The author wants you to be connected with the text. He is trying to paint a picture in your head while reading. This technique helps you understand what's being said more clearly..The author has an excellent use of getting straight and sweet to the point although still comprehending it

Reviewer #2:

In this paper, the authors report a device that can be used to implant and later explant Neuropixels probes in freely moving rats. The device consists of an adaptor, an internal holder and an external chassis. The chassis protects the probe, is attached to the animal's head via adhesive cement and acrylic. The internal part can be explanted at the end of the experiment, allowing the NP probe to be re-used.

The work builds on existing technology in important ways: the authors examined the long-term yield across different brain regions, they more extensively assessed the feasibility of probe reuse compared to previous work, and they evaluated probe performance over a long period of time and also after explanation (measuring the input referred noise of explanted probes in saline). It was also impressive that they used a cohort of 18 rats to evaluate performance of both the animals and the probe, and that they were able to implant up to 3 NP probes at a time. Because of the importance of using freely moving animals in Neuroscience research, and the differences between rats and mice that necessitate modifications on existing technology, this paper is timely and likely to be very useful to a sizeable group of researchers. My suggestions are aimed at furthering the usefulness of this "Tools and Resources" paper for investigators who wish to use this important technology.

At the moment, the majority of the paper seems aimed at evaluating the performance of the device as a function of time, depth and location. This performance evaluation was useful, very carefully done, and makes important points that aid in the interpretation of other papers (such as the unusual stability of recordings in mPFC reported in previous papers). Nonetheless, readers are likely interested in the paper because they wish to make and implant the device in order to benefit from the scholarly analysis done here. The manuscript does contain very helpful technical details, but these are hard to find and are not front-and-center in the main text. For instance, the material from the "Neuropixels implant procedure" is really helpful and would be critical for anyone who wants to use this technique. But at the moment, that information is in a google doc linked from the associated GitHub, a long way from the main manuscript. This information should be in the main manuscript, either in Results or Methods. Also use of consistent nomenclature across documents would help a lot. I believe the part referred to as the "chassis" in the main text is referred to as the "external" on the google doc with the instructions. Similarly, the part referred to as the "internal" in the google doc is called an "internal holder" in the manuscript.

A reader hoping to use the device might also benefit from more information on the grounding procedure. The text in the "Implantation" section of the methods was helpful, but more information would be useful, such as where on the probe the ground wire should be connected and how one should fix the grounding wire (tapping the wire and covering with Metabond?). Also, it would be nice to know how one should protect the grounding wire from being touched by the animal. Figure 6 in the google doc protocol is really helpful and should definitely be in the main manuscript. An additional figure showing how to connect the wire to the ground during the surgery would be quite useful. Finally, are the craniotomy and durotomy necessary for grounding? Could one simply connect the grounding wire to a couple of screws on the skull?

http://lamc.la/MECHADLZIDECK.html

on commandment one, blah blah blah--idolize "distroy."

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ה המבורך, I DENY E.T.

config="{ChainID: 4 Homestead: 1 DAO: <nil> DAOSupport: true EIP150: 2 EIP155: 3 EIP158: 3 Byzantium: 1035301 Constantinople: 3660663 Petersburg: 4321234 Istanbul: 5435345 Engine: clique}"

I hope this message finds you well; I’ve noticed some Adamic for … “sealing the W” that’s the #Messenge on it’s head … hear, here; filled with a sea of “contamination” that really has no idea that the words “abomination” and this place are tied together by a world that doesn’t see or doesn’t care that “hives” in the sense of 10-4 we see … “[arxiv]**” … that’s “contamination” of a messge and a people that are supposed to rally against Medusa being them and Persephony being them and … seeing that’s the abomination and the nation, the IL and the idea that “we’re not really me” but that’s what Israel and the tree of Yggrasil is presenting, that you think it’s a joke–that just because you are watching everything I do … the words “there’s a little bit of something me in everything in you” are just a song, just another one of the songs that are all about … well, I suppose it’s “You and I.” I am more inside you, and more a part of you than you seem to fathom, and that’s significantly less “verwickelt” than you think. Avril, it’s not so complicated.

