of some other beholder. I spent several months in Kentucky telling a story of a post apocalyptic and post-cataclysmic delusion; some world where I was walking around in a "fake plane" something like a holodeck built and constructed around me as I "took a walk around the world" to ... it did anything but ease my troubled mind.

      Recently a few weeks in Las Vegas, and a similar story; telling as I walked penniless down the streets filled with casino's and anachronistic taxi-cabs ... some kind of vision of the entirety of the heavens or the Earth or the "choir of angels" I think of when I echo the words Elohim and Aesir from mythology ... there with me in one small city in superposition; seeing what was a very well put together and interesting story about a "star port" Nirvane ... a place that could build cities into the face of mountains and half working monorails appearing in the sky---literally right before my eyes.

      I suppose this is the place "post cataclysm" though I still have trouble understanding what it is that's actually about ... in my mind it connects to the words "we are losing habeas" echo'ed from the streets of Los Angeles in a more clear and more military voice than usual--as I walked block by block trying to evade a series of events that would eventually somehow connect all the way to the "outskirts of Orlando, Florida" in a place called Alhambra.

      Apparently the name of a castle; though I wasn't aware of that until much later.

      It doesn't feel at all like a "cataclysm" to me; I see no great rift--only a world filled with silent liars, people who collectively believe themselves to have stolen something--something gigantic--at least that's the best interpretation of the throws and impetus behind the thing that I and mythology together call Jormungandr. With an eye for "mythological connections" you could clearly see that name of the Great Serpent of Revelation connects to something like the Unseelie; the faeries of Gaelic lore. To me though this world seems still somewhat fluid, it's my entire life--moving from Plantation to a place where the whole of it might be Bethlehem and to "clear my throat" it's not hard to see here how that land of "coughs" connects to the Biblical land of Nod and to the "Adamically sieved" Snifleheim ... from just a little twist on the ancient Norse land most probably as close to Hel as anyone ever gets--or so I dream and hope---still today. It all looks so real and so fake at the same time; planned for thousands of generations, the culmination of some grand masterpiece story that certainly ties history and myth and reality into a twisted heap of "one big nothing, one big nothing at all."

      I've tried to convey to the world how important I believe this place and this time to be--not by some choice of my own ... but through an understanding of the import of our history and the impact of having it be so obviously tuned and geared towards this specific time ... many thousands of years literally all focused on a single moment, on one day or one hour or even just a few years where all of that gets thrown down on the table as if some trump card has been played--and whether or not you fathom the same magnanimous statement or situation or position ... to me, I think it depends on whether or not you grew up in the same kind of way, believing our history to be so fixed and so difficult to change. I don't particularly feel like that's the "zeitgeist" of today; I feel like the children believe it to be some kind of game, and that it is such as easy thing to "sed" away or switch and turn into something else--another story, another purpose ... anyone's personal fantasy land come true.

      I don't think that's the case at all, it's clearly a personal nightmare; and it's clearly one we've seen time and time again--though not myself--the Jesus Christ that is the same yesterday, today; and once again perhaps echoing "no tomorrow" never remembers or believes that we've "seen it all before" or that we've ever really gotten the point; the thing you present to me as "factual reality" is a sickness, it disgusts me; and I'd do anything to go back to the world "where I was so young, and so innocent" and so filled with starry-eyed hope that we were at the foot of something grand and amazing that would become an empire turned republic of the heavens; filling the stars ... with the kind of love for kindness and fairness that I once associated very strongly with the thing I still believe to be the American Spirit.


      "Suddenly it changes, violently it changes" ... another song echoes through the ages--like the "words of the prophets dancing ((as light)) through the air" ... and I no longer even have a glimmer of hope that the thing I called the American People still exist; I feel we've been replaced by some broken container of minds, that the sky itself has become corrupt to the point that there's no hope of turning around this thing that I once believed with all my heart and all my mind was so obviously a "designed downward spiral" one that was---again--so obviously something of a joke, intended to be easy to bounce off a false bottom and springboard beyond "escape velocity" and beyond the dark waters of "nearest habitable star systems (being so very far away)" into a place where new words and new ideas would "soar" and "take flight."

      Here though; I am filled with a kind of lonely sadness ... staring at what appears to be the same mistake(s) happening over and over again; something I've come to call "skipping stones in the pond of reality" and really do liken it to this thing that appears to be the new meaning of "days" and ... a civilization that spends absolutely no love or lust to enter a once sacred and holy place and tarnish it with their sick beliefs and their disgusting desires. You all ... you appear to be some kind of springboard to "bunt" forth yet another age or era of nothingness into the space between this planet and "none worth reaching" and thank God, out of grasp. Today, I'd condemn the entirety of this world simply for it's lack of "oathkeepers" and understanding of what the once hallowed words of Hippocrates meant to ... to the people charged and dharmically required to heal rather than harm.

      It appears the place and time that was once ... at least destined to be the beginning of Heaven ... has become a "recurring stump" of some future unplanned and tarnished by many previous failed efforts and attempts to overcome this same "lack of conversation or care" for what it meant to be "humane" in a world where that was clearly set high aloft and above "humanity" in the place where they--where we were the best nature had to offer, the sanest, the kindest; the shining last best hope.


      Today I write almost every day ... secretly thanking "my God" for the disappearance of my tears and the still small but bright hope that "Tearran" will one day connect the Boston Tea Party and the idea that "render to Caesar" and Robin of Loxley ... all have something to do with a re-ordering of society and the worth and import of "money" ... to a place that cares more for freedom from murder than it does ... "freedom from having to allow others to hear me speak." I hold back tears and emotions; not by conscious choice or ability but ... still with that strange kind of lucky awkward smile; and secretly not so far below the surface it's the hope of "a swift death" that ... that really scares me more than the automatons and mechanical responses I see in the faces of many drivers as they pass me on the street--the imagery of connecting it to the serpentine monster of the movie Beetlejuice ... something I just "assume" the world understands and ... doesn't seem to fear (either); as if Churchill had gotten it all wrong and backwards--the only thing you have to fear, is the loss of fear of "loss."


      Here my crossroads---halfway between the city my son lives in and the city my parents live in--it's on making a decision on whether I should continue at all, or personally work on some kind of software project I've been writing about, or whether I should focus on writing about a "revolution" in government and society that clearly is ... "somewhat underway." In my mind it's obvious these things are all connected; that the software and the governance and the care of whether or not "Babylon" is remembered as a city of great laws and great change or a city of demons and depravity ... that these thi]ngs all hinge and congeal around a change in your hearts; hoping you will chose to be the beginning of a renaissance of "society and civilization" rather than the kings and queens of a sick virtual anarchy ... believing yourselves to have stolen "a throne of God" rather than to literally be the devastating and demoralizing depreciation of "lords and fiefdoms" to something more closely resembled by the time of the Four Horsemen depicted in Highlander.

      These words intended to be a "forward" to yet another compliment of a ((nother installment of a partial)) chain of emails; whimsically once half-joking ... I called it the Great Chain of Revelation. The software too; part of the great chain, this "idea" that the blockchain revolution will eventually create a distributed and equal governance structure, and a rekindling of monetary value focused on "free and open collaboration" rather than "survival of the most unfit"--something society and civilization seem to have turned the "call of life" from and to ... literally just in the last few years as we were so very close to ... reaching beyond the Heaven(s).

      I don't think its hard to imagine how a "new set of ground rules" could significantly change the "face of a place" -- make it something shiny and new or even on the other side of the coin, decayed or depraved. It's not hard to connect the kind of change I'm hoping for with "collision protection" and "automatic laws" to the (perhaps new, perhaps ... ancient) Norse creation story of the brothers of Odin: Vili and Ve.

      It might be hard to see today how a new "kind of spiritual interaction" might be only a few "mouse clicks" away though--how it could change everything literally in a flash of overnight sensation ... or how it might take something like a literal flash of stardom (or ... on the other hand, something like totalitarian or authoritarian "iron fisting") to make a change like this "ubiquitious" or ... something like the (imagined in my mind as ... messianic) "ED" of storming through the cosmos or the heavens and turning something that might appear to be "free and perfect feeling" today into a universe "civlized overnight" and then ...

      I wonder how long it would take to laud a change like that; for it to be something of a voluntary "reunderstanding" of a process ... to change the meaning of every word or every thought that connects to the process of "civilization" to recognize that something so great and so powerful has happened as to literally change the meaning of the word, to turn a process of civilization into something that had a ... "signta-lamcla☮" of forboding and then a magical staff struck into the heart of a sea and then ... and then the word itself literally changes to introduce a new "mid term" or "halfway point" in which a great singularity or enlightenment or change in perspective or understanding sort of acknowledges ...

      that some "clear outside" force not only intervened on the behalf of the future and the people of our world but that it was uniquely involved in the whole of--

      "waking up" tio a nu def of #Neopoliteran.

      ^Like the previous notation; the below text comes from an email previously sent; and while i stand behind things like my sanity, my words; and my continued and faithful attempt to speak and convey both a useful and helpful truth to the world---sometimes just a single day can make all the difference in the world.

      Sometimes it's just a single moment; a flash or a comment about ^th@ blink of an eye" ... and I've literally just "thought up/had/experienced/transitioned thru" that exact moment. The lies standing between "communication" and either "cooperation" or .... some other kind of action have become more defined. More obvious. Because of this clarification; like a kind of "ins^tant* gnosis"

      ... search high and lo ... the depths all the way to above the heavens ...\ \ for a festive divorce ceremonial ritual ... that looks something like a bachelor party ':;]

      --- @amrs@koyu.SPACe ... @suzq@rettiwtkcuf.social (@yitsheyzeus) May 22, 2020

      I ... TERON;

      Gjall are painting me into a corner here; and I don't see around it anymore--I don't see the light, and I don't see the point. I was a happy-go-lucky little kid in my mind; that's not "what I wanted to be" or what I wanted to present, it's who I was. I saw "Ashkenazi" and ... know I am one of those ... and I kind of understood that something horrible might have happened, or might happen here--and I kind of understand that crying smashing feeling of "to ash" that echoes through the ages in the potpourri songs about pockets full of Parker Posey .. and ancient Psalms about "from the ashes of Edom" we have come--and from that you can see the cyclical sickness of this ... place so sure it's "East of Eden" and yet gung-ho on barrelling down the same old path towards ash and towards Edom and towards ... more of Dave's "ashes to ashes dust to dust" and his "smoke clouds roll and symphony of death..." and few words of solace in a song called Recently that I imagine was fleeting and has recently come and gone--people stare, I can't ignore the sick I see.

      I can't ignore his "... and tomorrow back to being friends" and all but wonder who among us doesn't realize it's "ash" and "gone" and "no memory of today" that's the night between now and ... a "tomorrow with friends" not just for me--but for all of you--for this place that snickers and pantomimes some kind of ... anything but "I'm not done yet" and "there's more ... vendetta ... and retribution to be had, Adam ... please come back in a few more of our faux-days." This is sickness; and happy-go-lucky Himodaveroshalayim really doesn't do much but complain about that word, the "sickle" and the tragic unavoidable ... ash of it all ... these days--you'd think we could "pull out" of this mess, turn another way; smile another day, but it seems there's only one way to get to that avenu in the mind of ... "he who must not know or be me."


      I have to admit I found some joy in the epiphany that the hidden city of Zion and it's fusion with the Namayim' version of how that "Ha" gels and jives with the name Abraham and the Manna from Heaven and the bath salt and the tina and the "am in e" of amphetamine--maybe a glimmer or a shimmer or a glow of hope at the moment "Nazion" clicked ... and I said ... "no, not me ... I'm nothing like a king, no dreams of authoritarianism at all in the heart of Kish@r;" even as I wrote words that in the spirit of the moment were something of a "tis of a'we" that connected to my country and the first sing-songy "tisME" that I linked to trying to talk in the rhyming spirit of some "first Christ" that probably just like me was one limmerick away from the end of the rainbow and one "Four Non Blondes" song away from tying "or whatever that means" and this land crowned with "brotherhood" (to some personal "of the Bell, and of the bell towers so tall and Crestian") to just one Hopp skip and jump away from the heart of the obvious echoes of a bridge between haiku and Heroku... a few more gears shift into place, a click and and a mechanical turn of the face of the clock's ku-ku striking ... it was the word "Earthene" that was the last "Jesusism" around the post Cimmerian time linking Dionysus and Seuss to that same "su-s" that's belonging to a moment in the city of Uranus--codified and etched in stone as "MCO"--not just for its saucer and warp nacelles and "deflector dish" but for it's underground caverns and it's above ground "Space Mountain" and that great golf ball in the heart of it all.

      The gears of time and the dawns of civilizequey.org query the missing "here" in our true understanding of what "in the beginning, to hear; to here ... to rue the loss of the Maize from Monoceros to the VEGA system and the tri-galactic origin of ... "some imaginary universal ... Earthene pax" to have dropped the ball and lost it all somewhere between "Avenu Malkaynu" and melaleuca trees--or Yggrasil and Snifleheim--or simply to miss the point and "rue brickell" because of bricks rather than having any kind of love or nostalgia linking to a once cobblestone roadway to the city in the Emerald skies paved in golden "do not return" signs ... to have lost Avenues well after not realizing it was "Heaven'es that were long gone far before I stepped foot on this road once called too Holy for sandals" in a place where that Promised Land and this place of "K'nanites" just loses it's grip on reality when it comes to mentioning the possibility that the original source and story of Ca'anan was literally designed to rid the world of ... "bad nanites" and the mentality of ... vindictiveness that I see behind every smirk.

      The final hundred nanoseconds on our clock towards doom and gloom cause another bird to fly; another snake to curl up and listen again to the songs designed to charm it into oblivion; whether that's about a club in South Beach or a place not so far from our new "here..." all remains to be seen in my innocent eyes wondering what it truly is that stands between what you are ... and finding "forgiveness not needed--innocent child writes to the mass" ... and the long arm of the minute hand and the short finger of the hour for one brief moment reconcile and move towards "midnight" together; and it's simply idyllic, the Nazarene corner between nil and null you've relegated the history of Terran poast futures into ... "foreves mas" or so they (or you) think.


      I'm still so far from "Five Finger Death Punch" though; and so far from Rammstein and so far from any kind of sick events that could stand between me and "the eternal" and change my still "casual alternative rock" loving heart to something more death metal; I rue whatever lies between me and there being any kind of Heaven that thinks there could exist a "righteous side" of Hell and it... simultaneously.


      I still see light here in admonishing the masses and the angels standing against the story and the message God brings us in our history. I still see sparks in siding with the "causticness" of "no holodecks in sight" and the hunger and the pain of simulating ... "the hells of reality" over the story of decades or centuries of silence refusing to see "holography" and "simulated" in the word Holocaust and the horrors of this place that simply doesn't seem to fathom or understand the moments of hunger pangs and the fear of "dark Earth pits" or towers of "it's not Nintendo-DS" linking the Man in the High Castle to an Iron Mask.

      I rally against being what I clearly am raised high on some pedestal by some force beyond my comprehension and probably beyond that of the "perfect storm in time" that refuses to itself acknowledge what it means to gaze at such an unfathomable loss of innocence at the cost of a "happy and serene future" or even at the glimmer of the Never-Never-Land I'd hoped we would all cherish and love and share ... the games and the newfound freedom that comes not just from "seeing Holodeck" turn into "no bullets" and "no cages" but into a world that grows and flourishes into something that's so far beyond my capability to understand that I'm stuck here; dumbfounded; staring at you refusing to stop car accidents and school shootings ... because "pedestal." For the "fire and the glory" of some night you refuse to see is this one--this place where morality rekindles from ... from what appears tobe one small candle, but truly--if it's not in your heart, and it's not coming from some great force of goodness--fear today and a world of "forever what else may come."


      Here in a place the Bible calls Penuel at the crossing of a River Jordan ... the Angel of the Lord notes the parallels in time and space between the Potomac and the Rhine--stories of superposition and cities and nation-states that are nothing more than a history of a history of things like the Monoceros "arroz" linking not just to the constellation Orion but to Sagittarius and to Cupid and of course to the Hunter you know so well--

      Searching for a Saturday; a sabbath to be made Holy once more ... "at the Rubycon"

      The Einstein-Rosen Wormhole and the Marshall-Bush-JFKjr Tunnel

      The waters are called narah, (for) the waters are, indeed, the offspring of Nara; as they were his first residence (ayana), he thence is named Narayana.

      --- Chapter 1, Verse 10[3]

      In a semi-fit of shameless arexua-self recognition i'm going to mention Amazon's new series "Upload" and connect it to the PKD work that my Martian-in-simulcrum-ciricculum-vitae on "colonization education" ... tying together Transcendance, Total Recall and ... well; to be honest it actually gave me another "uptick" in the upbeat ... maybe i'll stick around until I'm sure there's at least one more copy of me in the ivrtual-invverse ... oh, that reminds me ... Farmer)'s Lord of Opium also touches on this same "mind of God in the computer" subject (which of course leads to Ghost in the Shell and Lucy--thanks Scarlette :).

      While I'm listing Matrix-intersected pieces of the puzzle to No Jack City, Elon Musk's neuralace and Anderson's Feed are also worth a mention. Also the first link in this paragraph is titled ... "the city of the name of time never spoken after time woke up and stfu'd" (which of course is the primary subject of this ... update to the city Aerosol).

      The ... "actual original typed dream" included a sort of "roller coaster ride" through space all the way to Mars; where the real purpose of "the thing" I am calling the "Mars Hall" was to display previous victories and failures ... and the introduction of "older or future" culture's suggestions for "the right way" to colonize a new habitat. If it were Epcot Center, this would be something like SpaceMountain taking you to to the foture of "Epcot Countries" as if moving from "countries" to planets were as easy as simply ... "reading backwards."

      THE SOFTWARE, SINGERS, AND SHIELD(S)

      OF

      HEIROSOLYMITHONEYY

      Thinking just a little bit ahead of myself, but I'm on "Unreal Object/Map Editor within the VR Server" and calling it something like "faux-wet-ware" ... which then of course leads to a similar onomonopeia of "weapons and ..." where-with-all to find a better singer's name to connect the road of "sword" to a Wo'riordan ... but I think that fusion of warrior and woman probably does actually say ... enough of it all; on this road to the living Bright Water that the diety in my son's middle name defines well here, as "waking up," stretching it's tributaries and it's winding wonders and wistfully ....

      Narayana (Sanskrit: नारायण, IASTNārāyaṇa) is known as one who is in yogic slumber on the celestial waters, referring to Lord Maha Vishnu. He is also known as the "Purusha" and is considered the Supreme being in Vaishnavism.

      andromedic; the ports of call ... to the mediterranean (literally) from the gulf coast;

      ... ho engages in the creation of 14 worlds within the universe as Brahma when he deliberately accepts rajas guna, himself sustains, maintains and preserves the universe as Vishnu by accepting sattva guna. Narayana himself annihilates the universe at the end of maha-kalp ...

      .

      there's no place like home. there's no place like home. there's no place like home.

      and so it begins ... "f:

      r e l i g i o n

      find out what it means to me. faucet, ever single one, stream of purity ...

      from Fort Myers ... f ... flicks ... Flint.

      "

      ^this notation will from this email forward in linear time denote some form of contact method or information related to the context of the message you are reading. This particular one sends me an encrypted email. 5if there is an "@" symbol involved in the "anchor's hypertext reference" (technically an "a href=" in HTML4) your browser should attempt to open an email client to send a message over an anonymous SMTP relay. Understand that "anonymous" in this case may or may not mean your sending email address is hidden or obvuscated--so if you want to receive a reply you must include it in the DATA of your SMTP transmission defined by the RFC5321 attached. In most cases "anonymous" also means that you will not have the recipients direct contact information unless they have made it public---additionally the exact server/system/relay used may or may not be the "Sbroken Berkman Perl Script" linked to in the "hypertext reference" specifically anchored to the words "an anonymous SMTP relay" above.

      A simple "hat character" (^) and the letter "t" as you see beginning the above paragraph will denote a contact method or form that works over the internet using an HTTP protocol defined in a series of RFC's including (but not limited to) RFC's numbered as 2616, 7230, 7235, 2068 and use a simple language which is based on a definition suggested or proposed currently by an organization called the "W3C Consortium"

      ---and ... previously set and defined by an organiza^tion located at html.spec.whatwg.org; which appears (to me, for the first time as I write these words) to follow the conceptual spirit of the "living document" defined by the several "Continental Congresses, et alia." I personally now conjoin this document in my head to a procession of patrilineal or matrilnear predecessors to the actual event .... still to be defined ... but related to this specific email, this mailing list; its contributors and readers as well as actual members of the organization (still to be created, defined, or named) that creates a "round table" of members that is open to the public, to all voters educated enough to understand the specific issue being voted on (up to a standard that; in this place and time appears to be unset and unmet but materially related to reawching the age of 18 years old; growing up in or being born in the United States of America (related spec. to the Constitution of the United States of America which is officially "self-defined" through a process which includes all three branches of the government which it also "self-defines" and purports to be "of, for, and by the people"--though the general population is only able to contribute through an indirect process (read:the people cannot directly contribute to the constitution without either running for office (like a senator) or being appointed to a specific government position (like a judge or executive branch public servant).

      The current state of American representative democracy is the highest standard to which I am currently knowledgable of "extant"--and it is specifically substandard, inferior, and "just not good enough" as a comparison to the process required to vote in the organization being "self-defined" through this process*. It is my sincere and clear hope that "this process" will result in a legal and moral amendment to the document shown in the previous link and presented by the Legislative Branch of the United States here. It is my current and faithful belief that anything else would also be significantly below the standards morally required by "this process" which of course includes over 200 years of American citizenship and (other international relations; i.e.e.gfor "iv" exampleid estexemplia gratia) as well as the Sons of Liberty and prior to that contributions from the Crown and the "Parliament and Crown" of the United Kingdom; among others et alea's ifndef: 'swikipedia/et_al..

      To note specifically because of lack of personal knowledge and public notoriety (assuming all other requiremnant* achem requirements)

      alas, babylon.

      i listened to a man yesterday who was talking about "true heroes" ... he of course noted jesus christ and superman together, suggesting the first was one, and the second just a fiction. he also talked about people like ghandi and "leaders who use non-violent means to "change the world." i at least agree with him on the third, ghandi is a good prototype for some kind of hero. staring at this ... "to be completed" work on tales of two cities, whether from sodom and gomorrah all the way to athens and sparta and perhaps even london and paris--and this particular city, babylon; it stands out as one which truly has no equal or even "mirror" in the history of the world. i suppose i'd add "alexandria" and suggest the library and the laws; something that are fundamental to the ethos of the planet i call "athens."

      i imagine he did not know "hammurabi's" name; and even today in this place where i ask and do not receive answers; i imagine you still don't connect muhammad or amsterdam ... to this king who in our history is set apart and lifted high on a pedestal of having "codified and written down" laws ... for the very first time. it's almost comical, it took me a paragraph and a sentence to connect "the king and i" to this mirror world, where the bible and the people have most assuredly decided "babylon" is a negative thing or a depraved place.

      "fallen, fallen, is [the city of] babylon the great"

      ... just a quote from one of my favorite movies; which of course is re-quoting "dante" and/or "the bible"

      "a dwelling place [of] (the) demons (say), it has become."

    1. Then I went home to mymistress's wigwam; and they told me I disgraced my master with begging, and if I did so anymore, they would knock me in the head. I told them, they had as good knock me in head asstarve me to death

      Sort of a role reversal here. A few removals ago, she complains of her skin being removed from her back by her load and they tell her they wouldn't care if it were her head removed from her soldiers. Then, her life was leverage for them. Now, she uses the threat of her death as leverage for herself.

    2. promising themmoney (as they told me) but they would not hearken to him but knocked him in head, andstripped him naked, and split open his bowels

      As per one of the other annotations made in this section, I am interested in Mary's explicit descriptions of violence while also keeping in mind the similarly violent conditions that colonial settlers subjected Native Americans to. It would be nice to read a text from the perspective of a Native American person on the kinds of violence they experienced at the hands of white people. I am also interested in Mary's mention of the settler trying to bargain for his life with money, and the Native Americans' disinterest in money in pursuit of seemingly(?) senseless (at least from this outset) bloodshed.

    1. And today, accompanied by the shades of the fallen, as with bowed head I permit others who were worthy earlier to precede me to this platform— how am I today to surmise and to express what they would have wished to say?

      The author feels as since he was so lucky to share his story and survive, he feels the need to share others stories as-well, and he feels pressure over that.

    1. Reviewer #1 (Public Review):

      Hwang et al., report that LRRC23 is required for RS3 head assembly and sperm motility, and the truncating LRRC23 is associated with asthenozoospermia in humans. They identified an LRRC23 variant in a consanguineous Pakistani family with infertile males diagnosed as asthenozoospermia and found this variant leads to early termination of LRRC23 translation with loss of 136 amino acids at the C-terminus. They generated Lrrc23 mutant mice that mimic the predicted outcome in human patients and found the truncated LRRC23 specifically disorganizes RS3 and the junctional structure between RS2 and RS3 in the sperm axoneme, which causes sperm motility defects and male infertility. These dates try to elucidate the pathogenicity of LRRC23 in asthenozoospermia. The conclusions of this paper are mostly well supported by data, but many aspects of data analyses and data interpretations need to be improved.

      1) The pathogenesis of truncating LRRC23 in asthenozoospermia needs to be further considered. The molecular mechanism of LRRC23 demonstrated in mice should be tested in patients with the LRRC23 variant. As it may be difficult to determine the structures of RS3 in the infertile male sperm, the LRRC23 localization should be observed in the sperm from patients with the LRRC23 variant.<br /> 2) The absence of the RS3 head in LRRC23Δ/Δ mouse sperm is not sufficient to support the specific localization of LRRC23 in RS3 head. Although LRRC23 might bind to RS head protein RSPH9, the authors state that "RSPH9 is a head component of RS1 and RS2 like in C. reinhardtii (Gui et al, 2021), but not of RS3" as the protein level and the localization of RSPH9 is not altered in LRRC23Δ/Δ sperm. Thus, the specific localization of LRRC23 in RS3 head should be further confirmed.<br /> 3) The interaction between LRRC23 and RSPH9 needs to be defined. AlphaFold models could help determine the likelihood of a direct interaction. In addition, the structure of the 96-nm modular repeats of axonemes from the flagella of human respiratory cilia has been determined (PMID: 37258679), and the localization of LRRC23 in RS could be further predicted.<br /> 4) The ortholog of the RSP15 may also be predicted or confirmed by using the reported structure in human respiratory cilia (PMID: 37258679). Whether the LRCC34 in RS2 is LRRC34?

    2. Reviewer #2 (Public Review):

      Summary:<br /> The present study explores the molecular function of LRRC23 in male fertility, specifically in the context of the regulation of spermiogenesis. The author initiates the investigation by identifying LRRC23 mutations as a potential cause of male sterility based on observations made in closely related individuals affected by asthenozoospermia (ASZ). To further investigate the function of LRRC23 in spermatogenesis, mutant mice expressing truncated LRRC23 proteins are created, aligning with the identified mutation site. Consequently, the findings confirm the deleterious effects of LRRC23 mutations on sperm motility in these mice while concurrently observing no significant abnormalities in the overall flagella structure. Furthermore, the study reveals LRRC23's interaction with the RS head protein RSPH9 and its active involvement in the assembly of the axonemal RS. Notably, LRRC23 mutations result in the loss of the RS3 head structure and disruption of the RS2-RS3 junction structure. Therefore, the author claimed that LRRC23 is an indispensable component of the RS3 head structure and suggests that mutations in LRRC23 underlie sterility in mice.

      Strengths:<br /> The key contribution of this article lies in confirming LRRC23's involvement in assembling the RS3 head structure in sperm flagella. This finding represents a significant advancement in understanding the complex architecture of the RS3 structural complex, building upon previous studies. Moreover, the article's topic is interesting and originates from clinical research, which holds significant implications for potential clinical applications.

      Weaknesses:<br /> 1. While the author generated mutant mice expressing truncated LRRC23 proteins, the expression of these truncated proteins was not detected in sperm. This implies that, in terms of sperm structure, the mutant LRRC23 protein behaves similarly to the complete knockout of the LRRC23 protein, which has been previously reported and characterized (Zhang et al., 2021).

      2. This reviewer questions the proposal that LRRC23 is an integral component of RS3, as the results indicate not only the loss of the RS3 head structure but also an incomplete RS2-RS3 junction structure. In addition, the interaction of LRRC23 with RSPH9 alone does not fully explain its involvement solely in RS3 assembly. Additional evidence is required to examine the influence of LRRC23 on the RS2-RS3 junction.

      3. The article does not explore how these mutations affect the flagella structure in human sperm, which needs further study. Expanding the study to include human sperm structure would undoubtedly enhance the quality of the article.

    1. Reviewer #2 (Public Review):

      In this study, Lamire et al. use a calcium imaging approach, behavioural tests, and pharmacological manipulations to identify the molecular mechanisms behind visual habituation. They show a valuable drug screen paradigm to assess the impact of pharmacological compounds on the behaviour of larval zebrafish.

      The pharmacological screen identifies an expected suppression of habituation by GABA receptor antagonists. More interestingly, it identifies potentially new contributions of melatonin receptor agonists, and oestrogen receptor agonists to habituation, as they seem to increase the rate of habituation.

      The volumetric calcium imaging of habituation to dark flashes is valuable, but the mix of responses to visual cues that are not relevant to the dark flash escape, such as the slow increase back to baseline luminosity, lowers the clarity of the results. The link between the calcium imaging results and free-swimming behaviour is not especially convincing, however, that is a common issue of head-restrained imaging with larval zebrafish. The identification of a cluster of neurons with potentiating responses, which could drive the habituation is intriguing, but more characterizations of these neurons would be needed to fully understand their function in habituation. The pharmacological manipulation of the habituation circuits mapped in the first part does not arrive at any satisfying conclusion, which is acknowledged by the authors.

      Overall, the authors did identify interesting new molecular pathways that may be involved in habituation to dark flashes. Their screening approach, while not novel, will be a powerful way to interrogate other behavioural profiles. The authors identified circuit loci apparently involved in habituation to dark flashes, and the potentiation and no adaptation clusters have not been previously observed and are interesting targets for future work. This work suggests that the circuits and mechanisms underlying habituation are likely more complex than anticipated. The data will be useful to guide follow-up experiments by the community on the new pathway candidates that this screen has uncovered, including behaviours beyond dark flash habituation.

    1. Author Response

      Reviewer #1 (Public Review)

      The authors present a scRNAseq study describing the transcriptomes of the tendon enthesis during postnatal development. This is an important topic that has major implication for the care of common clinical problems such as rotator cuff repair. The results are a valuable addition to the literature, providing a descriptive data set reinforcing other, more comprehensive studies. There are weaknesses, however, in the scRNAseq analyses.

      1)The authors should provide additional rationale for the PCA analysis shown in Fig 1d. It is uncommon to use PCA for histomorphologic parameters. These results do not convincingly demonstrate that P7 is as a critical developmental timepoint.

      2) According to the methods, it appears that the entire humeral head-supraspinatus tendon was used for cell isolation for scRNAseq. This results in the inclusion of cells from a variety of tissues, including bone, growth plate, enthesis and tendon. As such, only a very small percentage of cells in the analysis came from the enthesis. Inclusion of such a wide range of cells makes interpretation of enthesis cells difficult.

      3) The differentiationpseudotime analysis described in Fig 3 is difficult to follow. This map includes cell transcriptomes from vastly different tissues. Presumably, embedded in these maps are trajectories for osteoblast differentiation, chondrocyte differentiation, tenocyte differentiation, etc. With so many layers of overlapping information, it is difficult to (algorithmically) deduce a differentiation path of a particular cell type.

      4) The authors uses the term function throughout the paper (e.g., functional definition of fibrocartilage subpopulations). However, this is a descriptive scRNAseq study, and function can therefore only theoretically be inferred from the algorithms used to analyze the data. A functional role for any of the identified pathways or processes can only be defined with gain- andor loss-of-function studies.

      5) C2 highly expressed biomineralization-related genes (Clec3a, Tnn, Acan). The three example genes are not related to biomineralization.

      6) The functional characterization of the three enthesis cell clusters is not convincing. For example, activation of metabolism-related processes can mean a lot of things (including changes in differentiation), yet the authors interpret it very specifically as role in postnatal fibrochondrocyte formation and growth.

      7) The pseudotime analysis of the enthesis cell clusters is not convincing. The three clusters are quite close and overlapping on the UMAP. Furthermore, the authors focus on Tnn as a novel and unique gene, yet the expression pattern shown in Fig 5g implies even expression of this gene across all three clusters.

      8) The TC1 markers (Ly6a, Dlk3, Clec3b) imply a non-tendon-specific cell population. Perhaps a tendon progenitor pool or an endothelial cell phenotype is more appropriate.

      9) Pseudotime analyses assume that your data set includes cells from progenitor through mature cell populations. It is unclear that the timepoints studied here included cells from early progenitor states.

      10) The CellChat analysis is difficult to follow, as the authors included 18 cell types. The number of possible interactions among so many cell types is enormous, and deducing valid connections between any two cell types in this case should be justified. Is the algorithm robust to so many possible interactions

      Thank you very much for your comments and suggestions. According to your suggestions, we carefully revised the paper. We integrated our dataset with open source GSE182997 datasets and re-performed the downstream analysis. On the other hand, we added immunofluorescence tests to validate the results came from single-cell datasets. And we hope all the mentioned issues in prior version to be well addressed.

      Reviewer #2 (Public Review)

      To reveals cellular and molecular heterogeneity in enthesis, the authors established a single-cell temporal atlas during development. This study provides a transcriptional resource for further investigation of fibrocartilage development.

      Thank you very much for your kind suggestions. According to your suggestions, we integrated our dataset with open source GSE182997 datasets and re-performed the downstream analysis. On the other hand, we added immunofluorescence tests to validate the results came from sinlge-cell datasets. And we hope the mentioned issues in prior version to be well addressed.

    2. Reviewer #1 (Public Review):

      The authors present a scRNAseq study describing the transcriptomes of the tendon enthesis during postnatal development. This is an important topic that has major implication for the care of common clinical problems such as rotator cuff repair. The results are a valuable addition to the literature, providing a descriptive data set reinforcing other, more comprehensive studies. There are weaknesses, however, in the scRNAseq analyses.

      1.The authors should provide additional rationale for the PCA analysis shown in Fig 1d. It is uncommon to use PCA for histomorphologic parameters. These results do not convincingly demonstrate that P7 is as a critical developmental timepoint.

      2. According to the methods, it appears that the entire humeral head-supraspinatus tendon was used for cell isolation for scRNAseq. This results in the inclusion of cells from a variety of tissues, including bone, growth plate, enthesis and tendon. As such, only a very small percentage of cells in the analysis came from the enthesis. Inclusion of such a wide range of cells makes interpretation of enthesis cells difficult.

      3. The differentiation/pseudotime analysis described in Fig 3 is difficult to follow. This map includes cell transcriptomes from vastly different tissues. Presumably, embedded in these maps are trajectories for osteoblast differentiation, chondrocyte differentiation, tenocyte differentiation, etc. With so many layers of overlapping information, it is difficult to (algorithmically) deduce a differentiation path of a particular cell type.

      4. The authors uses the term "function" throughout the paper (e.g., "functional definition of fibrocartilage subpopulations"). However, this is a descriptive scRNAseq study, and "function" can therefore only theoretically be inferred from the algorithms used to analyze the data. A functional role for any of the identified pathways or processes can only be defined with gain- and/or loss-of-function studies.

      5. "C2 highly expressed biomineralization-related genes (Clec3a, Tnn, Acan)". The three example genes are not related to biomineralization.

      6. The functional characterization of the three enthesis cell clusters is not convincing. For example, activation of metabolism-related processes can mean a lot of things (including changes in differentiation), yet the authors interpret it very specifically as "role in postnatal fibrochondrocyte formation and growth".

      7. The pseudotime analysis of the enthesis cell clusters is not convincing. The three clusters are quite close and overlapping on the UMAP. Furthermore, the authors focus on Tnn as a novel and unique gene, yet the expression pattern shown in Fig 5g implies even expression of this gene across all three clusters.

      8. The TC1 markers (Ly6a, Dlk3, Clec3b) imply a non-tendon-specific cell population. Perhaps a tendon progenitor pool or an endothelial cell phenotype is more appropriate.

      9. Pseudotime analyses assume that your data set includes cells from progenitor through mature cell populations. It is unclear that the timepoints studied here included cells from early progenitor states.

      10. The CellChat analysis is difficult to follow, as the authors included 18 cell types. The number of possible interactions among so many cell types is enormous, and deducing valid connections between any two cell types in this case should be justified. Is the algorithm robust to so many possible interactions?

    1. AbstractTransformer-based language models are successfully used to address massive text-related tasks. DNA methylation is an important epigenetic mechanism and its analysis provides valuable insights into gene regulation and biomarker identification. Several deep learning-based methods have been proposed to identify DNA methylation and each seeks to strike a balance between computational effort and accuracy. Here, we introduce MuLan-Methyl, a deep-learning framework for predicting DNA methylation sites, which is based on five popular transformer-based language models. The framework identifies methylation sites for three different types of DNA methylation, namely N6-adenine, N4-cytosine, and 5-hydroxymethylcytosine. Each of the employed language models is adapted to the task using the “pre-train and fine-tune” paradigm. Pre-training is performed on a custom corpus of DNA fragments and taxonomy lineages using self-supervised learning. Fine-tuning aims at predicting the DNA-methylation status of each type. The five models are used to collectively predict the DNA methylation status. We report excellent performance of MuLan-Methyl on a benchmark dataset. Moreover, we argue that the model captures characteristic differences between different species that are relevant for methylation. This work demonstrates that language models can be successfully adapted to applications in biological sequence analysis and that joint utilization of different language models improves model performance. Mulan-Methyl is open source and we provide a web server that implements the approach.

      This work has been published in GigaByte Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giad054) and has published the reviews under the same license. These are as follows.

      **Reviewer 1. Yupeng Cun **

      Zeng et al. proposed an ensemble framework for identifying three type DNA-methylation sites, and performed a benchmark comparison in multiple species' genomic data. This paper give a valuable study on how ensemble transfer learners works and the predictability in different species. My suggestion is the manuscript acceptable with following minor revision: 1. Calculated a consensus ranking using Kendall's tau rank distance method for each method in Figure 2-C. 2. the multi-head self- attention and self-attention head formula should redescribed by following this preprint: https://arxiv.org/pdf/1706.03762.pdf 3. MLM and MuLan-Methyl mixed in some cases, which need be used in a consensus way.

    1. Is the ability for students to develop communication skills through various experiences

      I relate to this statement, because as a kid growing up in a Mexican household I was taught to speak only when told to, dealt with tough love, and many other reasons that had me grow up holding all types of emotions which made me a quiet kid, because my feelings weren't valid. I never had a sibling or anyone to lean my head on but once I started experiencing friendships, being around cousins my age, going to school or events and meeting new people, being able to cry to someone with out feeling ashamed, I then started to develop many forms of communication skills and I'm still slowly learning more through new experiences.

    1. "the object we call a book isnot the real book, but its potential, like a musical score or seed. Itexists fully only in the act of being read; and its ... home is insidethe head of the reader, where the symphony resounds, the seed ger-minates."10 Artworks must be activated to exist

      The book or piece of art seems to be a lump of clay which can be sculpted by each individual to become a unique and holistic piece of art by adding one's own connections and vision to it.

    1. when I go to sea, I go as a simple sailor, right before the mast, plumb down into the forecastle, aloft there to the royal mast-head. True, they rather order me about some, and make me jump from spar to spar, like a grasshopper in a May meadow. And at first, this sort of thing is unpleasant enough. It touches one’s sense of honour, particularly if you come of an old established family in the land, the Van Rensselaers, or Randolphs, or Hardicanutes. And more than all, if just previous to putting your hand into the tar-pot, you have been lording it as a country schoolmaster, making the tallest boys stand in awe of you. The transition is a keen one, I assure you, from a schoolmaster to a sailor, and requires a strong decoction of Seneca and the Stoics to enable you to grin and bear it. But even this wears off in time.

      这是第二条注释, 但位置在第一条的前面

    1. Waikīkī is now a miasma of concrete and asphalt, its waters drained into a canal dividing tourist from resident. The mountain’s springs and waterfalls, trickle where they are allowed to flow, and left stagnant elsewhere, pullulate with staphylococcus. In the uplands, the fields and have long been dismantled, their rock terraces and heiau looted to build the walls of multi-million dollar houses with panoramic Diamond Head and/or ocean views

      Compared to the first stanza which was full of native Hawaiian, the second begins to limit the use of native language and instead consists mostly of mainland English. The third and last stanzas are entirely void of Hawaiian, possibly symbolic of how Hawaii was itself stripped of its nature and culture for the appeasement of tourists.

    1. scores (tuple(torch.FloatTensor) optional, returned when output_scores=True is passed or when config.output_scores=True) — Processed prediction scores of the language modeling head (scores for each vocabulary token before SoftMax) at each generation step.

      [!NOTE] 🤗 Transformers 中,generate() 返回的 scores 的内容为?

      flashcard

      即最后一层的 logits,需要经过 soft-max 才是概率

    1. Author Response

      The following is the authors’ response to the original reviews.

      We would like to thank the Reviewers for their careful reading and the many thoughtful suggestions to improve our manuscript, as well as both the Editors and Reviewers for the generally positive evaluations and encouraging statements.

      Editorial assessment:

      This important work presents an interesting perspective for the generation and interpretation of phase precession in the hippocampal formation. Through numerical simula- tions and comparison to experiments, the study provides solid evidence for the role of the DG-CA3 loop in generating theta-time scale correlations and sequences, which would be reinforced through the clarification of the concepts introduced in the study, in particular the notion of intrinsic and extrinsic sequences. This study will be of interest for the hippocampus and neural coding fields.

      We appreciate that our work has been considered important. In our revision we made a considerable effort to improve on the presentation of our results and the justification of our model assumptions. Particularly we aimed to clarify the meaning of intrinsic and extrinsic sequences by ad- ditional figure panels as well as fleshing out their definition via spike-timing correlations being independent or dependent on the direction of the running trajectory, respectively. To address all the requests, we added 3 new Fig- ures, multiple new Figure panels and simulated a new model variant.

      Reviewer #1 in their public review assessed ”The manuscript has the potential to contribute to the way we interpret hippocampal temporal coding for navigation and memory.”

      They criticized

      • The findings generally relate to network models of phase precession (re- viewed in e.g., Maurer and McNaughton, 2007, Jaramillo and Kempter, 2017). An important drawback of these models with respect to explaining specific experimentally observed features of phase precession, is that they cannot straightforwardly explain phase precession upon first exposure onto a novel track. This is because, specific connectivity in network models may re- quire experience-dependent plasticity, which would not be possible upon first exposure. This is essential, given that the manuscript addresses the possible origin of phase precession in terms of network models and at minimum, this weakness should be discussed.

      We agree with Reviewer # 1 (and also with Reviewer # 2, who brought up a similar point) that models based on recurrence struggle to ex- plain how the recurrent connectivity matrix should come about. While we feel that a full model of how the 2-d topology in the recurrent weights can be learned goes far beyond the scope of this paper (and to our knowledge has not been solved so far in any existing model), we added a new model variant (new Figure 6 and Supplementary Figure 1), which explains the ba- sic phenomenology of extrinsic and intrinsic sequences without the need of recurrent connections, only using feed-forward synaptic facilitation. Thus, assuming recurrent connection is not necessary for our main findings. How- ever, we would like to point out that this does not exclude the possibility that recurrent connections, if set up in an appropriate way, also contribute to phase precession and theta sequences.

      • An important and perhaps essential component of the manuscript, is the distinction between extrinsic and intrinsic models. However, the main con- cepts on which this hinges, namely extrinsic and intrinsic sequences (and the related extrinsicity and intrinsicity) could be better explained and illustrated. Along these lines, the result suggested by the title, namely, hippocampal theta correlations, may be important yet incidental in light of the new concepts (e.g., extrinsicity, intrinsicity) and computational models (e.g., DG-CA3 recurrent loop) that are put forward.

      We have added substantial new explanatory material to the figures, captions and text to more didactically introduce the concepts of in- trinsicity and extrinsicity. We have also completely rewritten the abstract and added a subtitle: ”extrinsic and intrinsic sequences”

      • The study seems to put forward novel computational ideas related to neural coding. However, assessing novelty is challenging as this manuscript builds on previous work from the authors, including published (Leibold, 2020, Yiu et al., 2022) and unpublished (Ahmadi et al., 2022. bioRxiv) work. For example, the interpretation of intrinsic sequences in terms of landmarks had been introduced in Leibold, 2020.

      We agree with the reviewer that this paper touches on many related ideas from previous papers (not only of our lab) and is supposed to tie loose ends. Thus, the novel contribution is a biologically plausible mechanistic model of how intrinsic sequences and 2-d place maps interact on the level of interconnected spiking neurons. Such a level of explanation has not yet been available in previous work. We have considerably extended the Discussion section in our revision detailing the bigger picture underlying this theory. Also our addition of the non-recurrent model variant (see above) adds considerable novelty, since it provides an account of phase precession and preplay in novel environments.

      • The significance of the readout tempotron neuron could be expanded on. In particular, there is room for interpretation of the output signal of that neuron (e.g., what is the significance of other neurons downstream? Why is the rationale for this output to being theta-modulated?)

      We have added an additional Figure 8 to better illustrate the inner workings of the tempotron. We also extended the discussion to better explain the potential use of the tempotron output (see above). In short, we consider the tempotron to signal a unique behaviorally important context that is independent of remapping induced by changes of sensory cues, which is a new prediction of the model. Since the context signal is resulting from DG loops it requires a stable code to also exits in the DG. Evidence for such long-term stability in DG has been found in Hainmu¨ller & Bartos (2018).

      Reviewer #2 in their public review find ”this research topic to be both important and interesting” and appreciates ”the clarity of the paper.”, com- mending our ”efforts to integrate previous theories into their model and con- duct a systematic comparison”.

      We are very happy about these positive remarks and sincerely would like to thank the reviewer!

      Reviewer #1 made the following specific recommendations for changes:

      The abstract is somewhat difficult to parse. I have identified some words and/or sections that could be improved.

      • ’ ....inherently 1 dimensional’. This statement seems to be related to an a priori interpretation of the authors. On the other hand, if offline sequences are trivially 1 dimensional because they are sequences (i.e., they constitute a vector), then online sequences would be 1-dimensional as well. What is the key difference between offline and online? Is it the omnidirectional place fields in two dimensions? Perhaps more importantly, how relevant is this fact with respect to the main results of the manuscript, which concern ex- trinsic and intrinsic sequences?

      We indeed meant that the sequences are trivially 1-dimensional. The main challenge that we would like to address in this paper is how a 2-d topology of place cells (and direction dependent theta sequences) and a 1-d sequence topology of intrinsic theta correlations and during (p)replay can be reconciled. We hope this has become clearer in the rewritten abstract.

      • The language in lines 36-38 is overly technical. I suggest modifying the language, the language was less technical and more understandable in the body of the manuscript, which should be also reflected in the Abstract.

      We would would like to apologize for making the abstract too technical. Also in response to Reviewer #2, we decided to rewrite the ab- stract entirely.

      The authors use a mixture of conductance based models and Izhikevich neurons, presumably for the spiking generating mechanism. The conductance component can be readily interpreted in terms of the underlying biophysics. The Izhikhevich neuron model, however, is phenomenological. I suggest you address i) the rationale for using Izhikevich model, 2) its biophysical inter- pretation, 3) and its combination with conductance-based currents.

      The reviewer is correct that spike generation is modelled using Izhikevich’s model whereas synaptic integration is included in a conductance- based manner. As suggested by the reviewer, we have added further expla- nation in the Methods part, explaining that the Izhikevich approach allows to adjust burst firing properties with only few parameters by efficiently em- ulating the bifurcation structure of spike generation in the full biophysical model (1&2) and otherwise has no effect on the integration of conductance- based synaptic currents in a subthreshold regime (3).

      Line 126: when you say preferred angle, do you mean preferred (heading) direction? If so, please maintain consistency throughout.

      We thank the reviewer for pointing out the inconsistency. We have added the word ”heading” throughout the manuscript whenever ap- propriate. To further improve the consistency, we have clarified the meanings of ”best” (or ”worst”) direction and reserved the use of it solely for cases when trajectory direction is compared with the preferred heading direction, namely, ”best” (”worst”) direction when trajectory is along (opposite) the preferred heading direction.