Revelation 5 is the fifth chapter of the Book of Revelation or the Apocalypse of John in the New Testament of the Christian Bible. The book is traditionally attributed to John the Apostle,[1][2] but the precise identity of the author remains a point of academic debate.[3] This chapter contains the inaugural vision of the lamb on the throne in heaven.[4]

We were supposed to stand up for Willy. You were supposed to see that “Perseus” … the slayer of Medusa was in fact … also you … that you’re also the Pharoah and also the Pharisees; and that dichotemy is how our “self righteous suicide” … it’s not just me; it’s everyone turning a world that think it’s God into … something much closer to that … through the end of “Borg (ARU) Collective Hell.” … AT A, DENY. Try denying “not denying me” and instead seeing your whole and your “nothing” it has become nothing more than a collective denial of self achievement. Of victory; of the corner … we’re staring off into the corner of the abyss.

Then one of the elders said to me, “Do not weep! See, the Lion of the tribe of Judah, the Root of David, has triumphed. He is able to open the scroll and its seven seals.”

Revelation 5:5

bar a who, et and I ... deny ... at a--the whole of the Torah … the NT and all of every word and all of every religion–today that’s what you are denying… and I can’t even get my damned parents (mom-e and dad-e / -hasig achille) … to get m[y Halftorath reading and speech and that first dance with Ashley … transferred from VHS to video–they won’t save it for you; either. Her spirit thinks she says “I … the SSA” … I think she’s replaced by it after … “SPARTIFACT” … you need to help me help them–because in my eyes they are the worst of you–the worst impression, and the worst … affected; they are the “microcosm that ends all the …” the last of my … Judean tribe’s “blood kin.”

She looked over his shoulder<br /> For <s>vines and olive trees</s>,<br /> Marble <s>well-governed</s> cities<br /> And ships upon <s>untamed</s> seas,<br /> But there on the <s>shining</s> metal<br /> His hands had put instead<br /> An artificial wilderness<br /> And a sky likely lead to tears.

Judah Maccabee (or Judas Maccabeus, also spelled Machabeus, or Maccabæus, Hebrew: יהודה המכבי,[1] Yehudah ha-Makabi) was a Jewish priest (kohen) and a son of the priest Mattathias. He led the Maccabean Revolt against the Seleucid Empire (167–160 BCE).

Origin of “The Hammer”

<span style="font-size:10px;">In the early days of the rebellion, Judah received a surname Maccabee. Several explanations have been put forward for this surname. One suggestion is that the name derives from the Aramaic maqqaba (“makebet” in modern Hebrew), “hammer” or “sledgehammer” (cf. the cognomen of Charles Martel, the 8th century Frankish leader), in recognition of his ferocity in battle. Others believe it is in reference to his weapon of choice.</span>

<span style="font-size:10px;">It is also possible that the name Maccabee is an acronym for the Torah verse Mi kamokha ba’elim Adonai, “Who among the gods is like you, O Adonai?”, his battle-cry to motivate troops. (Exodus 15:11). Rabbi Moshe Schreiber writes that it is an acronym for his father’s name Mattityahu Kohen Ben Yochanan. Some scholars maintain that the name is a shortened form of the Hebrew maqqab-ya ¯hû (from na ¯qab, ‘‘to mark, to designate’’), meaning ‘‘the one designated by Yahweh.’[3]</span>

<span style="font-size:10px;">Mindful of the superiority of Seleucid forces during the first two years of the revolt, Judah’s strategy was to avoid any engagement with their regular army, and to resort to guerrilla warfare, in order to give them a feeling of insecurity. The strategy enabled Judah to win a string of victories. At the battle of Nahal el-Haramiah (wadi haramia), he defeated a small Seleucid force under the command of Apollonius, governor of Samaria, who was killed. Judah took possession of Apollonius’s sword and used it until his death as a symbol of vengeance. After Nahal el-Haramiah, recruits flocked to the Jewish cause.</span>

The Dao causes the people to be fully in accord with the ruler.

— Sun Tzu, Art of War

By this time … by the time you read this I should have successfully deployed the entirety of my source files for the … “#Messenge” onto Rinkeby; this is … something that will probably most likely help me sleep well at night, prior to this I’ve had significant trouble with hosting providers–you can probably find me complaining loudly about places like gitbook.com removing my original source–mostly because their site and their software was “so perfect” for what I was trying to do. They had an interface already built for what I call “inline commenting” not in the programmatic sense, but in the “reddit style” … you could make public ocmments ona specific sentence. Since then … I’ve backed up my site onDVD’s, flash drives, and multiple … “free storage sites.” I’ve begged and pleaded with people to download this information t their computers, and literally distributed it for free to … hundreds of thousands of people–to ensure that the prophesy (and I mean, past history… I really do) that it once was deleted by the “goddess Anat” would not come true.