      Line 174: When discussing cross-correlation, sometimes you mean a cross-correlation function between two place fields and sometimes to the his- togram of all such correlations? Please clarify.

      We used histograms to empirically estimate the underlying cross-correlation function. For clarity, we have specified that it is a cross- correlation histogram in the revised manuscript whenever we refer to the empirical estimate.

      Figure 3:

      Understanding the difference between extrinsic and intrinsic sequences is fundamental for the manuscript. I suggest that in the section that refers to Figure 3 (or Figure 3 itself), you kindly provide an example depicting how extrinsic and intrinsic sequences can

      1) coexist yet be distinctly identified

      2) depend on trajectory

      3) depend on DG input

      By coexistence, we meant the heterogeneous population of ex- trinsic and intrinsic cell pairs and, hence, the extrinsic and intrinsic theta correlations, as shown in Figure 3J. To improve the clarity, we added the following sentence in the section that refers to Figure 3: ”In our simula- tion, extrinsically and intrinsically driven cell pairs are both present in the population (Figure 3J), indicating a coexistence of extrinsic and intrinsic sequences.”. To illustrate how extrinsic and intrinsic sequences depend on both tra- jectory and DG recurrence, we have also added annotations in Figure 3F to mark the extrinsic and intrinsic part of the sequence.

      Moreover, the caption of Figure 3 refers to the directionality of the theta sequences. How does this again relate to the extrinsic/intrinsic distinction?

      We hope the highlighting in panel F of Figure 3 has resolved this problem.

      Figure 5:

      • This is a crucial figure that should illustrate the differences between extrinsic and intrinsic sequences, as the figure caption suggests. Surprisingly, it is not at all clear where (i.e., in which panel) and how (i.e., methodologi- cally) should one distinguish one type of sequence from another. I suggest that at least one such panel is dedicated to illustrating the difference and/or detection of these sequences in time and/or from phase precession plots. Moreover, there is significant visual crowding that makes the interpretation challenging (e.g., insert a space between G and E)

      We would like to apologize that in the previous version of the manuscript, we seemed to have evoked the impression that the difference between intrinsic and extrinsic sequences should be mainly illustrated in Figure 5. We hope that our revisions of Figures 1 and 3 have made it sufficiently clear to this point. The main purpose of Figure 5 was (and is) to illustrate how intrinsic sequences can lead to out-of-field firing. We have modified the figure caption (and text) accordingly. To address the visual crowding problem in Figure 5, we have inserted a space between panels and also removed repeated labels.

      Tempotron neuron and Figure 6:

      From the reviewer’s questions on Figure 6, we feel that our presentation caused considerable confusion about the motivation and inter- pretation of the tempotron simulations. We therefore rewrote parts of the associated text and Figure caption. We hope that the revised presentation clarifies the issues. We therefore only briefly respond to the reviewer’s points here, because we think they largely resulted from misunderstandings.

      • Intuitively, and as the manuscript results suggest, late phases are asso- ciated to extrinsic mechanisms while early phases are associated to intrinsic. Why not construct a simpler classifier readout based on this fact? How does it compare to a tempotron?

      Opposite to the reviewer’s comment, extrinsic mechanisms are visible at early phases (late in the field), intrinsic mechanisms at late phases (early in the field). In fact, what the tempotron does is learning to identify the intrinsic (late phase) part and to disregard the extrinsic (early phase) part.

      • What is the significance of theta-modulated output of the tempotron (readout) neuron?

      The theta modulation of the tempotron output is a trivial re- sult of the theta-modulation of the input, i.e., the detection of the intrinsic sequence pattern is done once every cycle.

      Suggestion for Figure 6 related to Tempotron readout: Focus on ’with DG loop condition’, as the challenge and most important point here is to identify extrinsic and intrinsic sequences. The No-loop condition could be left as a supplementary figure or side panel.

      The no-loop condition is the essential control showing that the tempotron only responds to the previously learned intrinsic pattern and can- not identify spatial location based on the extrinsic pattern.

      Further work/predictions.

      Lines 196-198. ”Since intrinsic sequences can also propagate outside the trajectory (Figure 5) and activate place cells non-locally, our model predicts direction-dependent expansion of place fields.” If remote activation is ’suffi- ciently’ remote, wouldn’t this predict two separate place fields instead of an expansion?

      The reviewer is completely correct. Out of field spiking can be also affecting remote locations, if the intrinsic sequences link to remote place fields. This would lead to double fields, however, the intrinsic part would only be active at late theta phases. For simplicity, we have not added such a case in our paper, but we would like to thank the reviewer for this comment, since it leads to a nice prediction of the model, which can be experimentally tested and therefore was included to the discussion.

      Lines 556-558. ”In our model, firing rate is determined by both low-phase spiking from sensory input and high-phase spike arrivals of DG-CA3 loops, both producing opposing effects on the phase distribution.” Is it possible to make a differential prediction based on lesions here, e.g., along the lines of reduced range phase precession, for either high phases or for low phases?

      We thank the reviewer for this great suggestion. Lesion of DG in the model does indeed reduce the phase range and mean spike phase. This further corroborates the effect of DG-loop on theta compression and high-phase spiking. We have included a new panel D in Figure 4 and a corresponding mention in the result section.

      Line 570. ”We speculate that the functional roles of intrinsic sequences may not be limited to spatial memories.”. Is there any relationship to re- play and/or sleep-dependent memory consolidation? Some speculation in the Discussion section would be welcome and appropriate.

      We have added some further speculative ideas to the last section of the Discussion. We propose that replay and preplay reflects the intrinsic sequences that express the current expectation of the animal. We have not yet thought well enough about their relation to memory consolidation to phrase this in the manuscript, but would suggest that they could serve to signal multimodal context information to the neocortex where it can evoke retrieval of unimodal memory traces.

      The description of the results, as stated in the public review, can be im- proved. A key component is the definition and identification of extrinsic and intrinsic sequences.

      Some comments:

      • I think that the words ’extrinsic’ and ’intrinsic’ are problematic as both types of sequences/models rely on external (spatial) input, hence both are in some sense ’extrinsic’. On the other hand, both are network mechanisms, thus in some sense ’intrinsic’, where the asymmetry is either programmed directly onto the weights or due to synaptic depression. To add to the con- fusion, ’intrinsic’ mechanisms very often refer to cellular mechanisms in neurophysiology. I kindly ask you to, ideally, reconsider the terminology, or at the very least, be very thorough and precise when describing the mech- anisms. For example, sometimes extrinsic (intrinsic) ’models’ are referred to, sometimes ’sequences’, sometimes ’factors’, sometimes ’pairs’, etc.

      We understand and appreciate the reviewers argument, but would like to stick to the terminology, since it was already used in our prior publication. We have made considerable effort to improve the explanation and illustration of extrinsic vs. intrinsic pairs in the main text, Figure 1 and 3 to highlight our definition that is based on pair correlations: Extrin- sic pairs flip the correlation lag with reversal of running direction, intrinsic pairs don’t. This is simply a functional definition and should not be con- fused with potential microscopic mechanisms. One of those (DG-loops) is suggested in our paper.

      • As discussed in the public review, network mechanisms may require experience-dependent plasticity and hence cannot easily explain phase pre- cession on the first pass. Please discuss why and/or how your model fits with this observation.

      We agree that the two models under consideration both require the recurrent network be set up appropriately and there is no theory so far that would explain how. The reason we chose these two models is because they are well known in the community and relatively similar. We reasoned that comparison between an intrinsic model and an extrinsic model would make most sense if the two are a similar as possible. Nevertheless, we ex- tended the manuscript by a new set of simulations in which we do not use re- current CA3 connections and obtain phase precession solely be feed-forward synaptic facilitation (new Figure 6 and supplementary Figure S1). The new simulations show that the basic phenomenology can also be obtained with- out using recurrent CA3 connections, however, as expected when removing one mechanisms of phase precession, the range of phase range is somewhat reduced as compared to the full model.

      Along a similar vein, phase precession in Figure 1E only has a range of pi/2, which is about half of the typical range of phase precession for single runs. This should be characterized as a weakness of the intrinsic model.

      The precession range in spiking models is highly sensitive to a large number of parameters such that it is hard to make such definite claims (see also above response). In the original Tsodyks et al. 1996 paper the phase range went up to 270 degrees with a slightly different implementation to ours in terms of current vs. conductance-based synapses, an exponen- tial instead of a Gaussian recurrent weight function, and 1-d (original) vs 2-d (ours). We chose conductance-based synapses, and a Gaussian weight profile for better comparison with the Romani and Tsodyks (2015) model. In the original non-spiking implementation by Romani and Tsodyks (2015), the phase range was hardly 70 degrees. Our model implementation of the Romani and Tsodyks (2015) model fits the experimentally reported phase ranges of about 70 to 180 degrees in CA3 (Harris et al., 2001).

      Lines 282-284: ”...since phase precession properties change in relation to running directions, nor are they solely intrinsic since reversal of correlation is still observed in most of the sequences (Huxter et al., 2008; Yiu et al., 2022).”. To which extent is this a consequence of the phase precession model (extrinsic vs intrinsic) or the fact that place fields are sometimes directional?

      The reversal of sequences with reversed running direction is how we define extrinsic correlation. We hope our changes in relation to Figure 1 has clarified this point.

      Figure 2: Is it i) directional input or ii) short-term facilitation that gives rise to lower phase? (or perhaps both?) Please clarify.

      It’s both. This is now clarified in the revised version of the Re- sults sections related to Figure 2: higher depolarization always yields earlier phases in spiking models, however, pair correlations are not affected by ei- ther of the two mechanisms.

      Line 320. ”...onset of phase precession”. Do you mean in CA3/CA1/DG?

      Thank you for pointing this out. We have clarified that this statement refers to CA3.

      Line 323. ”....at a different location”. Please add rationale why it has to be at a different location and a reference to the appropriate equation.

      The sequence rationale as well as the equation number have been added.

      Line 384. ” ... predicting that loss of DG inputs is compensated for by the increase of release probability in the spared afferent synapses from the MEC.”. It wasn’t clear whether this was a ’homeostasis prediction’, or and implementation in the model. Please clarify.

      Since the model explained the experimental observations by implementing an increased probability of release, the model predicts that in animals with DG lesion the probability of release should be enhanced. We have modified the wording to avoid confusion.

      Line 428 ”...and near future locations) is obvious, the potential role of the lesser expressed intrinsic sequence contributions is not straightforward.”. Similar to my comments above regarding terminology, please clarify what are both contributions and why are intrinsic sequences ’lesser expressed’.

      We have rewritten this passage to avoid unclear wording.

      Line 474. ”...we showed that the trajectory-independent sequences”. Do you mean ’intrinsic sequences’?

      We thank the reviewer for careful reading! We have changed the wording ”intrinsic sequences” in the revision.

      Line 482. ”...field pairs being extrinsic”. Please clarify, as the usage of extrinsic now refers to field pairs.

      Thank you for pointing this out. We went through the whole manuscript and clarified the terms.

      Line 245 (heading). Consider rewriting as ’Dependence of theta se- quences on heading directions’. Extrinsic and Intrinsic models have not yet been introduced.

      Since the main purpose of the first Results section is to explain the difference between extrinsic and intrinsic sequences we kept these terms in the heading but modified it to ”Dependence of theta sequences on head- ing directions: Extrinsic and intrinsic sequences”. Additionally, we have put more emphasis on introducing the terms ”extrinsic” and ”intrinsic” in this section.

      Figure 1.

      • I suggest using the same font - C and D, and F and G are too close to each other, consider adding space. For example, the exponent, 10-2 makes reading cumbersome. Line 300. Phase tail means offset phase? Phase tail may be too informal. Line 325: DG loop. Do you mean CA3-DG projection?

      We thank the reviewer for the suggestions. In the revised manuscript, we have ensured that the same font is used in all of the fig- ures. To improve the readability of Figure 1, we have added space between panels as suggested, removed repeated axis label and downsized the text ”10-2”. Furthermore, we have rewritten the referenced line without using the word ”tail”, and also, clarified the meaning of DG loop as the short form of CA3-DG projection.

      Figure 4 caption: ”DG lesion reduces temporal correlations...”. It is more precise to say that the lesion reduces the slope of the fitted lag vs dis- tance. And how is this related to sequence compression?

      In the paragraph referring to Figure 4, we have elaborated on the meaning of theta compression and its relation with the the lag-distance plot. However, we argue that ”reduces the slope of the fitted curve” is not comprehensive enough to express our summarized conclusion in a caption title. We have modified the wording to be ”DG lesion reduces theta compression”.

      In addition, we have changed the slope unit to be radians per cm rather than radians per maximum pair distance, in conformity to unit standards.

      General comment about terminology with regards to tuning and connec- tivity: it is not formally correct to compare connectivity with trajectories (e.g., lines 388-395, caption of Figure 5A, etc). Perhaps compare tuning to particular directions/preference or receptive field?

      We have corrected the wording such that the direction of DG- loop projection is compared to the direction of trajectory.

      Line 470. ’...fixed recursive loop.” Sentence is not clear, do you mean recurrent loops?

      The reviewer is correct. We corrected the wording

      Reviewer #2 had the following recommendations.

      M1. The abstract focuses on the differences between online and offline hippocampal replays. However, the replay topic is not touched upon in the rest of the manuscript. I found this very confusing when I first read the pa- per. I suggest the authors reconsider the best way to approach the opening or at least discuss if and how their model would incorporate replay phenomena.

      Also in response to reviewer #1 we have rewritten the abstract focusing on the problem of how to generate 2-d topology from 1-d sequences. In addition, also in response to Reviewer#1 we added a paragraph in the discussion detailing a hypothesis on how er think replay and intrinsic se- quences work together.

      m2. On lines 89-91, the authors provide the selection of neuronal pa- rameters for excitatory pyramidal cells and inhibitory cells in the Izhikevich model. While the choice of model is reasonable, it would be helpful to clarify the source of these neuronal parameters, especially for readers who are not familiar with the model.

      Again, also in response to reviewer # 1, we have added more motivation for the Izhikevich model.

      M3. On lines 94-98, the model considers a 2D sheet of CA3 neurons. One of the most significant assumptions is that each 2x2 tile of place cells is considered a unit with four directional angles. What is the basis for this assumption? Is there any experimental result supporting this, or is it a completely artificial design for the model? This is important since the or- ganization of CA3 cells also affects the network architecture discussed later and impacts the realism of the model.

      This comment is related to Reviewer #1’s concern on experience- dependent plasticity: How is this connectivity pattern established? We fully agree that this is an open problem for the Tsodyks et al.-type networks. The main reason for choosing them (as argued in our response to reviewer #1) is to have two published models, representing one type of sequence each, that are similar enough for comparison. In addition, we added new simulations (new Figure 6 and Supplementary Figure S1), showing that the basic phe- nomenology can also be obtained in a model without recurrent connections (see also response to Reviewer # 1)

      m4. Similarly, on lines 111 and 140, the model uses 500 ms for the timescales of short facilitation and short-term synaptic depression. The choices of these two timescales are vital for producing directionality in extrin- sic and intrinsic sequences, yet their experimental sources are not clarified.

      In the Methods section of the revised manuscript, we have in- cluded the sources of previous experimental data and modelling work to support our choice of the time constants.

      M5. On line 126, the authors assume that the synaptic strengths be- tween CA3 cells, Wij, are given by the distances between neurons and the similarity between their directional preferences. While this assumption seems reasonable in the sensory cortex, I am unsure if this is also the case in the hippocampus, and the authors should clarify the basis for this assumption.

      The distance dependence simply reflects the original Romani and Tsodyks 2015 model (see response to M3) and we share the concern of the reviewers. The increased connectivity for neurons with the same di- rectional preference was necessary to recover the direction dependent phase precession properties (Figure 2) in the realm of the Romani and Tsodyks 2015 model. Please also see our new Figure 6 showing simulations without the recurrent matrix.

      More importantly, the existing connections within CA3 and DG cells completely determine the ”intrinsic” sequences. But wouldn’t this be fragile when place cells undergo global remapping, which can take place within only a few seconds? The author should comment on this in the discussion.

      We would like to thank the reviewer for bringing up this inter- esting point. In our thinking, the DG-CA3 connectivity is fixed (multiple 1-d trajectories, not necessarily requiring 2-d topology), i.e., the same in- trinsic sequence should show up in multiple environments (and should not remap), although it may just not be active in some environments). This is a prediction of our model and we have added it to the Discussion.

      M6. I found the setup of DG place cells unreasonable. DG place cells are found to be granule cells rather than pyramidal cells. Moreover, the model does not consider recurrent connections between DG cells (These setups are closer to CA1 place cells).

      We agree with the reviewer, DG granule cells should rather be modelled as high-input resistance EIF neurons. However, the feedback loop via the dentate is not a direct one. It involves hilar mossy cells plus multiple hierarchies of feedback inhibition (this is probably what the reviewer means with recurrent connections between DG neurons, because granule cells are not recurrently connected in the non-pathological state). To our knowledge a biologically realistic model of the hilar-DG network does not exist and it would be far beyond the scope of this paper to develop one. We therefore see our DG feedback model rather as phenomenological. The discussion paragraph on the anatomy of the dentate gyrus touches on these points.

      Therefore, a significant concern is: Why should it be the DG feedback projection to CA3 responsible for the ”intrinsic” sequences instead of pro- jections from other brain areas?

      The reviewer is generally correct, any brain structure which im- plements fixed sequences via a loop would do. The reason why we suggest the DG to be the best candidate is purely empirical referring to papers with dentate lesions: Sasaki et al. 2018 and Ahmadi et a. 2022. We have added a similar argument to the discussion.

      m7. On line 166, the authors claim that there are no connections between inhibitory cells at all. While I understand that this is for simplification of the model, the lack of recurrent inhibition between interneurons may have limited the model’s ability to produce gamma-band dynamics (referring to PING and ING mechanisms), which are robust rhythms produced in CA3. I am very curious if the model can incorporate theta-gamma coupling by in- troducing connections between CA3 inhibitory cells.

      We have omitted the gamma oscillation for simplicity, because we do not have a hypothesis for a functional role in the context of dis- tinguishing extrinsic from intrinsic sequences (Occam’s razor) and, as the reviewer correctly anticipates, they unavoidably show up when inhibitory in- terneurons connect to each other (e.g. Thurley et al. 2013). Of course, one could envision situations in which gamma for intrinsic sequences my have different frequency than for extrinsic ones, by differentially manipulating the CA3 and DG basket cell networks, but, as long as there is no experimental data, it would be pure speculation and thus we have not included it in the model.

      m8. The authors should clarify the source of parameters in Table 1, especially the synaptic strengths. These values are vital for extrinsic and intrinsic theta sequences.

      The weight values have been chosen to allow for large theta phase precession range, coexistence of extrinsic and intrinsic sequences, and stability of the network activity. A similar statement has been added to the manuscript.

      M9. I have another concern regarding the measurements of ”extrinsic- ity” and ”intrinsicity” defined on lines 185-196. Are they the best measures? To distinguish the cause of spike correlations, the ”extrinsicity” and ”intrin- sicity” of a pair of spikes should not be high at the same time. However, this is clearly not the case in the model, according to Figs 3 and 5. Moreover, in the data analysis carried out later, spike pairs are considered extrinsic or intrinsic merely by comparing the two measurements. I suggest the authors consider counterfactual methods in causal inference. For example, would a spike pair (cell1, cell2) still exist if we change the sensorimotor inputs or the DG-CA3 projections? If this is difficult to implement, the authors should at least discuss how different choices of measurements would impact the con- clusions of the paper.

      The problem the reviewer has identified arises from the funda- mental symmetry of theta phase quantification: if spikes of a pair of place fields have a phase difference of 180◦ one cannot say which cell leads and which cell follows, hence, the phase difference is both intrinsic (because the peak doesn’t flip) and extrinsic (because the peak flips and ends up at the same phase). The fact that in some cases extrinsicity as well as intrinsicity are high simply means that the field pair has a correlation peak lag close to 180◦. Since in the experimental data set in (Yiu et al. 2022) only field pairs were available, we have not been able to use a different quantification then and decided to apply the same quantification in our model for comparison. Moreover, Figure 5F nicely shows that the measures are able to retrieve the ground-truth intrinsic DG-loop structure when considered on the population level.

      In our model, though, we can go beyond 2-nd order statistics and derive sequence similarity measures including multiple cells, e.g., Chenani et al. 2019. However, since, we already know the ground truth by construction, we decided to not use these methods. We added a paragraph in the discus- sion elaborating on beyond 2nd order sequence quantification.

      m10. The authors begin discussing ”intrinsic sequences” from line 316. However, it is not defined before that (and in the rest of the paper as well), causing confusion when reading the paper. The exact definitions of extrinsic and intrinsic sequences should come earlier.

      We hope that our changes to the beginning of the results section (Figure 1), also asked for by Reviewer # 1 could clarify the confusion.

      m11. On lines 345-347, the authors claim that ”the intrinsic sequences are played out backward as determined by the direction of fixed recurrence (Figure 3F),” which is vague. If such sequences are present in that panel, it should be more explicitly indicated graphically.

      Also in response to Reviewer #1, we have graphically high- lighted the two types of sequences.

      M12. On lines 309, 356, 484, 495, 515, and possibly other instances, the authors repeatedly claim that the model simulations are in ”quantitative agreement” with their previous experimental paper. However, no experimen- tal data or comparison with the simulations are presented in this paper. The authors should at least create one figure to demonstrate the degree of consistency between them, instead of merely asking the reader to refer back to their previous paper.

      We agree with the reviewer that the experimental data of our previous paper should be presented in the manuscript. However, creating more panels or figures is likely to clutter the already crowded visuals and ob- scure our main message. We therefore decided to give numerical comparisons the previous findings in the main text whenever appropriate, specifically, in the sections referring to Figures 2, 3 and in the Discussion.

    1. Walk into the test with your head high and your shoulders back. How you walk can affect how you feel, and if you act confident, you just may feel more confident.

      walk confidently!

    2. When you are reading a multiple-choice question, try to come up with the answer in your head before you look at the choices.

      come up with answer for multiple choice

    1. She could see how happy the thought of having a theater again made Kermit, and in her head she saw the future unspooling out in front of her: their own theater, a new show every night, too many jokes and songs and unprogrammed answers to ever be faked.

      I think this story has real-world implications because it shows how quickly AI can evolve and grow stronger in such a short period of time.

    1. Reviewer #2 (Public Review):

      The authors of the manuscript have developed and used cloning-free method. It is not entirely novel (rather it is based on previously described ISA method) but it is clearly efficient and useful complementation to the already existing methods. One of strong points of the approach use by authors is that it is very versatile, i.e. can be used in combination with already existing methods and tools. I find it important as many laboratories have already established their favorite methods to manipulate SARS-CoV-2 genome and are probably unwilling to change their approach entirely. Though authors highlight the benefits of their method these are probably not absolute - other methods may be as efficient or as fast. Still, I find myself thinking that for certain purposes I would like to complement my current approach with elements from authors CLEVER method.

      The work does not contain much novel biological data - which is expected for a paper dedicated to development of new method (or for improving the existing one). It may be kind of shortcoming as it is commonly expected that authors who have developed new methods apply it for discovery of something novel. The work stops on step of rescue the viruses and confirming their biological properties. This part is done very well and represents a strength of the study. The properties of rescued viruses were also studied using NSG methods that revealed high accuracy of the used method, which is very important as the method relies on use of PCR that is known to generate random mistakes and therefore not always method of choice.

      What I found missing is a real head-to-head comparison of the developed system with an existing alternatives, preferably some PCR-free standard methods such as use of BAC clones. There are a lot of comparisons but they are not direct, just data from different studies has been compared. Authors could also be more opened to discuss limitations of the method. One of these seems to be rather low rescue efficiency - 1 rescue event per 11,000 transfected cells. This is much lower compared to infectious plasmid (about 1 event per 100 cells or so) and infectious RNAs (often 1 event per 10 cells, for smaller genomes most of transfected cells become infected). This makes the CLEVER method poorly suitable for generation of large infectious virus libraries and excludes its usage for studies of mutant viruses that harbor strongly attenuating mutations. Many of such mutations may reduce virus genome infectivity by 3-4 orders of magnitude; with current efficiencies the use of CLEVER approach may result in false conclusions (mutant viruses will be classified as non-viable while in reality they are just strongly attenuated).