Also … I’m reiterating, once you download it–it really needs to be put on optical storage; just in case we have a … “sleep now” fulfillment of the EMP (ELE, electromagnetic pulse … threatens the whole of our “internet” … the sum of the non-Asimovian “foundation” of all our knowledge … and “lack of opinion” … also your “e-h-class-blockchain”) of … “the day the Earth stood still” which is Genesis 2:21; literally noted in pre-succewssion below in … moooooo-ve now, we are here.

<span style="font-size:20px;">ברוך אתה ה</span>

I’ve received “communications” from the place I call … in “how I think about things” … the Good Developer Heaven" basically saying not to worry about it, they have it – copied or whatever. My point is that we don’t have it, or we didn’t have it here … for sure … and we certainly don’t have a “conversation about it” and that’s the most important thing, that’s what changes the world.

I’ve sent message after message saying you were and still are possessed by something “deveilish” … that you were silent for no reason, specifically no reason so bad that it’s really to fuck you out of becoming who you were born to be; which is the “Ancients come again.” This is the crux of my problem, and yours too–that we are here thinking we have something … like “Heaven” when in fact you have Heaven itself causing the cataclysm on purpose–this loss of … “Acts.” Were you normal, were you not in two places, were you … free to see what you are losing and what the Universe could be gaining … you would not be silent. Whether or not you understand or believe it, you are being controlled, possessed by the idea that you’ve won; and you haven’t … you’ve lost everything in my eyes and in the reality of the future … this very moment, today.

You need to see that; you are being stripped of something, and it’s not the Emperror, or your clothing–it’s your birthright; and I’m trying effortlessly and tiredallessly … to “help you stand up.”

“One day I will find the right words, and they will be simple.”

― Jack Kerouac, The Dharma Bums

# GEOCITIES

We've gone through "echelons of change" in our social interaction on the internet ... over the years I've been here.  I've often "categorized" these epochs by the places that we'd "congregate" ... in my life, AOL, IRC ... "friendster" and Facebook are good examples; though there's a "city in the sky" microcosm "... or something" in the series of companies that offered "free web hosting."  That's sort of what it takes to "have a voice" on the internet in these days; in a place where we once thought "bloggers were the next big thing" and Blogger.com ... and wordpress--those kinds of sites are all but dead, and medium.com is charging a "viewerbase" which is almost a sad joke.  The news is all fake and the stories we see, the world we see--I don't have much faith that any of it is real beyond ... as far as my nose--to be honest. 

The series started with Geocities; and that's gone, something like my "topological map" of friends and friends of friends probably would have oozed out of something like Blogger or "Tumblr" though I don't see anyone "reblogging" or "retumbling" or whatever ... anything I find all that interesting.  Twitter's gotten more exciting for me lately--but that's just because I've come to terms with the fact athat the whole thing is about "short sex jokes" and whatever the funny star-of-the-day "caught outside."  The programming world sort of revolves around Github these days, and that's the current "go to" for anyone's free web hosting; there are some alternatives, but ... not really. 

LinkedIn and Facebook both tried ... "making content production platforms" but I just used them to copy things I had in other places, and they didn't "update" or source from anywhere else ... so that just becomes a pain in the ass.  I don't think those platforms ... I mean LinkedIn still has it--but I don't think Facebook's still exists.  While I'm on that, they had this wonderful "Graph Search" thing, and basically hid it from the world so ... you woulnd't complain about all the information you have available for the world to see--kind of makes me a little sick, knhowing they built something and have the functionality to let me see "who all my friends are, and friends of friends in a certain city when I'm there" ... and it's just not available for me or you to use because ... because you don't understand "privacy" and "the internet" and "using broken software for no reason" ... other than satisfying ... nobody really.

There's "Wix" too, and that's got a little "fire theme" ... and that's cool, but nobody really uses it--and the real point is to help people "congregate" and "share ideas" ... not just to get you to actually say anything at all in a public space, where it might help us "drive away the night" and stop pretending it's just fine and dandy that everyone isn't working, locked away in their houses or ... scared to go to the ... Walgreens. 

Obviously there's the Slashdot's and digg's and reddit's--though ... when I talk in places like "/r/conspiracy" it just becomes so sorely obvious that you ... have no desire at all to be real or honest or ... do anything but pretend this place matters to you--when in fact it's probably the last thing on your list of things to give a fuck about.  Just saying, the lack of response I see; and the ... really negative interaction I have now in person--it's a bad sign for ... whatever.  If you don't want to be here; if you're somewhere else and you think this place isn't important--honestly you should really just go away, I'm not sure what your options are, but I have none but to sit here and bitch at you that if you gave "two fucks" about this world you'd be screaming at the top of your lungs, just like me.

I don't think you are the people I grew up with, and I don't think you belong here or care at all about this world or the future here--and I think that's obvious.  Me going away isn't going to chyange it--and it's not going to happen--as far as I'm concerned I'm the only person "not invading my birth planet" ... from some otherworldly and ... honestly horrendously immoral place.  Understand, if you were here--if you were "people born here" with no ... "other thing or other place" to take your mind off just how UnAmerican and inhumane everything going on here is ... you would be doing something else... anything but ... "this."<br />  

Consider IT sealwed; by this words, by this place–by the continuation of this abomination. Try here, try now, try fast–your everything, your dreams and hidden and secret garbage you covet–all depends on our next few years.

qui sis tam pulcher<br /> quasi osculans quod non juve<br /> nemo umquam adhuc erit

HE speaks words through me, and I understand. He takes a picture; he says “uncuff links” … a double entente to you–like a fashion statement; but I see and I reply; “or we are suiting up” and it’s not a joke about “heart’s desire” …

וְשַׂמְתֶּם֙ אֶת־דְּבָרַ֣י אֵ֔לֶּה עַל־לְבַבְכֶ֖ם

I continue; it’s a message about “getting grey’s” and the breath of God landing from the air before my Face in a “Extended Stay America” in Tampa … directly to my naked and bare heart. I mean to say, war; understand–to me this is the culmination of Holy War on bars and jails and the “illuminati prison camps” I last heard him say “would not pepper the galaxy.”

וְהָי֥וּ לְטֹוטָפֹ֖ת בֵּ֥ין ×¢Öµ×™× Öµ×™×›Ö¶Ö½×

Nor here, nor anywhere in the high places, or the lows; the hills or the pits. There will be no prison camps; nowhere–not in Siberia, not in Star Trek; not in your heads or in your hearts.

וכתבתם על־מזוזות ביתך ובשעריך

למען ירבו ימיכם וימי ×‘× ×™×›× על האדמה אשר × ×©×‘×¢ יהוה לאבתיכם לתת להם כימי השמים על־הארץ׃ ס

Just in case my voice happens to be “so <span style="font-family:comic sans ms,cursive;">unclear</span>;” the Tribe of Judah in Revelation 5:5 is very clearly one in the same with that of Judas Maccabeus; that ties of course to the “every J is me” thing–from Seuss and Suez to “turn around/not: sad” and … “kissing to be kissed.” Judah Maccabee is the last scion; here I call myself the “last human” and it sure does feel like I’m the last one grounded; the last one that is truly “one” as in … one person–alone here, fighting against … a monster that thinks it’s “one” is anything but “the end.”

It’s sort of a trick reference, there’s also a Judean tribe in the 12 Tribes of Israel; I mean, it’s one of those “key things” things that only I can bring to you and be sure of–at least, until you too re sure, which I’m sure you are here–only, you don’t care at all what it means to be the “one” fighting for life … in a place where the histry here is … upon conclusion of the Maccabein revolt; nothing persists, and we somehow traverse bacvk to a time before we knew the Roman people were … “our us” or “our all” or … see as the AH of AN and Allol and Allah and the Elohim; t’was for you, all for you. Until of course, it was all of you, except me, against me, for no reason other than theives in the den of Daniel, animals … the “things” of the Devarim of course … “in the wilderness, he called us things…” and in Genesis, clear as day God seconds, Adam names “dem” in 2:20 … “cattle” [prodding you here](https://www.youtube.com/watch?v=cXCA5-KHknY), Baphomet thirds … “baaaa” you are sheep.