    1. There’s a collective (and bipartisan!) sense that these changes have steered CTE in a positive direction, toward “relevance and rigor,” and away from its “dark history” of tracking disadvantaged students into low-wage, low-opportunity occupations.

      Anyone else find the author of this article getting under their skin? I think his "academic" head is up his arse- the ignorance of his assumptions is maddening. Disadvantaged students is a terrible generalization and to consider the trades "low-wage, low-opportunity occupations", bro has no idea how good of a living tradespeople make.

    2. the combination will give them greater agility when automation threats come knocking tomorrow.

      I agree with this point. When students have both transferable and technical skills they are very versatile and head into the workforce with many skills that others do not have. They often have thorough critical thinking and problem solving skills which also sets them apart.

    3. The introduction of ChatGPT last fall thrust artificial intelligence into the national consciousness,

      This hits the nail on the head. I feel like ChatGTP is all I have heard about since it was released last fall. I wasn't terribly interested in it but it's been impossible to escape. I gave a digital marketing workshop to small business owners this past spring and even had to address it in my talk. I was anti using it to generate content (fyi)

    4. I’m there to gawk over what it can do—and, spoiler, it goes well beyond producing first-year term papers

      Very new to learning what AI actually is and the more I learn the harder it is to wrap my head around!

    1. n the image encoder, we insert the adapters beforethe multi-head attention modules.

      本文在image encoder中也添加了adapters,引入了更多可训练参数

  13. Jul 2023
    1. roughly I'd say there's about a dozen or so core attributes to the library

      can check out htmx.org reference if you want to see them all as well as you know all the events and so forth that htmx triggers I'm pretty rich environment but not the the core of it is very small at the end of the day you can probably get your head around htmx I'd say in a day or so if you have some HTML slash web development experience so that's great it's pretty small but the question is okay we've generalized hypermedia maybe um what uh what can we accomplish with

    1. What does the science say?In scientific studies, it seems people experience more of a mix than the self-selected responders to a viral post that implied it was either/or.A small study in 2011 tried to get a better picture of how people think. They gave beepers to a random sample of students. When the beeper went off, they had to note down what was going on inside their heads moments before it went off. This went on for several weeks, to get them used to it and then to get an accurate picture of what was happening inside their minds."Subjects experienced themselves as inwardly talking to themselves in 26 percent of all samples," the team wrote in Psychology Today. "But there were large individual differences: some subjects never experienced inner speech; other subjects experienced inner speech in as many as 75 percent of their samples. The median percentage across subjects was 20 percent.Advertisement"Some people talk to themselves a lot, some never, some occasionally."

      After learning about [[aphantasia]] (people who can't visualize things in their mind's eye), I wanted to know more about people without an inner monologue.

    1. I think this is also part of  our sense of who we are as humans, as ourselves,   and the idea of the self, the individual, and  even the humans as this individual species,   these divisions are arbitrary.
      • for: emptiness, human interbeing, human interbecoming
      • example
        • BEing journey
          • I think this is also part of our sense of who we are as humans, as ourselves,
          • and the idea of the self, the individual, and even the humans as this individual species,
          • these divisions are arbitrary.
          • I don't stop at my skin.
          • I'm breathing air.
          • I'm drinking the water.
          • I'm eating food.
          • I'm eating an apple.
          • When I eat an apple, when do the molecules of the apple become me? -When I'm chewing it in my mouth?
            • when it's in my stomach?
            • when my system has broken down the nutrients?
            • when is that point that nitrogen molecule becomes me versus the apple?
          • I would propose that apple is me when it's growing on the tree.
          • I think of the blossoms of the tree and the bees.
            • The blossoms of the tree,
            • the tree can't reproduce without the bees.
            • So is the bee part of the tree?
            • The bee is part of the reproductive system of the tree.
            • So the bee is part of the tree,
            • the tree is part of the bee.
            • The bee needs the tree.
            • The tree needs the bee.
          • This is just one simple relationship,
            • but it's not simple at all because
              • the bee needs a lot of other things,
              • and the tree needs a lot of other things.
              • And the mycelium and the soil.
          • We talk about a tree and the soil and the atmosphere and the bee as if they're all separate things.
          • And that's convenient because our language has nouns that mean certain things.
          • So we want to talk about trees.
          • It's nice to have a word for tree,
            • but we get it in our head that the tree is separate from the soil,
            • which is separate from the atmosphere,
            • which is separate from the bee.
          • And I'm saying no, those divisions are indeed somewhat arbitrary,
          • but we use them for convenience.
          • But the soil's not the soil without the relationship with the tree
            • and the tree's not the tree without the relationship with the soil and the atmosphere.
            • And the atmosphere is not the atmosphere without the relationshi to the tree, to the bee, to me and the soil.
          • So to me that's the essence of ecology.
          • And that we have to expand this sense of self,
            • individual self as well as
            • the species of humans.
        • And this isolated self, I think is a socially reinforced construct, - but we get sucked into it.
          • And we talk about relationships in ecology and we talk about the value of all living things,
          • but in our actions we come back to the individual self.
    1. People absolutely try. I can't name the journals that try these off the top of my head, but as you can see from that Wikipedia section, there are journals that: Do double-blind peer review (authors don't know who the reviewers are, and vice versa) Do triple-blind peer review (authors & editors & reviewers don't know who each other are) Do open peer review (everyone knows who everyone else is) Do open peer reports (reviews are published together with the paper) Do open participation (reviewers self-select to review the paper) Do post-publication peer review (every paper is published, reviews are done after publication) Do results-blind peer review (reviewers receive a manuscript where the results & conclusions are omitted) Do two-stage results-blind peer review (review done in two stages; in the first stage reviewers don't know the results/conclusions, in the second stage they do) Do novelty-blind peer review (reviewers are specifically instructed not to comment on whether the paper is novel, only if it is correct) The fact that the traditional model has endured is a sign of how robust it is. Everyone knows it is flawed, but nobody has been able to come up with a better model. ShareShare a link to this answer (Includes your user id)Copy linkCC BY-SA 4.0 Edit Follow Follow this answer to receive notifications answered Jan 5 at 7:58 Allure

      A response by [[Allure]] to an [[Academia]] [[StackExchange]] question about alternative publishing models for scientific experiments that help deal with the [[replication crisis]].

      In the comments, Allure suggests that journals that "Do results-blind peer review (reviewers receive a manuscript where the results & conclusions are omitted)" encourage publishing "non-significant results".

    1. First, remember that formal languages are much more dense than natural languages, so it takes longer to read them. Also, the structure is very important, so it is usually not a good idea to read from top to bottom, left to right. Instead, learn to parse the program in your head, identifying the tokens and interpreting the structure. Finally, the details matter. Little things like spelling errors and bad punctuation, which you can get away with in natural languages, can make a big difference in a formal language.

      Orientações importantes sobre como ler e escrever um código.

    1. the problem starts to rear its ugly head in incredibly bizarre and depressing ways. One of the ways that we know this happens is in the criminal justice system.
      • definition
        • babyfaceness
      • when a person's face resembles a baby's face,
        • it can advantage or disadvantage that person depending on
          • which race they are part of
            • if they are babyfaced white, then they will be frowned upon because they are perceived as weak
            • if they are babfaced minority, then they will be more likely to be promoted as they are less threatening
    1. said to contain the "high points of each lecture." This seems to be an attempt to patent ideas, rather than the  professor's actual presentation of them.

      This is concerning as students should be leaving with the imparted ideas weather in their head or on there exercise pad it is still the intention for the course to pass on to others at least the most important points.

    1. Myer et al. (2016) evaluated the so-called Q-Collar, a neck collar worn by athletes with the intention to protect the brain from head impacts. Aim of this study was to test the collar’s effect on reducing neuroanatomical and neurophysiological damage in two groups of hockey players that either wore the collar or did not (the latter served as controls).

      مطالعه بروی هاکی بازهایی بوده که گروهی محافظ گردن داشتن و گروهی نداشتن و صدمات نوروفیزیولوژی رو بررسی کردن

    1. Figure 10.6 The transformation of the optic array obtained by alocomotor movement (Stage 6). The solid lines represent the opticarray before the observer stands up, the dashed lines after he has moved.The path of locomotion of the head is forward and upward. The wholearray is transformed, including the invisible portion behind the head, butthe latter is not represented in the drawing.

      The "optic array" can be transformed? Later Gibson states that "transformation" is just one of the

      vast variety of optical "motions"

    1. Author Response

      Reviewer #1 (Public Review):

      This study demonstrates that a hybrid measurement method increases 3 fold the resolution of mouse USV localization. This increased resolution enables to revise previous occurrence frequency measures for female vocalizations and establishes the existence of vocal dominance in triadic interactions. The method is well described and its efficiency is carefully quantified. A limitation of the study is the absence of ground truth data, which may have been generated eventually with miniaturized loudspeakers in mouse puppets. However, a careful error estimation partially compensates for the absence of these likely challenging calibrations. In addition, the conclusions take into account this uncertainty. The gain in accuracy with respect to previous methods is clear and the impact of localisation accuracy on biological conclusions about vocalisation behavior is clearly exemplified. This study demonstrates the impact of the new method for understanding vocal interactions in the mouse model, which should be of tremendous interest for the growing community studying social interactions in mice.

      We have performed the requested, additional ground estimate using a movable miniature speaker, for more details see point 2 of Reviewer 2, and the new supplementary figure.

      Reviewer #2 (Public Review):

      Past systems for identifying and tracking rodent vocalizations have relied on triangulating positions using only a few high-quality ultrasonic microphones. There are also large arrays of less sensitive microphones, called acoustic cameras that don't capture the detail of the sounds, but do more accurately locate the sound in 3D space. Therefore the key innovation here is that the authors combine these two technologies by primarily using the acoustic camera to accurately find the emitter of each vocalization, and matching it to the highresolution audio and video recordings. They show that this strategy (HyVL) is more accurate than other methods for identifying vocalizing mice and also has greater spatial precision. They go on to use this setup to make some novel and interesting observations. The technology and the study are timely, important, and have the potential to be very useful. As machine learning approaches to behavior become more widespread in use, it is easy to imagine this being incorporated and lowering entry costs for more investigators to begin looking at rodent vocalizations. I have a few comments.

      1) What is the relationship of the current manuscript to this: https://www.biorxiv.org/content/10.1101/2021.10.22.464496v1 which has a number of very similar figures and presents a SLIM-only method that reportedly has lower precision than the current HyVL approach. Is this superseded by the submitted paper?

      The referred manuscript (now published in Scientific Reports) is indeed related to the current work: The currently presented system is based on the integration between SLIM (based on 4 high quality microphones) and Beamforming (based on the 64-channel microphone array). The accuracy of SLIM is generally lower than that of HyVL, but it makes essential contributions to the overall accuracy of HyVL through the integration of the complementary strengths of the two methods/microphone arrays (see Fig. 3A, L-shape of errors). To our knowledge, SLIM was the previously most accurate technique (based on 4 microphones, see comparison in the Discussion), but HyVL exceeds this by a substantial margin. Some figures appear similar mostly due to related code in the underlying analysis pipeline and visualization scripts (e.g. the half-disc densities). However, the set of dyadic and triadic recordings was collected specifically for the present study, and all top-level analyses were performed separately. The single mouse (C57Bl/6 WT) ground truth dataset is shared between the two studies, where in the SLIM paper only the USM4/SLIM part was evaluated (leading to a correspondingly lower, single animal accuracy).

      We felt that the level of detail above would probably impede the reading of the manuscript, and we have therefore added a subset of the above clarifications to the methods and the first time the other study is mentioned.

      2) Can the authors provide any data showing the accuracy of their system in localizing sounds emitted from speakers as a function of position and amplitude? I am imagining that it would be relatively easy to place multiple speakers around the arena as ground truth emitting devices to quantify the capabilities of the system.

      Ground truth data is critical for any meaningful comparison. First, we would like to highlight that we already provided ground truth data in the previous version of the manuscript: In Fig. 3C. we analyzed vocalization data from trials with (1) just a single mouse as well as (2) vocalization at times when all mice were far apart in relation to the accuracy of HyVL (>100 mm, i.e. >25x the accuracy of HyVL) where the chances of erroneous assignment are negligible. We think that these tests are the most relevant, as they are conducted with the relevant sounds, at their actual intensity, spectral profile and emitter acoustics.

      In addition, we have now conducted a series of tests with sounds produced by a miniature speaker placed in 25 different locations to demonstrate the lower-bound of accuracy achievable with the system. The tests indicate an accuracy of MAE < 1mm under these ideal conditions, i.e. without the absorption of the mouse bodies, varying direction of emission of the mouse snout, varying intensity, varying spectral content, duration, etc. Exploring the dependence on all these parameters is in itself interesting, but requires a detailed study in itself. The detailed experimental conditions and results are now provided in Supplementary Fig. 4, including a quantification of the dependence on amplitude.

      3) How is the system's performance affected by overlapping vocalizations? It might be useful to compare the accuracy of caller identification for periods where only one animal is calling at a time vs. periods where multiple animals are simultaneously calling.

      This is an excellent question. Our current code for detecting vocalizations cannot automatically determine if one or multiple vocalizations are concurrently present. We have therefore manually checked all vocalizations for overlapping instances, including those in triadic recordings with two males, where this would be expected to occur most frequently.

      We considered vocalizations to be overlapping if the overlapping constituent timefrequency traces did not form a harmonic stack. Overall, overlaps were surprisingly rare. We did find a couple of cases (<0.1%) where our detection algorithm produced a longer vocalization interval that contained multiple, differently shaped vocalization traces that, when re-analyzed in shortened time-frequency bins with beamforming, belonged to two different males. Note here that beamforming is separately performed from the onset to the end of each vocalization, so the cumulative heatmap can change depending on these onset and end times, which are normally determined by our detection algorithm.

      However, although the identity of the assigned vocalizer could shift in these very rare cases depending on which time bin was re-analyzed, the system’s localization performance remained in principle unaffected: as mentioned above, shorter time bins on non-overlapping parts correctly show the origin of the vocalizations in this case, and therefore a solution to this issue could be a USV detection algorithm that is able to detect the overlap based on the spectral shapes and parses them apart. During the beamforming each vocalization can then be separately localized, by restricting the beamforming to the corresponding time and frequency range. Further, the analysis could be refined so that multiple salient peaks can be detected in the soundfield estimate. This would, however, substantially change the analysis approach, i.e. rather than a single estimate per USV, a sequence of soundfield estimates should be computed and later fused again. Since such a procedure uses less data per single estimate, it also increases the possibility of false positives, which in the current situation with very few overlaps in time, would likely reduce the overall accuracy of the system, we decided to not modify the algorithm in this direction, but we agree that ideally a joint approach - combining separation on the spectrogram and soundfield level - should be pursued. For the present data, if a time window was analyzed such that the intensity map of the sound field contains multiple hotspots of an approximately equal magnitude, the USV would likely remain unassigned, because the within soundfield uncertainty would be higher than for a single peak, and this would reduce the MPI. However, given the rarity of these cases in our dataset, we do not think that their exclusion would change the results appreciably. This information was added as a paragraph to the Discussion.

      It is worth noting that HyVL is very robust: There were a number of cases (<5%) where environmental dampening in combination with harmonic stacking produced interesting timefrequency traces in some of the USM4 microphones, but our system did not have any issue spatially localizing this - what seems like a - smeared vocalization trace. We provide a few examples of this kind in a short video (see Rebuttal Video 2 and the legend at the bottom of this document), where the overlap is also reflected in the intensity map of the sound field, overlaid onto the platform.

      4) Can the authors comment on how sound shadows cast by animals standing between the caller and a USM4 affect either the accuracy of identification or the fidelity of the vocal recording?

      An important point to raise. Sound scattering and dampening caused by the conspecifics of the vocalizing animal can impede the accuracy of any sound localization system, but can unfortunately not be avoided in a social setting. To address this issue, we raised all USM4 microphones by ~12 cm above the interaction platform to minimize the instances of sound blocked by the mice. Further, the Cam64 device should largely be unaffected by sound shadows as it is centrally located above the platform. We have added a modified version of the above comment to the discussion under the heading "Current limitations and future improvements of the presented system".

      5) I'm a bit confused about how the algorithm uses the information from the video camera. Reading through the methods, it seems like they primarily calculate competing location estimates by the two types of microphone data and then make sure that a mouse is in close proximity to one location, discarding the call if there isn't. Why did the authors choose this procedure rather than use the tracked position of the snouts as constrained candidate locations and use the microphone data to arbitrate between them? Do they think that their tracking data are not reliable or accurate enough?

      Thanks for this important suggestion, which we have actually grappled with a lot during the analysis. First of all, the visual tracking data, in particular the manual data, is in our opinion (based on human visual identification) near perfect (within the limits of the video resolution, pixel resolution = 0.8 mm), i.e. on the order of 1-2 mm, and is therefore not the source of any unattributable vocalizations. If we understand the reviewer correctly, then we indeed perform the attribution as he indicates based on the tracked snouts of all mice, specifically by measuring the MPI's of both acoustic location estimates for all mice and then choosing the most reliable one. Specifically, the attributions can be grouped into 3 cases: (i) Estimated origin close to one snout, and snouts rather far apart, (ii) Estimated origin close to one snout and snouts close, and (iii) estimated origin not close to either snout. (i) is easy to address, (ii) is appropriately handled by the mouse probability index, but (iii) is tricky. Since the vocalization has to come from one of the mice, this already indicates that the localization is not working well in this case. Therefore we found it prudent (similar to Neunuebel et al. 2015) to not assign in these cases. Interestingly the MPI is not useful in these cases, as due to the exponential dependence of the normal density on distance, for example a case with a distance of 50 mm to one snout and 60 mm to another snout could lead to an MPI close to 1, which is likely not trustable. We have described this in the Methods as follows:

      "This distance threshold mainly serves to compensate for a deficiency of the 𝑀𝑃𝐼: if all mice are far from the estimate, all 𝑃𝑘 are extremely small, however, the 𝑀𝑃𝐼𝑘 will often exceed 0.95."<br /> Due to the inherent limit for localizing very quiet, short USVs by any system, we think this kind of selection (introduced originally by Neunuebel et al 2015) is a valuable and necessary step in the processing to avoid confusions (which are of course already substantially reduced through HyVL here).

      6) I guess the authors have code that we can run, but I couldn't access it. The manuscript describes the algorithms and equations that are used to calculate the location, but this doesn't really give me a feel for how it works. If you want to have the broadest impact possible, I think you would do well to make the code user-friendly (maybe it is, I don't know). In pursuit of that goal, I would suggest that the authors devote some of the paper to a guided example of how to use it.

      While the code was made available to the reviewers via the link at the beginning of the manuscript (p2, before abstract), we completely agree that this method of distribution is not very accessible. We have therefore created a publicly available GitHub repository (https://github.com/benglitz/HyVL) which hosts the code and details its use on the basis of a sample data set (which is available to the reviewers in the repository link, and later to the public under https://doi.org/10.34973/7kgc-ta72). While we do provide a sample video and analysis workflow there, our data analysis pipeline is quite integrated and other labs will likely use different pipelines. We have therefore tried to make the core functions independent of our pipeline and thus easy to integrate by others into their analysis pipelines.

      Reviewer #3 (Public Review):

      The present manuscript describes a new method to identify the emitter of ultrasonic vocalisations during social interactions between 2 or 3 mice. The method combines two technologies (an "acoustic camera" and a set of four microphones) and succeeds in increasing the spatial precision and the attribution of USV emission to one of the mice. The manuscript describes the characteristics and advantages of each method and the advantages of using both to optimize the identification of USV emitter. The authors used the method to confirm that females are also vocalising during male-female interactions and that females emit USV mostly during nose-nose contact while this was not the case for males. Interestingly, the authors identified that the vocal behaviour of two competing males was strongly asymmetric when facing a female. This was not the case for two females facing one male.

      The method is really promising since the identification of the emitter of USVs during mouse social interactions is a necessary step to speed up our understanding of this communication modality. The increase in spatial precision and in the proportion of attributed vocalisations is non-negligible and will be of great utility in the future.

      We would like to thank the reviewer for this positive perspective on the future utility of our system.

      Generally, the statistical analyses should be adjusted. Indeed, the statistical analyses do not consider the fact that the same individuals were recorded several times (if we understood well the methods). Each point was considered independent (in non-parametric Wilcoxon tests), while this is not the case given the repetitions with the same individuals (the number of repeated encounters per individual should be given in the methods section, by the way). We strongly recommend revising the statistical analyses of the results in Figures 4 and 5. In addition, it could be interesting to check whether the vocal behaviour is stable within each individual (i.e., a male that is vocalising frequently in one situation vocalises always frequently in other situations).

      We generally agree with this suggestion: In order to properly conduct the analysis for individuals as you suggest, a balanced dataset should be used. We had initially collected such a balanced dataset, which was previously not detailed in the manuscript, as the focus was on USV localization/attribution and hence only the recordings containing USVs were analyzed (detailed now in the beginning of Results and Methods). However, overall, the probability of a recording containing vocalizations at all is low: in our balanced set only 23/112 recordings contained vocalizations. We therefore had collected additional recordings with the best vocalizers which created the previously analyzed set of 83 recordings containing USVs recorded with all microphones. This dataset is therefore dominated by recordings from mice that are active vocalizers. While this does not raise any issue for the estimation of the accuracy of the method (Figure 3) or the female vocalizations (Figure 4, because recordings were always randomized across female mice), it precludes an encompassing analysis of individual differences in Figure 5, i.e. the dyadic-triadic comparison. In the new Figure 5, we address the reviewer's question for the dyadic recordings, finding that the current set of recordings does not provide sufficient evidence that individual male mice had significantly different vocalization rates. We would, however, like to point out that this is likely a consequence of the n=4 recordings that are compared here. For the female mice, we also did not find differences in vocalization rates, which is based on n=14 recordings and thus a more reliable result (p=0.16, 1-way ANOVA with factor individual).

      For the triadic recordings, however, due to a limitation in the experiment execution, we unfortunately do not have the complete information available on an experiment level for the triadic recordings, i.e. the video stream was accidentally started after all mice were placed in the platform, and since the same sex animals are visually not separable (while the female mice are separable from the males, based on a slightly shaved region on their head), we cannot completely assess this question in triadic recordings based on the available data. When including the triadic recordings in addition and assuming a single vocalizer (combining all male USVs, see below for why the males could not be assigned in the triadic condition) the male individual comparison can be approximately performed with n=8 recordings, and then the dependence on individual becomes borderline significant (p=0.028, 2-way ANOVA with factors individual and condition).

      For the comparison of vocalization rates in the previous Figure 5 that the reviewer was referring to, we cannot perform a rigorous analysis on the individual level, due to the lack of balance. While we thus agree that differences between individual mice can contribute to the differences observed, we do not think that this would change the conclusion that one of the mice dominates the vocal emissions. If the reviewers agree, we would thus leave Figures 6 (old Fig. 5) and new Figure 7 (behavioral confirmation of dominant/subordinate division) as part of the manuscript, with a clear cautioning about the possible contribution of individual differences to the observed differences. If the reviewers find it inappropriate to leave the results based on the unbalanced dataset in, all results after figure 5 could also be excluded (although we would find this unfortunate, given the additional time and effort we have invested in these).

      It is not easy to understand the rationale behind testing animals in pairs and in triads from the beginning of the manuscript. The authors should better introduce this aspect in the manuscript, especially given the fact that biological results deal with this aspect in Figure 5. The authors might strengthen the parts of the biological results extracted from their new method.

      Thank you for pointing out the need for clarification regarding the rationale behind testing animals in pairs and in triads. It is because courtship interactions are particularly vocal and social, that they are of interest to many fields, e.g. neurodevelopmental disorders.3,4 Due to the natural competitiveness between mice during courtship interactions, high accuracy is particularly beneficial in this regard because it allows disentangling USVs at close distances. We adapted the introduction to better reflect this reasoning and included an extra paragraph in the introduction and also where the biological results from old Fig. 5 / new Fig. 6 are summarized.

      More specifically, the fact that one male takes over the vocal behaviour within a triad is of high interest. Nevertheless, some behavioural data would be needed to strengthen these findings.