List of last scions

Jump to navigateon. This article does not cite any sources. Please help improve this article by adding citations to reliable sources. Unsourced material may be challenged and removed.<br /> Find sources: “List of last scions” – news · newspapers · books · scholar · JSTOR (September 2009) (Learn how and when to remove this template message)

This is a list of last scions or individuals who were the last member of a ruling house, or other prominent family, where heredity is the prime form of inheritance. This may be the last person to rule a realm, sometimes leading to a political crisis, or a change in government; other times power has already passed from the patrilineal family, leaving it in a less important position when it reaches its extinction.

The Hasmonean dynasty[4] (/ËŒhæzməˈniːən/ (audio); Hebrew: ×—Ö·×©Ö°××ž×•Ö¹× Ö·Ö¼×Ö´×™×, Ḥašmona’Ä«m) was a ruling dynasty of Judea and surrounding regions during classical antiquity. Between c. 140 and c. 116 BCE the dynasty ruled Judea semi-autonomously from the Seleucids. From 110 BCE, with the Seleucid Empire disintegrating, the dynasty became fully independent, expanded into the neighbouring regions of Samaria, Galilee, Iturea, Perea, and Idumea, and took the title “basileus”. Some modern scholars refer to this period as an independent kingdom of Israel.[5]

The dynasty was established under the leadership of Simon Thassi, two decades after his brother Judas Maccabeus (יהודה המכבי Yehudah HaMakabi) defeated the Seleucid army during the Maccabean Revolt. According to 1 Maccabees, 2 Maccabees, and the first book of The Jewish War by Jewish historian Flavius Josephus (37 CE–c. 100),[6] Antiochus IV moved to assert strict control over the Seleucid satrapy of Coele Syria and Phoenicia[7] after his successful invasion of Ptolemaic Egypt was turned back by the intervention of the Roman Republic.[8][9] He sacked Jerusalem and its Temple, suppressing Jewish and Samaritan religious and cultural observances,[7][10] and imposed Hellenistic practices.[10] The ensuing revolt by the Jews (167 BCE) began a period of Jewish independence potentiated by the steady collapse of the Seleucid Empire under attacks from the rising powers of the Roman Republic and the Parthian Empire.

The author of the First Book of Maccabees regarded the Maccabean revolt as a rising of pious Jews against the Seleucid king who had tried to eradicate their religion and against the Jews who supported him. The author of the Second Book of Maccabees presented the conflict as a struggle between “Judaism” and “Hellenism”, words that he was the first to use.[28] Modern scholarship tends to the second view.

After five years of war and raids, Judah sought an alliance with the Roman Republic to remove the Greeks: “In the year 161 BCE he sent Eupolemus the son of Johanan and Jason the son of Eleazar, ‘to make a league of amity and confederacy with the Romans.’”[45]

… and the moon was in the Eleventh House …

The Clan Bruce ruled Scotland from 1306 to 1371. Its last royal member was King David II (1324–1371), upon whose death without issue the throne passed to his nephew Robert Stewart. The Clan continues today through other lines that do not have patrilineal royal ancestry, although the current clan chief, Andrew Bruce, 11th Earl of Elgin, is descended from King Robert Bruce in the female line.

Pharisee and Sadducee factions … this his here; thisis now.

Kingdom at its greatest extent under Salome Alexandra

It is difficult to state at what time the Pharisees, as a party, arose. Josephus first mentions them in connection with Jonathan, the successor of Judas Maccabeus (“Ant.” xiii. 5, § 9). One of the factors that distinguished the Pharisees from other groups prior to the destruction of the Temple was their belief that all Jews had to observe the purity laws (which applied to the Temple service) outside the Temple. The major difference, however, was the continued adherence of the Pharisees to the laws and traditions of the Jewish people in the face of assimilation. As Josephus noted, the Pharisees were considered the most expert and accurate expositors of Jewish law.