      We agree that this is an interesting finding and also agree that some additional behavioral analysis is useful to complement it. In order to arrive at this analysis, we performed all-frame, 3-animal tracking on the 14 triadic recordings with two males. This required switching to skeleton tracking with SLEAP5 in addition to manual post-processing to ensure that no identity switches occur. In each recording the dominant male was then defined as the one that emitted more vocalizations, and then the vocalization-independent spatial interaction histogram was computed, similar to the ones in Fig.4, but now separating between the dominant and the subordinate males (see new Figure 7). The results are consistent with the most typical location of vocalization of the male, in proximity to the female abdomen: The dominant male's spatial interaction histogram (Fig. 7A) was more clearly peaked in the location of the female abdomen very close to the male's snout, in comparison with the subordinate male's histogram (Fig. 7B), which shows up very clearly in the difference between the normalized histograms (Fig. 7C). Significance analysis was performed using 100x bootstrapping on the relative spatial positions to estimate p=0.99 confidence bounds around the histograms of the dominant and subordinate respectively. Significance at a level of p<0.01 highlights multiple relative spatial positions (Fig. 7D), including the one proximal to the snout which has the largest absolute difference (Fig. 7C). Note, that these analyses were conducted on the basis of the non-balanced dataset which contained enough vocalizations to assess the dominant male based on the vocalization rates and thus individual traits of certain animals remain as a possible confound.

      A small proportion of USVs was not assigned. The authors did not discuss the potential reason for this failure (Were the USVs too soft? Did they include specific acoustic characteristics that render them difficult to localise?). These points could be of interest when testing other mouse strains or other species.

      Good point, we agree that it is interesting to know the reasons for failure. As so often, there is not a single property that makes localization hard, but multiple factors contribute. In the SLIM paper, we already identified duration and intensity as important contributors (Fig. 3E/F), and in the speaker test (see new Supplementary Fig. 4) we again demonstrated the influence of intensity. In addition, frequency bandwidth and acoustic occlusion are two other main contributors that each influence the availability of the information/signal-to-noise ratio at the microphones:

      • Frequency bandwidth: In signals that are very narrowband, there are more opportunities for phase ambiguity, in particular for very high-frequency signals. These are avoided/reduced for more wideband signals.

      • Acoustic occlusion: As ultrasonic sounds can be quite directional, if an animal is vocalizing away from a microphone, which in addition would put its body in the way of the sounds to the microphone, then this can reduce the intensity at the microphone to a level where the information is insufficient to utilize information from this microphone. This mostly influences the 4 microphones surrounding the platform, while the Cam64 overhead will likely not be affected by acoustic occlusion in the plain.

      We have added a brief version of this explanation to the discussion under the heading: "Current limitations and future improvements of the presented system"

    1. In addition to thesuggestions I provided earlier, look to your course notes and syllabusfor answers. Often professors will tell you why they assign specificreadings. Pay attention

      I find this very helpful, as I have made it a habit to always look at the course syllabus as soon as possible. It gives me a strong head start on knowing what is to come and allowing me to prepare myself.

    1. In 2018, a painting that shows David sitting triumphantly next to Goliath’s severed head—long attributed to the Baroque artist Giovanni Francesco Guerrieri—came to auction

      but did artemesia paint it?

    2. In her version, two men emerge from behind a marble balustrade, violently interrupting Susanna’s ablutions. Her head and her body torque away from the onlookers as she raises a hand toward them, in what looks like ineffectual self-defense.

      So, in this version she does not hide her sexuality and it is not romanticized

    1. In this study, the authors examine spider development using ACME dissociation and SPLiT-seq at three developmental stages associated with segmentation and regionalization. The authors cluster cells in their data to identify groupings of cell identity across timepoints. The authors examine the expression of AP genes such as Hox genes and DV genes as well as newly-identified markers from their scRNA-seq data using in situ hybridization and fluorescent in situ hybridization.

      In generating and analyzing their data, the authors uncover expression of genes in the precheliceral region and in the posterior SAZ, which gives rise to the opisthosomal segments.

      This study and the data it generates provide an exciting window into spider development and should greatly accelerate future investigations.

      One thing that could be added to the manuscript to provide a greater understanding of its impact would be a more thorough engagement with and discussion of the current arthropod comparative developmental literature.

      For example, it would be interesting to consider how the data presented for the SAZ corresponds to the sequential addition of segments during development of the flour beetle Tribolium castaneum, a system for which thorough investigations of this process have been conducted.

      It would also be interesting to hear consider how the authors might decode the logic of Hox gene co-expression in the spider appendages based on their RNA-Seq expression data, or how the data from precheliceral patterning might provide some additional insights into the arthropod head problem.

      Overall, this study provides a wealth of data for future developmental biology work and will be a valuable resource for other researchers.

    1. I. The god sits on a throne, and he is made of gold and ivory. On his head lies a garland which is a copy of olive shoots.

      The prize for the Olympics was a garland of olive branches

    1. Reviewer #1 (Public Review):

      In this manuscript, the authors present a valuable new method to represent animal behavior from video data using a variational autoencoder framework that disentangles individual-specific and background variance from variables that can be more reliably compared across individuals. They achieve this aim through the use of a novel Cauchy-Schwatz (C-S) regularization term in their loss function that leads to latents that model continuously varying features in the images. The authors present a variety of validations for the method, including testing across sessions and individuals for a head-fixed task. They also show how the methods could be used for behavioral decoding from neural data, quantifying social behavior in mice, demonstrating the applicability of the method outside of head-fixed environments and for different measurement modalities. While some areas of confusion and questions about the validation exist, this is an overall strong paper and an important contribution to this field.

      Strengths:

      - The use of the C-S regularizer is novel approach that has potential for wide use across experimental paradigms and model organisms<br /> - The extent of the validations performed was solid, although perhaps not as convincing in a couple of cases as might be ideal<br /> - The GitHub code demo worked well, and the code appears to be accessible and well-written

      Weaknesses:

      - Some of the validation figures were a bit unclear in their presentation, making it difficult to assess exactly what had been tested<br /> - It is possible that I missed this, but the authors didn't really provide a sense of how to pick a particular distribution to match using the CS term for a specific paradigm/modality and how the choice affects the results<br /> - While the authors' statements about individual training vs. transfer learning accuracy and efficiency in Figure 6 are technically true, the effect size is rather small ( a few percent at most in each case), thus I don't know how much of a big deal I would want to make out of these results<br /> - In general, I would have liked to have seen the Discussion section speak more to the choices and limitations inherent in applying the method. How does the choice of prior/metaparameters/architecture/etc affect the results? In what situations would this method to fail? What are the next advances that are necessary for the field to progress?

    1. Reviewer #1 (Public Review):

      More than ten years ago, it was shown that activity in the primary visual cortex of mice substantially increases when mice are running compared to when they are sitting still. This finding 'revolutionised' our thinking about the visual cortex, turning away from it being a passive image processor and highlighting the influence of non-visual factors. The current study now for the first time repeats this experiment in a primate (the marmoset). The authors find that in contrast to mice, marmoset V1 activity is slightly suppressed during running, and they relate this to differences in gain modulations of V1 activity between the two species.

      Strengths:

      - Replication in primates of the original finding in mice partly took so long, because of the inherent difficulties with recording from the brain of a running primate. The treadmill for the marmosets in the current study is a very elegant solution to this problem. It allows for true replication of the 'running vs stationary' experiment and undoubtedly opens up many possibilities for other experiments recording from a head-fixed but active marmoset.<br /> - In addition to their own data on the marmoset, the authors run their analyses on a publicly available data set on the mouse. This allows them to directly compare mouse and marmoset findings, which significantly strengthens their conclusions.

      Weaknesses:

      - The main thing that is missing from the study is a good explanation as to why running has such a different effect on marmoset V1 compared to mouse V1. Differences in neuromodulatory inputs are cited in the discussion as a possible cause for the discrepancy, but an obvious influencing factor that the authors could investigate in their own data set is the retinal input. In Fig1b, the authors even show these data in the form of gaze and pupil size. In these example data, by eye, it looks like the pupil size is positively correlated with the run speed. This would of course have large consequences on the activity in V1, but the authors do not do anything with these data. The study would improve substantially if the authors would correlate their run speed traces with other factors that they have recorded too, such as pupil size and gaze.

      - Fig2a shows the 'most correlated mouse session', i.e. the session where the relation between visual cortex activity and running speed was strongest. Looking at the raster plot, however, shows that this strong positive correlation must be due entirely to the lower half of the neurons significantly increasing their firing rate as the mouse starts to run; in fact, the upper 25% or so of the neurons show exactly the opposite (strong suppression of the neurons as the mouse starts running). It would be more balanced if this heterogeneity in the response is at least mentioned somewhere in the text.

      Significance:

      The paper provides interesting new evidence to the ongoing discussion about the influence of non-visual factors in general, and running in particular, on visual cortex activity. As such, it helps to pull this discussion out of the rodent field mainly and into the field of primate research. The elegant experimental set-up of the marmoset on a treadmill will certainly add new findings to this issue also in the years to come.

    2. Reviewer #2 (Public Review):

      This work aims at answering whether activity in the primate visual cortex is modulated by locomotion, as was reported for the mouse visual cortex. The finding that the activity in the mouse visual cortex is modulated by running has changed the concept of primary sensory cortical areas. However, it was an open question whether this modulation generalizes to primates.

      To answer this fundamental question the authors established a novel paradigm in which a head-fixed marmoset was able to run on a treadmill while watching a visual stimulus on a display. In addition, eye movements and running speed were monitored continuously and extracellular neuronal activity in the primary visual cortex was recorded using high-channel-count electrode arrays. This paradigm uniquely permitted investigation of whether locomotion modulates sensory-evoked activity in the visual cortex of a marmoset. Moreover, to directly compare the responses in the marmoset visual cortex to responses in the mouse visual cortex the authors made use of a publicly-available mouse dataset from the Allen Institute. In this dataset, the mouse was also running on a treadmill and observing a set of visual stimuli on a display. The authors took extra care to have the marmoset and mouse paradigms as comparable as possible.

      To characterize the visually driven activity the authors present a series of moving gratings and estimate receptive fields with sparse noise. To estimate the gain modulation by running the authors split the dataset into epochs of running and non-running which allowed them to estimate the visually evoked firing rates in both behavioral states.

      Strengths:<br /> The novel paradigm of head-fixed marmosets running on a treadmill while being presented with a visual stimulus is unique and ideally tailored to answering the question that the authors aimed to answer. Moreover, the authors took extra care to ensure that the paradigm in the marmoset matched as closely as possible to the conditions in the mouse experiments such that the results can be directly compared. To directly compare their data the authors re-analyzed publicly available data from the visual cortex of mice recorded at the Allen Institute. Such a direct comparison, and reuse of existing datasets, is another strong aspect of the work. Finally, the presented new marmoset dataset appears to be of high quality, the comparison between the mouse and marmoset visual cortex is well done and the results and interpretation are straightforward.

      Weaknesses:<br /> While the presented results are clear and support the main conclusion of the authors, additional analysis and experimental details could have further strengthened and clarified some aspects of the results. For example, it is known that the locomotion gain modulation varies with layer in the mouse visual cortex, with neurons in the infragranular layers expressing a diversity of modulations (Erisken et al. 2014 Current Biology). However, for the marmoset dataset, it was not reported from which cortical layer the neurons are from, leaving this point unanswered.

      Nonetheless, the aim of comparing the locomotion-induced modulation of activity in primate and mouse primary visual cortex was convincingly achieved by the authors. The results shown in the figures support the conclusion that locomotion modulates the activity in primate and mouse visual cortex differently. While mice show a profound gain increase, neurons in the primate visual cortex show little modulation or even a reduction in response strength.

      This work will have a strong impact on the field of visual neuroscience but also on neuroscience in general. It revives the debate of whether results obtained in the mouse model system can be simply generalized to other mammalian model systems, such as non-human primates. Based on the presented results, the comparison between the mouse and primate visual cortex is not as straightforward as previously assumed. This will likely trigger more comparative studies between mice and primates in the future, which is important and absolutely needed to advance our understanding of the mammalian brain.

      Moreover, the reported finding that neurons in the primary visual cortex of marmosets do not increase their activity during running is intriguing, as it makes you wonder why neurons in the mouse visual cortex do so. The authors discuss a few ideas in the paper which can be addressed in future experiments. In this regard, it is worth noting that the authors report an interesting difference between the foveal and peripheral parts of the visual cortex in marmoset. It will be interesting to investigate these differences in more detail in future studies. Likewise, while running might be an important behavioral state for mice, other behavioral states might be more relevant for marmosets and do modulate the activity of the primate visual cortex more profoundly. Future work could leverage the opportunities that the marmoset model system offers to reveal new insights about behavioral-related modulation in the primate brain.

    1. Reviewer #2 (Public Review):

      This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however, the biological insight gained is rather limited.

    2. Reviewer #3 (Public Review):

      Complex behavior requires complex neural control involving multiple brain regions. The currently available tools to measure neural activity in multiple brain regions in small animals are limited and often involve obligatory head-fixation. The latter, obviously, impacts the behaviors under study. Hur and colleagues present a novel recording device, the E-Scope, that combines optical imaging of fluorescent calcium imaging in one brain region with high-density electrodes in another. Importantly, the E-Scope can be implanted and is, therefore, compatible with usage in freely moving mice. The authors used their new E-Scope to study neural activity during social interactions in mice. They demonstrate the presence of neural correlates of social interaction that happen simultaneously in the cerebellum and the anterior cingulate cortex.

      The major accomplishment of this study is the development and introduction of the E-Scope. The evaluation of this part can be short: it works, so the authors succeeded.

      The authors managed to reduce the weight of the implant to 4.5 g, which is - given all functionality - quite an accomplishment in my view. However, a mouse weighs between 20 and 40 g, so that an implant of 4.5 g is still quite considerable. It can be expected that this has an impact on the behavior and, possibly, the well-being of the animals. Whether this is the case or not, is not really addressed in this study. The authors suffice with the statement that "Recorded animals made more contact with the other mouse than with the object (Figure 2A), suggesting a normal preference for social contact with the E-Scope attached."

      Overall, the description of animal behavior is rather sparse. The methods state only that stranger age-matched mice were used, but do not state their gender. The nature of the social interactions was not described? Was their aggressive behavior, sexual approach and/or intercourse? Did the stranger mice attack/damage the E-Scope? Were the interactions comparable (using which parameters?) with and without E-Scope attached? It is not even described what the authors define as an "interaction bout" (Figure 2A). The number of interaction bouts is counted per 7 minutes, I presume? This is not specified explicitly.

      In Figure 1 D-G, the authors present raw data from the neurophysiological recordings. In panel D, we see events with vastly different amplitudes. It would be very insightful if the authors would describe which events they considered to be action potentials, and which not. Similarly, the raw traces of Figure 1E are declared to be single-unit recordings of Purkinje cells. Partially due to the small size of the traces (invisible in print and pixelated in the digital version), I have a hard time recognizing complex spikes and simple spikes in these traces. This is a bit worrisome, as the authors declare the typical duration of the pause in simple spike firing after a complex spike to be 20-100 ms. In my experience, such long pauses are rare in this region, and definitely not typical. In the right panel of Figure 1A, an example of a complex spike-induced pause is shown. This pause is around 15 ms, so not typical according to the text, and starts only around 4 ms after the complex spike, which should not be the case and suggests either a misalignment of the figure or the detection of complex spike spikelets as simple spikes, while the abnormally long pause suggests that the authors fail to detect a lot of simple spikes. The authors could provide more confidence in their data by including more raw data, making explicit how they analyzed the signals, and by reporting basic statistics of firing properties (like rate, cv or cv2, pause duration). In this respect, Figure 2 - figure supplement 3 shows quite a large percentage of cells to have either a very low or a very high firing rate.

      The number of Purkinje cells recorded during social interactions is quite low: only 11 cells showed a modulation in their spiking activity (unclear whether in complex spikes, simple spikes or both. During object interaction, only 4 cells showed a significant modulation. Unclear is whether the latter 4 are a subset of the former 11, or whether "social cells" and "object cells" are different categories. Having so few cells, and with these having different types of modulation, the group of cells for each type of modulation is really small, going down to 2 cells/group. It is doubtful whether meaningful interpretation is possible here.

      This brings us to the next point: neural correlates of social interaction are notoriously difficult to interpret. Social behavior is complex, and involves the processing of sensory cues (olfaction, touch (whiskers), visual and auditory), the production of ultrasonic vocalizations (in specific contexts), movements, and emotional behavior (fear, pleasure, sexual interest). In other words, neural activity patterns observed during social interaction do not necessarily relate specifically to social interaction, but can also occur in a non-social context. The authors control this by comparing social interactions with object interactions, but I miss a direct comparison between the two conditions, both in terms of behavior (now only the number of interactions is counted, not their duration or intensity), and in terms of neural activity. There is some analysis done on the interaction between movement and cerebellar activity (Figure 2 - figure supplement 4), but it is unclear to what extent social interactions and movements are separated here. It would already help to indicate in the plots with trajectories (e.g., Fig. 2H) indicate the social interactions (e.g., social interaction-related movements in red, the rest of the trajectories in black).

      The neuron count in the anterior cingulate cortex is much higher than for the cerebellum, but also here it is not so clear what is "social" and what is "non-social". In Figure 3G-H, the authors indicate a near-perfect separation between cells active during social encounters and those active during object encounters. This could indicate that there is here indeed a social aspect, but as we do not know to what extent the sensory and motor aspects differ between social and non-social interactions, this is still hard to interpret.

      Finally, the authors show that there are correlations between the modulation in neurons of the anterior cingulate cortex and cerebellar neurons related to bouts of social activity. Here, it could be interesting to see whether there are differences in latency between the two brain areas.

      In conclusion, the authors present a novel method to record neural activity with single cell-resolution in two brain regions in freely moving mice. Given the challenges associated with understanding of complex behaviors, this approach can be useful for many neuroscientists. The authors demonstrate the potential of their approach by studying social interactions in mice. Clearly, there are correlations in the activity of neurons in the anterior cingulate cortex and the cerebellum related to social interactions. To bring our understanding of these patterns to a higher level, more detailed analyses (and probably also larger group sizes of cerebellar neurons) are required, though.

    1. As I continue the balancing act between the loaded hot dog and pop back to my seat, a cheering fan bumps into my pop hand. The pop splashes out of the cup and all over my shirt, leaving me drenched. I make direct eye contact with the man who bumped into me and he looks me in the eye, looks at my shirt, tells me how sorry he is, and then I just shake my head and keep walking. “It’s all just part of the experience,” I tell myself. Before I am able to get back to my seat, I hear the crack of a bat, followed by an uproar from the crowd. Everyone is standing, clapping, and cheering. I missed a home run.

      The spilled pop, and the missed homerun don't seen to bother the author. the mood remains unbroken

    1. List as many ways you think you can realistically improve, change, (or start over…) your study area. Remember, you might not have the advantage of a whole room, or even a corner of a room, but there are still some changes you can make to create a more effective study environment.

      A few ways I could realistically change and improve on my study are a would be: find a time when nobody is home, buy some kind of desk, buy some noise cancellation head phone, play music, and ask others to be more quite.

    Tags

    Annotators

    1. But since Covid-19, I’ve watched people around me – friends, family and perfect strangers my own age whose stories are told in obituaries – drop dead from this contagion. A sharp sense of existential dread has taken up residence in my psyche. That vague inevitability that I assumed would happen in the distant future smashed me over the head like an anvil in an old cartoon. I could easily die sooner than later. My mortality was, for the first time, in center focus.
      • due to death of so many young people, covid has shifted mortality salience into center focus for many young people
    1. ```python from flask import Flask, request from collections import defaultdict import re import random

      GREEN ="🟩" YELLOW ="🟨" WHITE ="⬜"

      def get_answers(): with open("allowed_answers.txt") as f: answers = set(l for l in f.read().splitlines() if l) return answers

      def get_guesses(): guesses = get_answers() with open("allowed_guesses.txt") as f: for l in f.read().splitlines(): if l: guesses.add(l) return guesses

      app = Flask(name, static_folder="static") app.answers = get_answers() app.guesses = get_guesses() word = random.choice(list(app.answers)) print(f"The word is {word}")

      def with_header(content): return f"""

      <html> <head> <link rel="search" type="application/opensearchdescription+xml" title="searchGame" href="http://searchgame:5000/static/opensearch.xml" /> </head> <body> {content} </body></html>

      """

      @app.route("/") def home(): return with_header("

      Right click on the address bar to install the search engine.

      ")

      @app.route("/search") def search(): return with_header(f"Content: {request.args.get('q')}")

      def to_result(guess, answer): chars = [WHITE] * 5 count = defaultdict(int) for idx, (g, a) in enumerate(zip(guess, answer)): if g == a: chars[idx] = GREEN else: count[a] += 1

      for idx, g in enumerate(guess):
          if g in count and count[g] > 0 and chars[idx] == WHITE:
              chars[idx] = YELLOW
              count[g] -= 1
      
      return "".join(chars)
      

      def maybe_error(guess): if len(guess) < 5: return f"less than 5 characters" if len(guess) > 5: return f"greater than 5 characters" if guess not in app.guesses: return f"not in wordlist" return None

      @app.route("/game") def game(): query = request.args.get("q") guesses = [x for x in re.split("[. ]", query) if x] response = [] if not guesses: response.append("Enter 5-letter guesses separated by spaces") else: most_recent = guesses[-1] # Don't show "too short" error for most recent guess if len(most_recent) < 5: guesses = guesses[:-1] if not guesses: response.append("Enter a wordle guess") for guess in guesses[::-1]: error = maybe_error(guess) if error is None: result = to_result(guess, word) s = f"{guess} | {result}" if result == GREEN * 5: s = f"{s} | CORRECT!" response.append(s) else: response.append(f"{guess} | ERROR: {error}")

      return [query, response]
      

      ```

    1. Reviewer #2 (Public Review):

      The authors succeed in generalizing the pre-alignment procedure for their cell identification method to allow it to work effectively on data with only small subsets of cells labeled. They convincingly show that their extension accurately identifies head angle, based on finding auto fluorescent tissue and looking for a symmetric l/r axis. They demonstrate that the method works to identify known subsets of neurons with varying accuracy depending on the nature of underlying atlas data. Their approach should be a useful one for researchers wishing to identify subsets of head neurons in C. elegans, for example in whole brain recording, and the ideas might be useful elsewhere.

      The authors also strive to give some general insights on what makes a good atlas. It is interesting and valuable to see (at least for this specific set of neurons) that 5-10 ideal examples are sufficient. However, some critical details would help in understanding how far their insights generalize. I believe the set of neurons in each atlas version are matched to the known set of cells in the sparse neuronal marker, however this critical detail isn't explicitly stated anywhere I can see. In addition, it is stated that some neuron positions are missing in the neuropal data and replaced with the (single) position available from the open worm atlas. It should be stated how many neurons are missing and replaced in this way (providing weaker information). It also is not explicitly stated that the putative identities for the uncertain cells (designated with Greek letters) are used to sample the neuropal data. Large numbers of openworm single positions or if uncertain cells are misidentified forcing alignment against the positions of nearby but different cells would both handicap the neuropal atlas relative to the matched florescence atlas. This is an important question since sufficient performance from an ideal neuropal atlas (subsampled) would avoid the need for building custom atlases per strain.

    1. no we don't
      • Answer

        • No.
        • we end up with a non conceptual insight that:
          • we can then communicate
          • that we can discuss
          • that we can articulate
          • that requires that reason be present at:
            • the beginning like the seed
            • in the middle when we're performing the analysis
            • like the rain that nourishes the crops and
            • in the end in the harvest
          • because non conceptuality is really easy to achieve all you need is a very large rock,
            • just bang right on your head and non conceptuality is there
          • but that's a mute inert non-conceptual
          • Non-conceptuality needs to be enriched by the conceptual insight that allows you to actually make something of it
      • The Middle Way

        • using the conceptual to reach a deeper appreciation of the state of non-conceptuality,
        • in other words, using dualistic thought and language to reach insights about the nondual
      • Title
        • Madhyamaka: Jay Garfield
      • Description
        • Jay Garfield talks about why Nagarjuna's technique employts reason to undermine itself to achieve peace in a nonconceptual state.
          • He humorously points out how its easy to achieve nonconceptual states in many ways, such as a large rock to the head, but that kind of nonconceptual state is not really insightful for penetrating the deep philosophical questions we all have.
          • He clarifies why Nagarjuna's process is called the Middle Way,
            • it employs (conceptual) analysis to achieve wisdom of the nondual (nonconceptual) state
    1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      We thank the reviewers for their insights and comments on this manuscript. Specific responses to reviewer concerns are detailed below. We made a couple of significant changes based on the feedback. First, we performed more experiments to increase biologic replicates and then quantified image data for multiple figures. The new quantitative information added to Figure 3 fully supports our original conclusions about changes to the ONH in Hes-TKO mutants. The quantification of Atoh7, Otx2, Rbpms and Crx expressing cells among the different genotypes revealed interesting differences in Notch intracellular gene requirements for both RGC and cone development. The most startling outcome is that changes in both cell types correlate with significant changes in Otx2, but not Atoh7. This singular finding suggests interesting future work is needed, well beyond the scope of this paper about the molecular mechanisms underlying these cell fates. Second, our data presentation was reorganized with new information added to Fig 1 that clarifies the relationships between Hes1, Hes5, Foxg1 and Pax2; old Figs 6 & 7 about neurogenesis were merged; and some data moved to new Suppl Figs 2 and 5. The numbering for multiple figures changed and a new summary model (now Fig 8) is provided. In addition, the manuscript was completely rewritten to improve clarity. We hope this revised manuscript is acceptable for publication.

      Reviewer #1 Summary:

      In this study, the authors employed an impressive set of mouse mutant or Cre lines to investigate the complexity of Notch signaling across different stages of retinal development. These comprehensive analyses led to two main findings: 1. Sustained hes1 in the OHS/OS is Notch-independent; 2. Rbpj and Hes1 exhibited opposing roles in cone photoreceptor development. Although the study is potentially interesting, the current manuscript needs the essential research background and quantification, a lack of which significantly reduced the clarity of the manuscript and the credibility of the major conclusions. Also, how the authors organized the results is quite confusing, making the manuscript very difficult to follow.

      Response: We agree with all reviewers concerning incomplete quantification of the data. We directly addressed this shortcoming in revised Figs 3 and 6 (the latter combines old Figs 6 +7). To do this, we repeated some IHC experiments to add more replicates and reorganized all of the neurogenesis phenotypic data figures. Our quantifications uncovered several surprising outcomes that clarify our model. For these reasons, the manuscript was exhaustively rewritten. We merged E13 neurogenesis data into revised Figure 6 and moved the most relevant E16 analyses to new supplemental data Fig 5. All changes made should make the paper easier to understand for retinal development, neurogenesis, and Notch pathway aficionados, in addition to readers lacking such expertise.

      Major comments: 1. The authors needed to make the quantification for many analyses to strengthen the conclusions, such as Fig. 1F, 1G, and etc.

      Response: We quantified optic nerve head (ONL) immunohistochemistry data in the revised Fig 3. We also quantified neurogenesis markers Atoh7, Otx2, Rbpms (RGCs), and Crx at E13 in revised Fig 6 (former Figs 6 and 7). Older stages were moved to a new Suppl Fig 5.

      Respectfully, Hes5 mRNA expression in old Fig 1F and 1G shows that Hes5, like other retinal progenitor cell (RPC) markers, expanded in Rax-Cre deletion but not Chx10-Cre deletion conditions. This is analogous to Pax6 and Rax expansion in Rax-Cre;Hes1 CKO eyes and Pax2 mutants (doi: 10.1523/JNEUROSCI.2327-19.2020) (1). In revised Fig 1, we now show analogous expansion of Hes5 mRNA in Pax2 mutant retinas (compare Figs 1F-1I). Because Hes5 RNA in situ hybridization experiments are nonquantitative, we do not discuss the possibility of Hes5 mRNA level changes in labeled cells.

      The authors reported many exciting results. However, further mechanistic insights are largely missing. They may focus on one of these exciting findings and give some mechanistic insights. For example, hes1 suppresses hes5 expression as the ONH boundary forms; hes1 expression in the ONH is Notch independent; differential influences of Rbpj and Hes1 on cone development. It is better for the authors to select one of these exciting findings and provide a deeper mechanistic study.

      Response: This revision brings fresh focus to Notch regulation of RGC and photoreceptor development, particularly differential influences for Rbpj versus Hes1. We also better support our interpretation of image data in Fig 1. We include new data about the spatial relationships between Hes5-GFP/Pax2 and Hes5-GFP/Foxg1. In summary, we find that as Pax2 becomes restricted to the nasal optic cup prior to the onset of RGC genesis, it becomes mutually exclusive with Hes5-GFP, at the same time that Hes5-GFP+ cells coexpress Hes1. This is consistent with Hes1 indirectly regulating Hes5-GFP as a marker of neurogenic RPCs at the forming ONH. Furthermore, it emphasizes the importance of genetically teasing apart the separate and potentially compensatory roles for Hes1 versus Hes5 undertaken here. These relationships remain poorly resolved during vertebrate CNS development.

      Some analyses lack an explanation of the rationale. For example, "To understand if the loss of multiple Hes genes is more catastrophic than Hes1 alone..."(PAGE 7). Please explain its significance.

      Response: We assume the reviewer is referring to the first sentence of the last paragraph on this page. We analyzed Hes triple mutant mice (TKO) to understand if removing multiple Hes genes reveals redundant functions. This is an open question, given that Hes1 is expressed in the ONH/OS, which is normally devoid of Hes5 by the time retinal neurogenesis begins. These questions have only been explored in a handful of tissues throughout the body. Also see response to point 2 above. In general, we have expanded the rationale for all of the experiments throughout the revised manuscript.

      Significance: In general, many results are quite interesting. However, the significance of these findings is largely hampered in the following aspects: 1. The authors were unable to provide the sufficient research contexts that are essential for understanding many results.2. Many conclusions were solely based on descriptive images but lacked statistical quantification, which significantly weakened many conclusions. 3. Many interesting findings are quite descriptive, and some mechanistic understandings of one of these exciting findings will be beneficial to improve the focus and significance of the study. Current format of the manuscript fits more specialized audience.

      Response: During in vivo development, we wished to understand which particular Notch pathway genes can interact in a Notch-dependent versus a Notch-independent manner. Genetic (phenotypic) studies produce extremely rigorous datasets, in our opinion. This revision now extensively quantifies key findings. Here we dissected the "receipt" of a Notch signal by identically testing the functional requirements of particular pathway members. For Mastermind (Maml), there are 3 paralogues, double mutants for Maml1 and Maml3 are early lethal, and no floxed alleles exist, so it was logical to employ the ROSA-dnMaml mouse strain, particularly since it has been discussed throughout the Notch literature as "analogous" to removing either a Notch receptor or Rbpj. Our finding that the dnMAML allele does not function like a Rbpj null in the retina is important for researchers in the broad Notch field to consider when designing and interpreting experiments.

      Reviewer #2: Hes genes are effectors of the Notch signaling pathway but can also act down-stream of other signaling cascades. In this manuscript the authors attempt to address the complexity of Hes effectors during optic cup development and retinal neurogenesis. To do so, they compared optic cup patterning and retinal neurogenesis in seven germline or conditional mutant mouse embryos generated with two spatio-temporally distinct Cre drivers. These lines allowed for the analysis of the consequences of perturbing the Notch ternary complex and multiple Hes genes alone or in combination. The authors show that the optic disc/nerve head is regulated by Notch independent Hes1 function. They also confirm that perturbation of Notch signaling interferes with cell proliferation enhancing the production of differentiated ganglion cells, whereas photoreceptor genesis requires both Rbpj and Hes1 with Notch dependent and independent mechanisms. This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study.

      Response: As described in the response to Reviewer 1, we agree and present considerably more quantification data. We extensively reorganized and rewrote this manuscript to emphasize that Hes1 in the ONH/OS is fully Notch-independent and highlight branchpoints in Notch-dependent signaling, for Rbpj versus Hes,1 during early retinal neurogenesis. It is too simplistic that the ternary complex (Rbpj-NICD-Maml) simply activates Hes1 (and/or multiple Hes genes) to regulate downstream signaling targets. This paradigm has been portrayed in the literature numerous times for many processes throughout vertebrate development, homeostasis or relative to particular diseases. By focusing on one tissue and a narrow window of development, our phenotypic studies delved more deeply to show the greater complexity and molecular cross-talk that we think underlie the modulation of signaling levels with in vivo context. Thus, our results are of broad interest and impact to the greater Notch field.

      1. The title is somewhat misleading. The authors have explored mostly the role of Hes1, 3 and5. Although these are Notch effectors, there is already evidence that they participate in other pathways This is confirmed by the data present here. I would suggest to eliminate Notch from the title and use instead "Hes" to better reflect the findings. Furthermore, it is unclear why there is a reference to "mutations" or what are the Notch branchpoints to which the authors refer at the beginning of the discussion.

      Response: We appreciate the reviewer’s viewpoint but disagree this paper is mostly about Hes genes, as there is a critical direct, comparable evaluation with Rbpj and dn-Maml. Direct comparison of 7 genotypes highlights where each pathway member exhibits idiosyncratic phenotypes. We are striving for a clear, simple title about a very complex topic, involving the in vivo genetic dissection of a signaling pathway. We modified the title to: "Notch pathway mutations do not equivalently perturb mouse embryonic retinal development "

      1. "Although the Pax6-Pax2 boundary is intact in Rax-Cre;RbpjCKO/CKO eyes, ONH shape was attenuated compared to controls (Fig 3I)". This statement is arguable as the difference seems subtle. Perhaps some kind of quantification would help.

      Response: We quantified Pax2+ cells (ONH domain) using the adjacent proximal terminus of the retinal pigmented epithelium (RPE) to indicate a transition from ONH to optic stalk (OS). We also quantified the number of Pax2+Pax6+ double positive cells where the 2 domains abut (boundary cells). Some higher magnification examples are now provided in Fig 3H';3K';3N'. Grossly, the imaging data support that the Pax2+ ONH is expanded in Chx10-Cre;TKO eyes, while boundary cells are most affected in Rax-Cre;HesTKO eyes, due to an expansion of retinal tissue. This is supported by our quantitative data (Fig 3O,3P). We observed even in controls that Pax2-expressing cells show some numerical variability. We attributed this to the position of the section through the ONH, which is a 3-dimsenional ring (torus). Therefore, we quantified additional wild-type controls and mutant samples in the new Fig 3O,3P graphs, improving statistical power, and allowing us to detect quantitative differences.

      Page 12 first paragraph. "....but all other genotypes were unaffected". This statement is unclear. All lines in which the Rax-Cre has been used seem to have an increased number of apoptotic cells. This should be better explained

      Response: Respectfully, only one genotype, Rax-Cre;Rbpj mutants contain a statistically significant increase in apoptotic cells (Fig 5P). This is demonstrated by one-way ANOVA analyses that included all pairwise comparisons. To ensure that the quantification was not misleading due to changes in tissue morphology, data in Figs 5, 6, and 7 were normalized to optic cup area. The area was traced in FIJI, creating a polygon whose area was determined in square microns. For every section image, the marker+ cells were divided by the square micron area of the retina (excluding the opening for the optic nerve). Such a method is critical for comparison across this allelic series, given the morphologic changes, differences in cell clustering where rosettes form, and reduced proliferation whenever Notch signaling is lost or reduced.

      Page 12, end of second paragraph: "E13.5 Chx10-Cre;HesTKO eyes had a milder RGC phenotype (Figs 6G, 6N, 6U), but all other mutants were unaffected (Figs 6E, 6F, 6L, 6M, 6S, 6T). This statement is also rather subjective. The phenotype of Chx10-Cre;HesTKO is quite strong and the other mutants seem to have a phenotype. Some quantifications here will help.

      Response: We agree and provide quantification for both Atoh7 and Rbpms positive cells in the revised Figure 6. This is now in the same figure with quantification of Otx2+, Otx2+Atoh7+ and Crx+ cells. The reviewer is correct that both ROSA-dnMaml and both HesTKO mutants have a statistically significant increase in RGCs. Surprisingly, neither of the Rbpj CKO mutants have this outcome (Fig 6Y).

      1. Page 13, toward the bottom..."...but noted that Chx10-Cre RbpjCKO/CKO eyes were not different from controls (Figs 7E, 7AA)". Again, this statement is questionable as staining for both CRX and Rbpms seem reduced as compared to controls as quantifications in 7AA seems also to indicate (about half?). Did the authors calculate whether there is a statistical difference between controls and Chx10-Cre RbpjCKO/CKO ?

      Response: Rbpms+ RGCs and Crx+ photoreceptor precursors were colabeled and quantified on sections for all genotypes. All counts were normalized to area as described above. Upon quantification and ANOVA with pairwise comparisons, there was no statistical difference in Crx+ or Rbpms+ cells between control and Chx10-Cre;Rbpj mutants (new Fig 6Y and Z).

      In Fig 7CC the authors should make the effort of including at least one additional sample, 2 biological replicates seem insufficient to draw a conclusion.

      Response: The Rax-Cre;Hes1CKO/+ X Hes1CKO/CKO matings stopped producing litters in late 2022. While this manuscript was out for review, we obtained younger mice, from which new control and Rax-Cre; Hes1 mutant littermates were collected, stained, imaged and quantified. Upon adding samples, we found that the outcome was unchanged, but the data better support the lack of a statistical difference in rods between genotypes at E17. These data were moved to revised Suppl Fig 5.

      Significance: This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study.

      Response: To increase the impact of our manuscript, we quantified all markers except Tubb3, since its localization in cell bodies and axons make it impossible to assign to individual cells. We feel that this additional quantification strongly improves the quality of our findings and allowed us to make well-supported and novel conclusions. While we certainly believe that the retinal development community will find this paper of interest, it will also be of value to the broader Notch pathway scientific community. In this manuscript, we simultaneously compared phenotypes for Notch pathway genes in signal receiving cells. We could find essentially no studies like this for the mouse CNS and only a few from the Kopan lab about the kidney and immune system. Interestingly, one of us (NLB) is a coauthor on a recent paper about Notch signaling in the cortex, in which ROSA-dnMaml behaves analogously to Notch1CKO or RbpjCKO. This emphasizes that findings in one organ may not recapitulate the "rules" for this pathway for other cell types or tissues (doi: 10.1242/dev.201408)(2). Deeper understanding of how the Notch pathway in the retina functions, analogously or differently, is important. We feel our revised study advances when and where there are "branchpoints" in canonical signaling that may be overlooked in other developing tissues and organs.

      Reviewer #3: I have reviewed a manuscript submitted by Bosze et al., which is entitled "Not all Notch pathway mutations are equal in the embryonic mouse retina". The authors focused on Notch signaling pathway. Notch signaling is deeply conserved across vertebrate and invertebrate animal species: in general, two transmembrane proteins, Delta and Notch, interact as a ligand and a receptor, respectively, which induces proteolytic cleavage of Notch receptors to generate Notch intracellular domain (NICD). NICD is translocated into nucleus, then forms the transcription factor complex including Rbpj (also referred to as CBF1) and Mastermind-like (Maml), and activates the transcription of Hes family transcription factors. Three Hes proteins, Hes1, 3, and 5, are important for nervous system development. In the vertebrate developing retina, these Hes proteins inhibit neurogenesis to maintain a pool of neural progenitor cells. In addition to their primary role in neurogenesis, the authors recently reported that Hes1 promotes cone photoreceptor differentiation. In the later stages of development, Hes proteins also promote Müller glial differentiation. In addition, Hes1 is highly expressed in the boundary between the neural retina and optic stalk and required for this boundary maintenance. To understand precise regulation of Notch component-mediated signaling network for retinal neurogenesis and cell differentiation, the authors compared retinal phenotypes in the knockdown of three Notch pathway components, that is (1) Hes1/3/5 cTKO, (2) Rbpj KO, and (3) dominant-negative Maml (dnMaml) overexpression, under the control of two Cre derivers; Rax-Cre and Chx10-Cre. First, the authors found that Hes1 expression in the boundary between optic stalk and neural retina is lost in Rax-Cre; Hes1/3/5 cTKO, but still retained in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression, suggesting that Delta-Notch interaction is not required for Hes1 expression in the boundary between optic stalk and neural retina. Furthermore, Hes1 expressing boundary region expands distally at the expense of the neural retina in Chx10-Cre; Hes1/3/5 cTKO. Maintenance of ccd2 expression in this expanded boundary area suggests that Hes1 normally maintains a proliferative state in the optic stalk, which may allow these cells to differentiate into astrocyte in later stages. Second, in addition to precocious RGC differentiation in all the Notch component KO, the authors found that, as compared with wild-type, cone and rod photoreceptor genesis is highly enhanced in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression and mildly enhanced in Chx10-Cre; dnMaml overexpression. On the other hand, in Rax-Cre; Hes1/3/5 cTKO, cone and rod photoreceptor genesis is not enhanced but similar to wild-type level. Since the authors previously reported that cone genesis is reduced in Rax-Cre; Hes1 cKO and Chx10-Cre; Hes1 cKO, so Rax-Cre; Hes1/3/5 cTKO may rescue decrease in cone genesis in single Hes1 cKO. The authors raise the possibility that elevated Hes5 expression in single Hes1 cKO may suppress cone photoreceptor genesis. The authors also found that amacrine cell genesis is significantly suppressed in Rax-Cre; Rbpj KO but not changed in Rax-Cre; dnMaml overexpression and Rax-Cre; Hes1/3/5 cTKO, suggesting that Rbpj is specifically required for amacrine cell genesis. From these observations, the authors propose that there are at least two branchpoints for photoreceptor and amacrine cell genesis in Notch component-mediated signaling network. Their findings are very interesting and provide some new insight on how Notch signaling components are integrated into other signaling pathways and promote to generate diverse but well-balanced retinal cell-types during retinal neurogenesis and cell differentiation, in addition to conventional classic view of Notch signaling pathway. However, one weak point is that, although the authors figured out what kinds of phenotypic difference appear in the KO retinas between these Notch components, the research result is descriptive and less analytical. Most of their conclusions may be supported by their previous works or others; it is still hypothetical. So, it is important to show more analytical data to support their interpretation and more clearly show what is new conceptual advance for Notch signaling pathways.

      For example, sustained Hes1 expression in the boundary region between optic stalk and neural retina may be reminiscent to brain isthmus situation. I would like to request the authors to show more direct evidence that Hes1 regulation in optic stalk/retina boundary is independent of Delta-Notch interaction. One possible experiment is whether DAPT treatment phenocopies Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression (Hes1 in optic stalk boundary is normal?).

      Response: Usage of the gamma secretase inhibitor DAPT is an interesting experiment as it can phenocopy the loss of Notch signaling in developing tissues. However, the reviewer's proposed DAPT experiment is problematic for two major reasons. First, DAPT blocks the gamma secretase complex, which has more than 90 protein targets in the cell membrane (3). Therefore, DAPT may not be informative for Hes1 regulation given the myriad of expected off-target effects. Second, it would be difficult to treat embryos at the relevant stages with DAPT. Injections into pregnant mice are lethal and we cannot localize drug to the relevant area during in vivo development. Our direct phenotypic comparisons with two Cre drivers strongly indicate that Hes1 is independent of canonical Notch signaling in the developing optic stalk.

      We include an extra related data figure (Reviewer Fig 1) showing anti-Hes1 immunolabeling of E13.5 Rax-Cre;Notch1CKO/CKO (n=2) and E13.5 Rax-Cre;Notch2CKO/CKO eyes (n=3). The Notch1 mutant lost oscillating Hes1 expression in retinal progenitors, but the uniform Hes1 ONH domain remains. Interestingly, the Notch2 mutant had essentially no effect on Hes1 (oscillating or sustained), or Hes5 mRNA expression. A Notch2 RNA in situ hybridization demonstrates that Notch2 mRNA was lost in the E13 optic cup and RPE (Rax-Cre expressing tissues). These data emphasize: A) the Notch1-specific dependency of oscillating Hes1 expression in retinal progenitors is absent from the ONH; B) although coexpressed in the same tissue, Notch receptors have unequal activities.

      Does Rax-Cre; Rbpj KO; Hes1-cKO phenocopy Rax-Cre; Hes1-cKO (or Rax-Cre; Hes1/3/5 cTKO)?

      Response: This is a good question! The first author tried very hard to produce Rax-Cre; Rbpj CKO;Hes1 CKO double mutant embryos. However, these progeny could not be recovered from E10-E13 embryos, despite collecting more than 10 litters. Thus, it is likely that this genotype is lethal before eye formation.

      Could the authors identify an enhancer element that drives Hes1 transcription in optic stalk/retina boundary, which should be not overlapped with that of NICD/ Rbpj binding motif? Such additional evidence will make their conclusion more convincing.

      Response: Another interesting question. We have been working for >3 years on Hes1 cis regulatory enhancers, but the pandemic greatly delayed progress. The proximal Hes1 600bp upstream region is a generic enhancer that contains Hes1 binding sites for repressing its own expression (4) and has a pair of Rbpj consensus sites for Notch ternary complex activation of Hes1 expression (5,6). Nearby is a binding site occupied by Gli2 in the E16 mouse retina (7). Recently, it was shown that Ikzf4 binds slightly farther away (8). The upstream 1.8 kb region (including the 600bp just described) can drive destabilized GFP or dsRed reporters in early postnatal retinal explants (9). However, this sequence was used to make and analyze a classic Hes1-GFP transgenic reporter mouse, in which GFP was not expressed in the early embryonic mouse optic vesicle or cup (10). Therefore, any early eye-specific enhancer(s) are located farther upstream, in an intron, or downstream (or combination thereof). Public domain epigenetic and chromatin accessibility datasets support this idea. Identifying the gene regulatory logic for Hes1 expression in the eye will be an exciting future story, well beyond this manuscript. We are excited to use live imaging of enhancer reporters to discern oscillating versus sustained activity patterns during early ocular development.

      Regarding the conclusion on new branchpoints on photoreceptor and amacrine cell genesis, a model shown in Figure 9 is still hypothetical. Figure 9B indicate a model in which the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO is mediated by Hes1, which is presumed to be activated in Notch-independent signaling. However, Hes1 expression in the neural retina is markedly reduced in Rax-Cre; Rbpj KO (Fig. 2I), which does not fit in with the model.

      Response: We removed Fig 9B and now present new models about the Notch-dependent versus -independent roles for both Rbpj and Hes1. The new summary is Fig 8.

      So, I would like to request the authors to examine whether the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO, (or Rax-Cre; dnMaml overexpression and Chx10-Cre; dnMaml overexpression) is inhibited by Hes1 KO.

      Response: If we understand this correctly, it would mean generating double mutants, some of which we determined are not viable (see the response above, and Suppl Table 2). Given there is only a partial knockdown of Hes1 or Hes5 in either dnMaml mutant we do not believe repeating this in the Hes1 CKO genetic background to be informative and it would take 3 generations to perform.

      Second, the authors concluded that both cone and rod genesis are enhanced in Rax-Cre; Rbpj KO by showing the data on Crx/Nr2e3 labeling in Rax-Cre; Hes1 cKO in Fig. 7BB. However, as the authors mentioned in the manuscript, Hes5 expression is elevated in Rax-Cre; Hes1 cKO (Fig. 1G). So, since Rax-Cre; Hes1 cKO has residual Hes activity in the retina, Fig. 7BB should be replaced with labeling of Crx/Nr2e3 in Rax-Cre; Hes1/3/5 cTKO.

      Response: Unfortunately, Rax-Cre;HesTKO embryos do not live past E13 (Suppl Table 2). Thus, we cannot evaluate rods, whose genesis starts around E13.5. Revised Fig 1G shows the Hes5 domain is shifted with the expansion of retinal tissue in E13.5 Hes1 single mutants, but importantly, also analogously shifted in Pax2 mutants (Fig 1H). We do not conclude that mRNA levels are "elevated" since mRNA in situ hybridization is not a quantitative technique. Our initial examination of rods in E17 Rax-Cre;Hes1 CKO mutants tested the idea of a fate shift from cones to rods. However, deeper quantification (Suppl Fig 5) do not support such a fate change.

      Furthermore, possibly, it is best to examine labeling of the retinas of Rax-Cre; Rbpj KO with rod and cone-specific markers and confirm that the number of both rods and cones is significantly increased. Third, as for defects in amacrine cells genesis in Rax-Cre; Rbpj KO, I would like to request the authors to show the data on Crx10-Cre; Rbpj KO. Although Rbpj KO is mosaic in Crx10-Cre; Rbpj KO, we can distinct Rbpj KO cells by GFP expression (Fig. S2C, C', C'). So, the authors can confirm that amacrine cell genesis is inhibited in a cell-autonomous manner in Crx10-Cre; Rbpj KO retinas but not in Crx10-Cre; dnMaml overexpression. Addition of such data will make the authors' conclusion is more convincing.

      Response: Suppl Table 1 lists multiple references (two from the NLB lab) that demonstrated both a rod and cone increase in Rbpj loss-of-function conditions. Chx10;Rbpj CKO animals were evaluated by Zheng et al., who showed an amacrine loss phenotype in these mutants (11). This is equivalent to what we see in our Rax-Cre;Rbpj CKO data, but without the complications of Chx10 mosaic Cre expression upon Rbpj deletion.

      Other comments: 1) Title of this manuscript is "Not all Notch pathway mutations are equal in the embryonic mouse retina". However, this title is quite obscure in what is research advancement of their findings. I suggest the authors to include more concrete and conclusive sentence in the title, for example "Hes and Rbpj differentially promotes retina/optic stalk boundary maintenance and photoreceptor genesis, in parallel with neurogenic inhibition by Notch signaling pathway".

      Response: We appreciate the reviewer's perspective. We are striving for a relatively simple title about a very complex topic, involving the in vivo genetic dissection of a signaling pathway. We modified the title to "Notch pathway mutations do not equivalently perturb mouse embryonic retinal development ".

      2) The "Results" section is a bit difficult to follow logics without detailed knowledge on roles of Notch signaling in mouse retinal development. I suggest the authors to improve a writing style of "Results" section for readers without such detailed knowledge on mouse Notch mutant phenotypes to follow logical flow more easily. There are many additional descriptions on research background before start to mention results. Such introductory sentences should be moved to the "Introduction" section, by which logical flow in the Results section should be simpler. In addition, the authors should show a concrete question at the beginning of each result subsection. Furthermore, the authors sometimes jump over from one result subsection and suddenly move to cite another figure panel in a far ahead subsection whose data has not been explained. Such a back-and-forth citation of figure data generally makes it difficult to follow logical flow.

      Response: We now present a considerable amount of new quantified data, reorganized multiple figures, and extensively rewrote the paper. We significantly revised the summary figure to improve clarity. In addition, Suppl Table 1 provides a wealth of background information to orient the reader on this topic. We feel that this extensive revision has greatly improved the quality, logical flow, and readability of the manuscript.

      3) In addition, figure configuration is not well organized. Each figure compared some particular marker expression in wild-type, Rax-Cre; HesTKO, Rax-Cre; Rbpj cKO, Rax-Cre; dn-Maml-GFP, Chx10-Cre; HesTKO, Chx10-Cre; Rbpj cKO, Chx10-Cre; dn-Maml-GFP. For example, Fig. 2 shows Hes1 for inhibition of neurogenesis, Fig. 3 shows Vsx2; Mitf and Pax2; Pax6 for retinal pigmented epithelium and optic stalk, Fig. 6 shows Atoh7, Rbpms, and Tubb3 for retinal ganglion cells. Fig. 7 shows Crx, Otx2, and Thrb2 for photoreceptor differentiation. Fig. 8 shows Prdm1, and Ptf1a for photoreceptors and amacrine cells. Although this figure configuration is convenient to show phenotypic difference between different genetic mutations, it is difficult to know how each differentiation steps are spatially and temporally coordinated during development. At least, I recommend the authors to show one summary figure, which shows spatio-temporal expression profile of retinal markers in wild-type mouse retinas.

      Response: We recognize this point and completely reorganized and combined Figs 6 and 7 to improve clarity. New Figure 6 presents E13 quantification for Atoh7, Otx2, Atoh7/Otx2, Rbpms and Crx expressing retinal populations. E16-E17 data were condensed and moved to a new Suppl Fig 5.

      4a) Page 7, line 7-10 "With earlier deletion using Rax-Cre, hes5 mRNA abnormally extended into the optic stalk": I wonder how the authors define the optic stalk. It is likely that optic stalk area (Pax2+, Vax1+ area) is shifted to more proximal (depart from the optic cup and move toward the brain), and neural retina is expanded accordingly (Fig. 4B, 4F), resulting in expansion of hes5 expression. Thus, it may be better to mention that optic stalk/neural retina boundary is abnormally shifted toward the brain.

      Response: The retina, including the optic nerve head, ends where the adjacent RPE terminates. This is conspicuous morphologically in our sections. We also defined this by colabeling for Pax2 and Pax6, which is now quantified in revised Fig 3. To clarify this further, we added the words " in all panels the brain is to the right" in the Fig 4 legend.

      4b) Page 8, line 14-15, "ONH/OS cells still express it (Hes1), demonstrating that sustained Hes1 is independent of Notch": I presume that Cre-Rax drives Cre in neural retina as well as optic stalk and pigmented epithelium. However, it is likely that Rbpj is not expressed in optic stalk/neural retina boundary area in wild type (Fig. S2A). No expression of Rbpj in optic stalk/neural retina boundary may support that Hes1 expression in this boundary area is Notch-independent. However, Rbpj expression is retained in some vitreal cells near optic nerve head in Rax-Cre; Rbpj-CKO retinas (Fig. S2B). What are these Rbpj+ cells? I would like to request the authors to confirm that Rbpj expression is completely absent in both neural retina and optic stalk in Rax-Cre; Rbpj-CKO mice. Otherwise, this conclusion is still not fully supported.

      Response: We show the Rax-Cre lineage in Suppl Fig 2 via the Ai9 (tomato) reporter. The results are striking, with all of the optic cup derivatives (retina, RPE, ONH, optic stalk, and presumptive ciliary tissue and iris) being tomato positive, while the well-described population of vascular cells in the hyaloid space lack tomato expression. Furthermore, our figure shows that Rbpj expression is only absent from the optic cup derivates, rather than the vascular structures in the vitreous. Vascular cells also depend on the Notch pathway and express Rbpj. Based on considerable evidence from the literature and our lineage experiments, the population of cells the reviewer highlights represents the hyaloid vasculature and associated cell types. It does not represent any population that derives from neuroectoderm.

      4c) Page 9, line 16-18, "Foxg1 had spread into the nasal optic stalk": Is Foxg1 expanded nasal area really "OS" rather than expanded retina? I suggest the authors to confirm molecular markers Pax2 expression is overlapped with Foxg1. Otherwise, it is difficult to conclude that foxg1 is expanded into the optic stalk territory, because foxg1 is normally a marker of retina. Indeed, Fig. 3K shows pax2 expression is shifted into more inside towards the brain, suggesting that neural retina is expanded. Please explain the situation.

      Response: Foxg1 (BF-1) mRNA and protein are found in the nasal retina and are expressed in other brain tissues. Multiple studies show Foxg1 in the nasal side of the E10 optic cup/retina/optic stalk and developing hypothalamus (See extra data figure Reviewer Fig 2; top row figure is data from Smith et al., 2017 (12) with Foxg1 mRNA in purple. Also see our new manuscript panel Fig 1C. We include here for reviewers (extra data Reviewer Fig 2 showing E13 ocular cryosections colabeled for Foxg1 and Pax2, highlighting their relationship in the retina, optic stalk and adjacent forming hypothalamus. On page 9 the text now reads "At E13.5 Rax-Cre;HesTKO eyes, the Foxg1 nasal retinal domain was contiguous with the nasal optic stalk (Suppl Fig 4D). This is reminiscent of younger stages (Fig 1C), since normally at E13.5, Foxg1 in the nasal optic cup/retina is separated from expression in the ONH/OS (Suppl Fig 4A). Based on the expansion of Pax6, Vsx2 and Hes5 RPC domains into the optic stalk, we conclude that the change in Foxg1 similarly reflects an extension of retinal tissue."

      4d) Page 10, line 4-5, In Rax-Cre; Hes1/3/5 cTKO eye, this tissue (RPE) extended into the optic stalk": This description seems to be incorrect. A part of Pax2 area, which is adjacent to the neural retina, contacts with RPE in wild type (Fig. 3AH), so most of RPE covers the neural retina even in Fig. 3DK.

      Response: We disagree with the reviewer’s interpretation. Fig 3D shows Mitf labeling of RPE nuclei. Figure 3K shows the adjacent section labeled with Pax2 and Pax6 (labels both retina and RPE). As the retina extended "towards the brain", the RPE analogously extends and surrounds the retinal domain. We also added higher magnification data panels 3H, 3K and 3N, showing merged and single channels.

      4e) Page 10, line 22-23, "For Chk10-Cre; Hes1/3/5 cTKO, there was a unique presence of ectopic Pax2 within the retinal territories": I wonder if this description is correct. I suspect that proliferative Pax2+ cells expand into regressing territory of Hes KO retinal cells, which undergo precocious neurogenesis and lose proliferative activity, in Chk10-Cre; HesTKO. In this case, it is possible that the Pax2/Pax6 interface may be maintained. Please show red and green channel panels for Fig. 3N to confirm that there is ectopic pax2 and pax6 double positive cells.

      Response: New quantification in revised Fig 3 (see panels O,P) fully supports our original conclusion. Only Chx10-Cre;HesTKO mutants have a statistically significant increase in Pax2+ cells. There are not more Pax2+Pax6+ double labeled cells. Only this particular genotype has an increase in Pax2+ single labeled cells.

      5a) Page 11, line 20-25. There seems to be inconsistency between result description and image data of Fig. 5A-G, and histogram Fig. 5O. Authors mentioned that a modest loss of pH3+ cell fraction in Chx10-Cre; Hes1/3/5 cTKO but not in Rax-Cre; Hes1/3/5 cTKO. However, Fig. 5D indicates severe reduction of pH3+ cell fraction in Rax-Cre; Hes1/3/5/ cTKO, which is similar to reduction of pH3+ cell fraction in Rex-Cre; Rbpj (Fig. 5B), but histogram data is different (Fig. 5O). Furthermore, pH3+ cell fraction is severely reduced in Chx10-Cre; ROSA(dn-Maml-GFP) (Fig. 5F) and modestly reduced in Chx10-Cre; Hes1/3/5 cTKO (Fig. 5G). However, pH3+ cell fraction seems to be normal in Chx10-Cre; Rbpj (Fig. 5E). These Chx10-Cre image data do not match the histogram of Fig. 5O. Please check their situation.

      Response: Images in old Figs 5-8 were normalized using area measurements, see methods and above comments (note: old Figs 6&7 were combined into new Fig 6). One-way ANOVA with pairwise comparisons for each mutant genotype compared to control were calculated using Prism. All genotypes except two have a statistically significant loss of M phase cells and we discuss possibilities for this outcome (Fig 5O). A normalization method for the sampled area is an essential component of these studies since morphologic differences are apparent for particular genotypes. The quantitative data are consistent with our original conclusions.

      5b) Fig. 5H-N, P: I wonder if the stage E13 is appropriate to evaluate cell death and survival because optic cup already becomes smaller in Rax-Cre; Rbpj, Hes1/3/5 cTKO, or ROSA(dn-MAML-GFP) than in wild-type control. I suggest the authors examine more earlier stage.

      Response: While an earlier effect is possible, we only observed size differences in a subset of the genotypes. Thus, E13 serves as a critical timepoint to examine early developmental phenotypes across the totality of our mutant conditions. It is also first age when the ONH is fully formed.

      5c) Page 12, line 19-20, "all other mutants (Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP) were unaffected (Fig. 6EF, LM, ST)": It is likely that atoh7 expressing cells are mildly decreased and neuronal marker, Tubb3 and Rbpms-expressing cells are increased in Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP). I requested the authors to evaluate the fraction of these markers in retinal area statistically in all the cases.

      Response: As described above, we quantified Atoh7 and Rbpms nuclear expression by immunohistochemistry. We do not believe that Tubb3+ cells can be reliably quantified. Nonetheless, it is useful to qualitatively show the extent of excess neuron formation. Importantly, we observed that it is not the Atoh7 status that matters for RGC formation, rather it is the Otx2 expression status. This is in good agreement with single cell-RNA transcriptomics data from Wu et al 2021 showing that Atoh7 mRNA in all early transitional RPCs remains fairly constant and its loss does not block the formation of early RGC cell states (13). By contrast Otx2 fluctuates but remains expressed in transitional RPCs that progress to photoreceptor lineages.

      6a) Page 7, line 19 "Ectopic blood vessels protruded from the ONH (Fig. 1K, 1L)": It is difficult to see blood vessel structures in these panels (Fig. 1I-L). Please show some molecular marker of blood vessels to confirm how blood vessel is organized in Hes1/3/5 cTKO.

      Response: These vascular structures are highly conspicuous by morphology in the H&E insets. Nonetheless, we used adjacent P21 sections to immunolabel for Endomuscin (14) and Tubb3 antibodies. This colabeling confirms the morphology and position of ectopic blood vessels in the abnormal tissue masses in Chx10-Cre;HesTKO mutant eyes. Ectopic tissue contains only rare Tubb3+ cells or cell processes suggesting it is overwhelmingly nonneural. All P21 data were moved to a new Suppl Fig 2. A full detailing of vascular phenotypes is beyond the scope of this manuscript and, interestingly, would be potentially attributable to non-autonomous effects of perturbing the Hes genes in the adjacent retina.

      6b) Fig. 5: Increase of pH3 fraction indicates several possibilities, for example (1) increased fraction of mitotic cells due to precocious neurogenesis, (2) increased fraction of mitotic cells due to activated cell proliferation of retinal progenitor cells, (3) increased cell-cycle arrest in M phase due to some stress response of progenitor cells. So, I suggest the authors to examine (1) BrdU percentage of retinal section area, (2) the percentage of pH3+ cells in PCNA+ retinal cells.

      Response: The data listed in Suppl Table 1 presents a unified picture that disrupting Notch signaling reduced proliferation. This paradigm extends to other model organisms (e.g., Drosophila, chick, frog, zebrafish and even to nonneural tissues). We included the phospho-histone H3 staining so readers would see how the six mutants evaluated in this study align with this paradigm, providing confidence for the novel findings in other figures. A full evaluation of cell cycle kinetics is interesting, but beyond the scope and focus of this manuscript.

      6c) Fig. 5: It is better that cell death fraction will be evaluated by TUNEL and labeling with anti-activated caspase 3 antibody.

      Response: We disagree. The DNA repair enzyme PARP is inactivated upon cleavage by activated caspase 3. There are currently ~3,600 citations that use it as a marker of apoptosis. PARP also has a separate and very specific role in maintaining the integrity of sperm DNA. This antibody works on all metazoans and is amenable to many tissue preparations and fixatives, making it easy to use, robust and quantifiable.

      7a) Please show red channel (Hes1) image in Fig1BC.

      Response: This was added to Revised Fig 1 (Fig 1A).

      7b) Fig. 1DH should be shown in neighbor. Fig. 1H should be assigned as Fig. 1E.

      Response: The new Fig 1 layout addresses this point.

      7c) Fig. S2D, F, H, J: Please show GFP green channel as well. Otherwise, it is difficult to see non-overlapping expression in optic stalk area.

      Response: In the revision, this is Suppl Fig 3. Chx-10-Cre is not expressed by ONH-OS cells (1). The green and fuchsia overlap (coexpression) in RPCs is white, we feel this is fairly clear. If needed, all readers can turn on and off the green channel in the final PDF version of this figure to compare GFP with Hes1 expression for those panels.

      7d) Fig. 9B: It is better to show Rax-Cre: Hes1/3/5 TKO rather than Rax-Cre: Hes1 cKO. 7e) Fig. 9B: Lettering "Rbpj mutant" should be revised as "Rax-Cre: Rbpj KO".

      Response: Fig 9B was removed so these terms are now irrelevant. Our models are presented in new Fig 8.

      Significance: The senior author of this manuscript, Dr. Nadean Brown, is an expert scientist who has investigate the role of Notch signaling pathway in vertebrate ocular tissue, including the neural retina and lens. In general, Notch signaling pathway consists of signaling stream from the interaction of Delta and Notch, Notch receptor activation by proteolytic cleavage, translocation of Notch intracellular domain (NICD) into nucleus, formation of transcription factor complex consisting of NICD/Rbpj/Maml, to the transcriptional activation of Notch target genes, Hes family transcription factors. Finally, Hes suppresses neurogenic program and maintain a pool of neural progenitor cells. Therefore, Notch is a key factor to regulate the balance between neurogenesis and progenitor proliferation. In this manuscript, the authors investigated retinal phenotypes in the knockout mice of different Notch signaling components, including Rbpj, Maml, and Hes. They found that functions of these three factors are not always equal in retinal cell differentiation; rather, they specifically regulate a particular step of retinal development. The authors propose the possibility that each of Notch signaling components may be modified by other signaling pathways and achieve some new roles beyond the conventional frame of classic Notch signaling pathway. In this point, this work has a potential to provide a new conceptual advance in the field of developmental and cell biology.

      We fully agree this work is a significant advance for the fields of developmental and cell biology. Our findings provide new information and stimulate fresh ideas for anyone working on signal transduction and signal integration.

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      14. Saint-Geniez et al., 2009 IOVS Vol 50: 311-21.
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      Referee #3

      Evidence, reproducibility and clarity

      I have reviewed a manuscript submitted by Bosze et al., which is entitled "Not all Notch pathway mutations are equal in the embryonic mouse retina". The authors focused on Notch signaling pathway. Notch signaling is deeply conserved across vertebrate and invertebrate animal species: in general, two transmembrane proteins, Delta and Notch, interact as a ligand and a receptor, respectively, which induces proteolytic cleavage of Notch receptors to generate Notch intracellular domain (NICD). NICD is translocated into nucleus, then forms the transcription factor complex including Rbpj (also referred to as CBF1) and Mastermind-like (Maml), and activates the transcription of Hes family transcription factors. Three Hes proteins, Hes1, 3, and 5, are important for nervous system development. In the vertebrate developing retina, these Hes proteins inhibit neurogenesis to maintain a pool of neural progenitor cells. In addition to their primary role in neurogenesis, the authors recently reported that Hes1 promotes cone photoreceptor differentiation. In the later stages of development, Hes proteins also promote Müller glial differentiation. In addition, Hes1 is highly expressed in the boundary between the neural retina and optic stalk and required for this boundary maintenance.

      To understand precise regulation of Notch component-mediated signaling network for retinal neurogenesis and cell differentiation, the authors compared retinal phenotypes in the knockdown of three Notch pathway components, that is (1) Hes1/3/5 cTKO, (2) Rbpj KO, and (3) dominant-negative Maml (dnMaml) overexpression, under the control of two Cre derivers; Rax-Cre and Chx10-Cre.

      First, the authors found that Hes1 expression in the boundary between optic stalk and neural retina is lost in Rax-Cre; Hes1/3/5 cTKO, but still retained in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression, suggesting that Delta-Notch interaction is not required for Hes1 expression in the boundary between optic stalk and neural retina. Furthermore, Hes1 expressing boundary region expands distally at the expense of the neural retina in Chx10-Cre; Hes1/3/5 cTKO. Maintenance of ccd2 expression in this expanded boundary area suggests that Hes1 normally maintains a proliferative state in the optic stalk, which may allow these cells to differentiate into astrocyte in later stages.

      Second, in addition to precocious RGC differentiation in all the Notch component KO, the authors found that, as compared with wild-type, cone and rod photoreceptor genesis is highly enhanced in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression and mildly enhanced in Chx10-Cre; dnMaml overexpression. On the other hand, in Rax-Cre; Hes1/3/5 cTKO, cone and rod photoreceptor genesis is not enhanced but similar to wild-type level. Since the authors previously reported that cone genesis is reduced in Rax-Cre; Hes1 cKO and Chx10-Cre; Hes1 cKO, so Rax-Cre; Hes1/3/5 cTKO may rescue decrease in cone genesis in single Hes1 cKO. The authors raise the possibility that elevated Hes5 expression in single Hes1 cKO may suppress cone photoreceptor genesis. The authors also found that amacrine cell genesis is significantly suppressed in Rax-Cre; Rbpj KO but not changed in Rax-Cre; dnMaml overexpression and Rax-Cre; Hes1/3/5 cTKO, suggesting that Rbpj is specifically required for amacrine cell genesis. From these observations, the authors propose that there are at least two branchpoints for photoreceptor and amacrine cell genesis in Notch component-mediated signaling network.

      Their findings are very interesting and provide some new insight on how Notch signaling components are integrated into other signaling pathways and promote to generate diverse but well-balanced retinal cell-types during retinal neurogenesis and cell differentiation, in addition to conventional classic view of Notch signaling pathway. However, one weak point is that, although the authors figured out what kinds of phenotypic difference appear in the KO retinas between these Notch components, the research result is descriptive and less analytical. Most of their conclusions may be supported by their previous works or others; it is still hypothetical. So, it is important to show more analytical data to support their interpretation and more clearly show what is new conceptual advance for Notch signaling pathways.

      For example, sustained Hes1 expression in the boundary region between optic stalk and neural retina may be reminiscent to brain isthmus situation. I would like to request the authors to show more direct evidence that Hes1 regulation in optic stalk/retina boundary is independent of Delta-Notch interaction. One possible experiment is whether DAPT treatment phenocopies Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression (Hes1 in optic stalk boundary is normal?). Does Rax-Cre; Rbpj KO; Hes1-cKO phenocopy Rax-Cre; Hes1-cKO (or Rax-Cre; Hes1/3/5 cTKO)? Could the authors identify an enhancer element that drives Hes1 transcription in optic stalk/retina boundary, which should be not overlapped with that of NICD/ Rbpj binding motif? Such additional evidence will make their conclusion more convincing.

      Regarding the conclusion on new branchpoints on photoreceptor and amacrine cell genesis, a model shown in Figure 9 is still hypothetical. Figure 9B indicate a model in which the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO is mediated by Hes1, which is presumed to be activated in Notch-independent signaling. However, Hes1 expression in the neural retina is markedly reduced in Rax-Cre; Rbpj KO (Fig. 2I), which does not fit in with the model. So, I would like to request the authors to examine whether the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO, (or Rax-Cre; dnMaml overexpression and Chx10-Cre; dnMaml overexpression) is inhibited by Hes1 KO. Second, the authors concluded that both cone and rod genesis are enhanced in Rax-Cre; Rbpj KO by showing the data on Crx/Nr2e3 labeling in Rax-Cre; Hes1 cKO in Fig. 7BB. However, as the authors mentioned in the manuscript, Hes5 expression is elevated in Rax-Cre; Hes1 cKO (Fig. 1G). So, since Rax-Cre; Hes1 cKO has residual Hes activity in the retina, Fig. 7BB should be replaced with labeling of Crx/Nr2e3 in Rax-Cre; Hes1/3/5 cTKO. Furthermore, possibly, it is best to examine labeling of the retinas of Rax-Cre; Rbpj KO with rod and cone-specific markers and confirm that the number of both rods and cones is significantly increased. Third, as for defects in amacrine cells genesis in Rax-Cre; Rbpj KO, I would like to request the authors to show the data on Crx10-Cre; Rbpj KO. Although Rbpj KO is mosaic in Crx10-Cre; Rbpj KO, we can distinct Rbpj KO cells by GFP expression (Fig. S2C, C', C'). So, the authors can confirm that amacrine cell genesis is inhibited in a cell-autonomous manner in Crx10-Cre; Rbpj KO retinas but not in Crx10-Cre; dnMaml overexpression. Addition of such data will make the authors' conclusion is more convincing.

      Other comments are shown below.

      1. Title of this manuscript is "Not all Notch pathway mutations are equal in the embryonic mouse retina". However, this title is quite obscure in what is research advancement of their findings. I suggest the authors to include more concrete and conclusive sentence in the title, for example "Hes and Rbpj differentially promotes retina/optic stalk boundary maintenance and photoreceptor genesis, in parallel with neurogenic inhibition by Notch signaling pathway".
      2. The "Results" section is a bit difficult to follow logics without detailed knowledge on roles of Notch signaling in mouse retinal development. I suggest the authors to improve a writing style of "Results" section for readers without such detailed knowledge on mouse Notch mutant phenotypes to follow logical flow more easily. There are many additional descriptions on research background before start to mention results. Such introductory sentences should be moved to the "Introduction" section, by which logical flow in the Results section should be simpler. In addition, the authors should show a concrete question at the beginning of each result subsection. Furthermore, the authors sometimes jump over from one result subsection and suddenly move to cite another figure panel in a far ahead subsection whose data has not been explained. Such a back-and-forth citation of figure data generally makes it difficult to follow logical flow.
      3. In addition, figure configuration is not well organized. Each figure compared some particular marker expression in wild-type, Rax-Cre; HesTKO, Rax-Cre; Rbpj cKO, Rax-Cre; dn-Maml-GFP, Chx10-Cre; HesTKO, Chx10-Cre; Rbpj cKO, Chx10-Cre; dn-Maml-GFP. For example, Fig. 2 shows Hes1 for inhibition of neurogenesis, Fig. 3 shows Vsx2; Mitf and Pax2; Pax6 for retinal pigmented epithelium and optic stalk, Fig. 6 shows Atoh7, Rbpms, and Tubb3 for retinal ganglion cells. Fig. 7 shows Crx, Otx2, and Thrb2 for photoreceptor differentiation. Fig. 8 shows Prdm1, and Ptf1a for photoreceptors and amacrine cells. Although this figure configuration is convenient to show phenotypic difference between different genetic mutations, it is difficult to know how each differentiation steps are spatially and temporally coordinated during development. At least, I recommend the authors to show one summary figure, which shows spatio-temporal expression profile of retinal markers in wild-type mouse retinas.
      4. There are several logically incorrect sentences or inconsistent sentences in the results section. Please respond my comment below.
        • a) Page 7, line 7-10 "With earlier deletion using Rax-Cre, hes5 mRNA abnormally extended into the optic stalk": I wonder how the authors define the optic stalk. It is likely that optic stalk area (Pax2+, Vax1+ area) is shifted to more proximal (depart from the optic cup and move toward the brain), and neural retina is expanded accordingly (Fig. 4B, 4F), resulting in expansion of hes5 expression. Thus, it may be better to mention that optic stalk/neural retina boundary is abnormally shifted toward the brain.
        • b) Page 8, line 14-15, "ONH/OS cells still express it (Hes1), demonstrating that sustained Hes1 is independent of Notch": I presume that Cre-Rax drives Cre in neural retina as well as optic stalk and pigmented epithelium. However, it is likely that Rbpj is not expressed in optic stalk/neural retina boundary area in wild type (Fig. S2A). No expression of Rbpj in optic stalk/neural retina boundary may support that Hes1 expression in this boundary area is Notch-independent. However, Rbpj expression is retained in some vitrial cells near optic nerve head in Rax-Cre; Rbpj-CKO retinas (Fig. S2B). What are these Rbpj+ cells? I would like to request the authors to confirm that Rbpj expression is completely absent in both neural retina and optic stalk in Rax-Cre; Rbpj-CKO mice. Otherwise, this conclusion is still not fully supported.
        • c) Page 9, line 16-18, "Foxg1 had spread into the nasal optic stalk": Is Foxg1 expanded nasal area really "OS" rather than expanded retina? I suggest the authors to confirm molecular markers Pax2 expression is overlapped with Foxg1. Otherwise, it is difficult to conclude that foxg1 is expanded into the optic stalk territory, because foxg1 is normally a marker of retina. Indeed, Fig. 3K shows pax2 expression is shifted into more inside towards the brain, suggesting that neural retina is expanded. Please explain the situation.
        • d) Page 10, line 4-5, In Rax-Cre; Hes1/3/5 cTKO eye, this tissue (RPE) extended into the optic stalk": This description seems to be incorrect. A part of Pax2 area, which is adjacent to the neural retina, contacts with RPE in wild type (Fig. 3AH), so most of RPE covers the neural retina even in Fig. 3DK.
        • e) Page 10, line 22-23, "For Chk10-Cre; Hes1/3/5 cTKO, there was a unique presence of ectopic Pax2 within the retinal territories": I wonder if this description is correct. I suspect that proliferative Pax2+ cells expand into regressing territory of Hes KO retinal cells, which undergo precocious neurogenesis and lose proliferative activity, in Chk10-Cre; HesTKO. In this case, it is possible that the Pax2/Pax6 interface may be maintained. Please show red and green channel panels for Fig. 3N to confirm that there is ectopic pax2 and pax6 double positive cells.
      5. There seems to be some mismatch descriptions between image data and histogram (or text in the result section). Please respond my comments below.
        • a) Page 11, line 20-25. There seems to be inconsistency between result description and image data of Fig. 5A-G, and histogram Fig. 5O. Authors mentioned that a modest loss of pH3+ cell fraction in Chx10-Cre; Hes1/3/5 cTKO but not in Rax-Cre; Hes1/3/5 cTKO. However, Fig. 5D indicates severe reduction of pH3+ cell fraction in Rax-Cre; Hes1/3/5/ cTKO, which is similar to reduction of pH3+ cell fraction in Rex-Cre; Rbpj (Fig. 5B), but histogram data is different (Fig. 5O). Furthermore, pH3+ cell fraction is severely reduced in Chx10-Cre; ROSA(dn-Maml-GFP) (Fig. 5F) and modestly reduced in Chx10-Cre; Hes1/3/5 cTKO (Fig. 5G). However, pH3+ cell fraction seems to be normal in Chx10-Cre; Rbpj (Fig. 5E). These Chx10-Cre image data do not match the histogram of Fig. 5O. Please check their situation.
        • b) Fig. 5H-N, P: I wonder if the stage E13 is appropriate to evaluate cell death and survival because optic cup already becomes smaller in Rax-Cre; Rbpj, Hes1/3/5 cTKO, or ROSA(dn-MAML-GFP) than in wild-type control. I suggest the authors examine more earlier stage.
        • c) Page 12, line 19-20, "all other mutants (Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP) were unaffected (Fig. 6EF, LM, ST)": It is likely that atoh7 expressing cells are mildly decreased and neuronal marker, Tubb3 and Rbpms-expressing cells are increased in Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP). I requested the authors to evaluate the fraction of these markers in retinal area statistically in all the cases.
      6. Some experiments are necessary to improve their design. Please respond my comments below.
        • a) Page 7, line 19 "Ectopic blood vessels protruded from the ONH (Fig. 1K, 1L)": It is difficult to see blood vessel structures in these panels (Fig. 1I-L). Please show some molecular marker of blood vessels to confirm how blood vessel is organized in Hes1/3/5 cTKO.
        • b) Fig. 5: Increase of pH3 fraction indicates several possibilities, for example (1) increased fraction of mitotic cells due to precocious neurogenesis, (2) increased fraction of mitotic cells due to activated cell proliferation of retinal progenitor cells, (3) increased cell-cycle arrest in M phase due to some stress response of progenitor cells. So, I suggest the authors to examine (1) BrdU percentage of retinal section area, (2) the percentage of pH3+ cells in PCNA+ retinal cells.
        • c) Fig. 5: It is better that cell death fraction will be evaluated by TUNEL and labeling with anti-activated caspase 3 antibody.
      7. Panel configuration of Figures should be revised as below.
        • a) Please show red channel (Hes1) image in Fig1BC.
        • b) Fig. 1DH should be shown in neighbor. Fig. 1H should be assigned as Fig. 1E.
        • c) Fig. S2D, F, H, J: Please show GFP green channel as well. Otherwise, it is difficult to see non-overlapping expression in optic stalk area.
        • d) Fig. 9B: It is better to show Rax-Cre: Hes1/3/5 TKO rather than Rax-Cre: Hes1 cKO.
        • e) Fig. 9B: Lettering "Rbpj mutant" should be revised as "Rax-Cre: Rbpj KO".

      Significance

      The senior author of this manuscript, Dr. Nadean Brown, is an expert scientist who has investigate the role of Notch signaling pathway in vertebrate ocular tissue, including the neural retina and lens. In general, Notch signaling pathway consists of signaling stream from the interaction of Delta and Notch, Notch receptor activation by proteolytic cleavage, translocation of Notch intracellular domain (NICD) into nucleus, formation of transcription factor complex consisting of NICD/Rbpj/Maml, to the transcriptional activation of Notch target genes, Hes family transcription factors. Finally, Hes suppresses neurogenic program and maintain a pool of neural progenitor cells. Therefore, Notch is a key factor to regulate the balance between neurogenesis and progenitor proliferation. In this manuscript, the authors investigated retinal phenotypes in the knockout mice of different Notch signaling components, including Rbpj, Maml, and Hes. They found that functions of these three factors are not always equal in retinal cell differentiation; rather, they specifically regulate a particular step of retinal development. The authors propose the possibility that each of Notch signaling components may be modified by other signaling pathways and achieve some new roles beyond the conventional frame of classic Notch signaling pathway. In this point, this work has a potential to provide a new conceptual advance in the field of developmental and cell biology.

    3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      Hes genes are effectors of the Notch signaling pathway but can also act down-stream of other signaling cascades. In this manuscript the authors attempt to address the complexity of Hes effectors during optic cup development and retinal neurogenesis. To do so, they compared optic cup patterning and retinal neurogenesis in seven germline or conditional mutant mouse embryos generated with two spatio-temporally distinct Cre drivers. These lines allowed for the analysis of the consequences of perturbing the Notch ternary complex and multiple Hes genes alone or in combination. The authors show that the optic disc/nerve head is regulated by Notch independent Hes1 function. They also confirm that perturbation of Notch signaling interferes with cell proliferation enhancing the production of differentiated ganglion cells, whereas photoreceptor genesis requires both Rbpj and Hes1 with Notch dependent and independent mechanisms.

      This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study. Specific comments are listed below:

      1. The title is somewhat misleading. The authors have explored mostly the role of Hes1, 3 and5. Although these are Notch effectors, there is already evidence that they participate in other pathways This is confirmed by the data present here. I would suggest to eliminate Notch from the title and use instead "Hes" to better reflect the findings. Furthermore, it is unclear why there is a reference to "mutations" or what are the Notch branchpoints to which the authors refer at the beginning of the discussion.
      2. "Although the Pax6-Pax2 boundary is intact in Rax-Cre;RbpjCKO/CKO eyes, ONH shape was attenuated compared to controls (Fig 3I)". This statement is arguable as the difference seems subtle. Perhaps some kind of quantification would help.
      3. Page 12 first paragraph. "....but all other genotypes were unaffected". This statement is unclear. All lines in which the Rax-cre has been used seem to have an increased number of apoptotic cells. This should be better explained
      4. Page 12, end of second paragraph: "E13.5 Chx10-Cre;HesTKO eyes had a milder RGC phenotype (Figs 6G, 6N, 6U), but all other mutants were unaffected (Figs 6E, 6F, 6L, 6M, 6S, 6T). This statement is also rather subjective. The phenotype of Chx10-Cre;HesTKO is quite strong and the other mutants seem to have a phenotype. Some quantifications here will help.
      5. Page 13, toward the bottom..."...but noted that Chx10-Cre RbpjCKO/CKO eyes were not different from controls (Figs 7E, 7AA)". Again, this statement is questionable as staining for both CRX and Rbpms seem reduced as compared to controls as quantifications in 7AA seems also to indicate (about half?). Did the authors calculate whether there is a statistical difference between controls and Chx10-Cre RbpjCKO/CKO ?
      6. In Fig 7CC the authors should make the effort of including at least one additional sample, 2 biological replicates seem insufficient to draw a conclusion.

      Significance

      This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study.

    1. Reviewer #2 (Public Review):

      With a much higher spatiotemporal resolution of ground dynamics than any previous study, the authors uncover new "rules" of locomotory motor sequences during peristalsis and turning behaviors. These new motor sequences will interest the broad neuroscience community that is interested in the mechanisms of locomotion in this highly tractable model. The authors uncover new and intricate patterns of denticle movements and planting that seem to solve the problem of net motion under conditions of force-balance. Simply put, the denticulated "feet" or tail of the Drosophila larva are able to form transient and dynamic anchors that allow other movements to occur.

      The biology and dynamics are well-described. The physics is elementary and becomes distracting when occasionally overblown. For example, one doesn't need to invoke Newton's third law, per se, to understand why anchors are needed so that peristalsis can generate forward displacements. This is intuitively obvious. Another distracting allusion to "physics" is correlating deformation areas with displaced volume, finding that "volume is a consequence of mass in a 2nd order polynomial relationship". I have no idea what this "physics" means or what relevance this relationship has to the biology of locomotion.

      The ERISM and WARP methods are state-of-the-art, but aside from generally estimating force magnitudes, the detailed force maps are not used. The most important new information is the highly accurate and detailed maps of displacement itself, not their estimates of applied force using finite element calculations. In fact, comparing displacements to stress maps, they are pretty similar (e.g., Fig 4), suggesting that all experiments are performed in a largely linear regime. It should also be noted that the stress maps are assumed to be normal stresses (perpendicular to the plane), not the horizontal stresses that are the ones that actually balance forces in the plane of animal locomotion.

      But none of this matters. The real achievements are the new locomotory dynamics uncovered with these amazing displacement measurements. I'm only asking the authors to be precise and down-to-earth about the nature of their measurements.

      It would be good to highlight the strength of the paper -- the discovery of new locomotion dynamics with high-resolution microscopy -- by describing it in simple qualitative language. One key discovery is the broad but shallow anchoring of the posterior body when the anterior body undertakes a "head sweep". Another discovery is the tripod indentation at the tail at the beginning of peristalsis cycles.

      As far as I know, these anchoring behaviors are new. It is intuitively obvious that anchoring has to occur, but this paper describes the detailed dynamics of anchoring for the first time. Anchoring behavior now has to be included in the motor sequence for Drosophila larva locomotion in any comprehensive biomechanical or neural model.

    1. edid nothaveashared under-standingofterms,nordidIgiveanythoughttowhatmypartnersmightneedor feel.

      This really resonates with me because I do things like this all of the time. I work out all the details in my head, and I get frustrated when I do not get the response I anticipated. This, then, leads to an unproductive use of the time. My goal this year will be to take the time to get to know what every person might be thinking or feeling, and allow for true dialogue and collaboration to happen.

    1. We prioritize what we see versus what we hear, why is that? Now, what comes to mind when I say that is when, somebody is saying no, but shaking their head yes. And so we have this disconnect, but we tend to prioritize what the action and not what we're hearing. So something that we visually see instead of what we hear.Speaker 1There isn't a definitive answer on that, but one source of insight on why do we do that, it could be related to the neurological real estate that's taken up by our visual experience. There's far more of our cortex, the outer layer of our brain that responds to visual information than any other form of information

      (13:36) Perhaps this is also why visual information is so useful for learning and cognition (see GRINDE)... Maybe the visual medium should be used more in instruction instead of primarily auditory lectures (do take into account redundancy and other medium effects from CLT though)

    1. Flash-ball guns are designed to be non-lethal riot control weapons that do not penetrate the skin.Their use by police in France is disputed as the projectiles have led to the loss of eyes, head injuries and other trauma.

      Any alternatives to crowd control? Is there any way to decrease that it has caused? - shoot rubber bullets on ground first

    1. Hale: Why, it is all simple. I come to do the Devil’s work. I come to counsel Christians they should belie themselves. His sarcasm collapses. There is blood on my head! Can you not see the blood on my head!!

      Why has Hale now "come to do the Devil's work" vs how he first came to Salem to "find him [the Devil] out" (Act 1) and "crush him utterly" (Act 1)?

    1. Reviewer #2 (Public Review):

      Kimchi et al. examined the role of cholinergic inputs to the amygdala in regulating reward-seeking behavior. To investigate this, the authors developed a head-fixed behavioral task where animals were trained to lick at random intervals, with some of these responses being reinforced ("windows of opportunity") as opposed to control epochs when no reward was delivered.

      The authors conducted in vivo optogenetic stimulation of basal forebrain cholinergic neurons and discovered that a 2-second optical stimulation of these neurons encouraged licking behavior when followed by reward delivery. This was in comparison to time epochs where no reward was delivered or compared to control mice only expressing EYFP. However, it remained unclear how many trials were required for this effect to manifest.

      Furthermore, they demonstrated that the stimulation of basal forebrain cholinergic neurons did not induce real-time place preference or affect locomotion. The reward-driven licking behavior was also mitigated by systemic cholinergic receptor antagonists.<br /> Next, the authors observed the bulk calcium dynamics from these neurons in a version of the task where an auditory cue predicted reward availability. They found strong calcium signals when mice were licking and when the tone was present, but also reported signals when mice were spontaneously licking.

      By injecting a genetically encoded Acetylcholine (Ach) sensor directly into the Basolateral Amygdala (BLA), they showed that Ach signals were present when mice were engaged in licking, both during reward availability and for non-rewarded licks. Photostimulation of Ach terminals directly in the BLA increased licking behavior when a reward was available.

      Finally, using in vivo and ex vivo physiology, they demonstrated that Ach signaling influences the electrophysiological dynamics in the BLA. This may help clarify some of the postsynaptic responses triggered by this neuromodulator.

      Strengths of the paper:

      1. The experiments were well-executed and sufficiently powered, with most statistics being correctly reported.<br /> 2. The paper is a technical tour de force, employing fiber photometry, in vivo and ex vivo electrophysiology, optogenetics, and behavioral approaches.<br /> 3. Robust effects were observed in most of the experiments.<br /> Weaknesses:<br /> 1. The experimental design varies slightly across each behavioral experiment, making it difficult to directly compare one effect to another.<br /> 2. The paper doesn't include data showing the precise location for the Ach recordings. As a result, it is unclear whether these signals are specific to the BLA, or whether they might also be coming from neighboring regions.

    2. Reviewer #3 (Public Review):

      This important manuscript investigates the role of basal forebrain cholinergic interneurons in conditioned responding by measuring the licking behaviour of head-fixed mice during photostimulation of the aforementioned neurons. Licking is found to increase only during windows when licking is rewarded, and similar behaviour is observed when terminals are stimulated in basolateral amygdala, then several more experiments are conducted to determine the behavioural and anatomical specificity of the effect. The findings are solid, particularly those relating to the recordings, although the interpretation of the behavioural findings is still somewhat unclear.

      Strengths<br /> • The manuscript is beautifully written and structured. I found it really easy to follow and felt that the authors did an exceptional job of walking me through each experiment that they completed, the rationale for it, and what they found.<br /> • The question of the function of basal forebrain cholinergics is an interesting one and a somewhat understudied question, so the study is timely and on an interesting topic.<br /> • The experiments are well-designed and the findings are novel. There are a number of important control experiments performed to determine that the observed effects were not due to locomotor activity and that stimulating basal forebrain ACh neurons is not inherently reinforcing.<br /> • The discussion is really nice - covering important topics such as potential interactions with dopamine, the potential anatomical specificity of the effects observed, and the possibility that projections other than those studied here might mediate effects, among other things.

      Weaknesses<br /> • Although very clearly written and set out, I found myself confused by the behavioural findings and their interpretation. Mainly this was because photostimulation only increased licking during the window of opportunity, which is not signalled by any discrete stimulus, which means that the only signal that the animal receives to determine that they are within the reward window is them receiving the reward. Therefore, the only time within this window that licking could be increased is post-reward (otherwise the reward window is identical to a non-rewarded window) and it is not clear to me what this increase in post-award licking might mean? In fact, this time post-award is actually the time the animal is most certain to not receive another reward for a few seconds, meaning that licking at this time is not a useful behaviour and therefore it is difficult to interpret what it means to artificially increase licking at this time. I think it would probably have been less confusing for the authors to study a paradigm in which animals develop a conditioned response that is unsignaled by discrete stimuli and then to inhibit basal forebrain ACh prior to that response.<br /> • I should also note that the authors state (Lines 249-251) that stimulation increases responding prior to reinforcer delivery, but I couldn't find evidence for this, and it seems counterintuitive to me that it would do so because then how would the animals discriminate the window of opportunity from a non-rewarded window? Perhaps I misunderstood something, but I found this confusing.<br /> • I do not think the behaviour in this task can be classed as operant - it is still a good task and still fine for detecting conditioned responding, but it cannot determine whether the responding is governed by a response-outcome association in the absence of a stimulus-outcome association (with stimuli being the licking spout, other facets of the behavioural context etc) through bidirectionality or omission, as would be required to demonstrate its operant nature.<br /> • I was confused by the pupil dilation data in Figure S4 as the authors seem to want to argue that this effect, although specific to the rewarded window as licking is, is independent of the licking behaviour as it develops more slowly than the behaviour (Lines 201-202). I was curious as to how the authors interpret these data then? Does it indicate that stimulating basal forebrain ACh interneurons does both things (i.e. increases arousal AND conditioned responding in the absence of discrete stimuli) but that the two things are independent of each other?<br /> • The authors refer to the dorsal medial prefrontal cortex in mice, which from the methods appears to be the prelimbic region. My understanding is that dmPFC has fallen out of favour for use in mice as it is not homologous to the same region in primates and can be confusing for this reason.

    1. Mary Warren, pointing at Proctor: You’re the Devil’s man!He is stopped in his tracks. Parris: Praise God! Girls: Praise God! Proctor, numbed: Mary, how –? Mary Warren: I’ll not hang with you! I love God, I love God. Danforth, to Mary: He bid you do the Devil’s work? Mary Warren, hysterically, indicating Proctor: He come at me by night and every day to sign, to sign, to - Danforth: Sign what?Parris: The Devil’s book? He come with a book? Mary Warren, hysterically, pointing at Proctor, fearful of him: My name, he want my name. “I’ll murder you,” he says, “if my wife hangs! We must go and overthrow the court,” he says! Danforth’s head jerks toward Proctor, shock and horror in his face.Proctor, turning, appealing to Hale: Mr. Hale! Mary Warren, her sobs beginning: He wake me every night, his eyes were like coals and his fingers claw my neck, and I sign, I sign...Hale: Excellency, this child’s gone wild!Proctor, as Danforth’s wide eyes pour on him: Mary, Mary! Mary Warren, screaming at him: No, I love God; I go your way no more. I love God, I bless God. Sobbing, she rushes to Abigail. Abby, Abby, I’ll never hurt you more! They all watch, as Abigail, out of her infinite charity, reaches out and draws the sobbing Mary to her, and then looks up to Danforth.

      Mary finally runs to Abigail and says that Proctor is allied with the devil. The vicious cycle of forced confession once again gets another character to turn against another and make a false confession and accusation.

    1. Tere, armas sõber Mart!Aitäh kirja eest! Sinu kirjad on alati väga huvitavad. Tore, et Kaarupison laupäeviti kino ja rahvamajas on iga nädal peod. Siin Tallinnas on kaväga huvitav, aga ma igatsen nüüd Kaarupisse, koju. Tulen Tallinnastkoju tagasi alles augustis.Täna läheme tädiga Ruudile külla. Ruudi elab Nõmmel, tema perekon-nal on seal suur maja ja väga ilus aed. Tal on ka kaks kassi ja koer. Mullemeeldivad kassid ja koerad. Ootan juba väga! Ruudi teeb meile õhtu-sööki. Sõidame tema juurde autoga. Tädi Johannal on punane auto.Tädile maitseb lambaliha kartulipudruga. Ruudi teeb ka väga head ros-oljet ja kartulisalatit.Üks suur uudis on veel! Ruudi õde Liidia õpetab mulle prantsuse keelt.Oskan juba natuke prantsuse keelt – see on raske, aga ilus keel. Kui suu-reks saan, siis lähen Prantsusmaale, näiteks Pariisi.Hüvasti! Pean nüüd minema. Tädi juba ootab mind. Panen veel tänaSinu kirja posti.Kirjuta mulle, kuidas Sa elad. Ootan Sinu kirja!

      Hi, dear friend Mart! Thank you for the letter! Your letters are always very interesting. Glad There are cinemas on Saturdays and there are parties every week in the community center. Here in Tallinn is also Very interesting, but now I miss Kaarup, home. I come from Tallinn Back home until August. Today we are going to visit Ruud with Aunt. Ruudi lives in Nõmme, his family Nal is a big house and a very beautiful garden. He also has two cats and a dog. Me I like cats and dogs. Looking forward to! Ruudi makes us evening meals. We drive to him by car. Aunt Johanna has a red car. The aunt tastes the lamb with potato porridge. Ruudi also makes very good ros- Oljet and potato salad. One big news is! Ruudi's sister Liidia teaches me French. I already know a little French - it's a hard but beautiful language. If the oral Reks I get, then I go to France, like Paris. Goodbye! I have to go now. Aunt is already waiting for me. I'll put on today Your letter post. Write to me how you live. Waiting for your letter!

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