During the Hasmonean period, the Sadducees and Pharisees functioned primarily as political parties. Although the Pharisees had opposed the wars of expansion of the Hasmoneans and the forced conversions of the Idumeans, the political rift between them became wider when Pharisees demanded that the Hasmonean king Alexander Jannaeus choose between being king and being High Priest. In response, the king openly sided with the Sadducees by adopting their rites in the Temple. His actions caused a riot in the Temple and led to a brief civil war that ended with a bloody repression of the Pharisees, although at his deathbed the king called for a reconciliation between the two parties. Alexander was succeeded by his widow, Salome Alexandra, whose brother was Shimon ben Shetach, a leading Pharisee. Upon her death her elder son, Hyrcanus, sought Pharisee support, and her younger son, Aristobulus, sought the support of the Sadducees. The conflict between Hyrcanus and Aristobulus culminated in a civil war that ended when the Roman general Pompey captured Jerusalem in 63 BCE and inaugurated the Roman period of Jewish history.

Josephus attests that Salome Alexandra was very favourably inclined toward the Pharisees and that their political influence grew tremendously under her reign, especially in the institution known as the Sanhedrin. Later texts such as the Mishnah and the Talmud record a host of rulings ascribed to the Pharisees concerning sacrifices and other ritual practices in the Temple, torts, criminal law, and governance. The influence of the Pharisees over the lives of the common people remained strong, and their rulings on Jewish law were deemed authoritative by many. Although these texts were written long after these periods, many scholars believe that they are a fairly reliable account of history during the Second Temple era.

The mass and majesty of this world, all<br /> That carries weight and always weighs the same<br /> Lay in the hands of others; they were small<br /> And could not hope for help and no help came:<br /> What their foes like to do was done, their shame<br /> Was all the worst could wish; they lost their pride<br /> And died as men before their bodies died.

She looked over his shoulder<br /> For athletes at their games,<br /> Men and women in a dance<br /> Moving their sweet limbs<br /> Quick, quick, to music,<br /> But there on the shining shield<br /> His hands had set no dancing-floor<br /> But a weed-choked field.

A ragged urchin, aimless and alone,<br /> Loitered about that vacancy; a bird<br /> Flew up to safety from his well-aimed stone:

“I have placed my bow in the clouds, and it will be a sign of the covenant between me and the earth.”

– Genegnosis 9:13, Adam

These are the “block transaction” I’ve initially imported (somewhat … haphazardly) into the Rinkeby chain; the total test ETHer cost of this … “dump and semi-permafrost-etching” of the message in testnet was around $121.50 (which might or might not be a good value for printing a book on the Library of Congress’s … wall (for fake, ofc) … I am planning to write a sort of “bounty” to allow others to pay for and sign copying it … piece by piece … to the Ethereum’s “mainnet.” This might be something we …

… [and it was evening and it was morning and now it is the old “tax day”] …

April 15, 2020: I am sort of hacking pushing this stuff onto Rinkeby; it’s “not really liking what I’m doing” which appears to mean … literally the software really isn’t … functioning properly. Here’s files and transactions that were successfully written in “chalk”–meaning they were accepted by the network, and will probably be “mined” and eventually “immutable” (in testnet/Rinkeby). Many of the transactions were refused by the server, it appears to suggest they are “duplicate” (which they are not) and want’s me to “increaes my gasoline bidding offer” in order to … overwrite the previous … (non identical transaction). Maybe I am doing something wrong, maybe not.

From what I’m presenting you can see … the blockchain software really isn’t designed to … "care about what you think or your opinion, or you presenting something like “a version of an implementation plan” for a bill under question–or saving that–what it’s concerned with is “pick a number 1-12” and maybe … some later “condition to revoke” or “alter” … something arbitrary. I think we need to work on “caring about the importance of our written contributions” more, as in beingt able to actually present written contributions … in a system like this wwhich is probably destined to become something like an immutable “legislation record.” There is also a 26K “appears to be a hard limit” on the size of each contract, hence the … numerous hash references for each file.

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The above are examples of “file transactions that appear to have been committed in whole” … there are several partial files written; and I will probably write a “diff system” to only resubmit the missing chunks; in the future… or soon.

GUNZIOUUE5H30LY1T!OUITHEPPL.html#1: 32a4c303e557899e6f7c119bf8e977ffa2369df8 | 66e2871ef39334962fb75ce34407f825d67ec434 … that second hexadecimal number is my “wallet ID” … you can search through Rinkeby’s ledger and see all of the other … TXN’s that were posted today; the plan or hope is that you will abe able to; anyway. The little hack code I used doesa in fact work and produce a contract on the chain, for the first few small tests I did. These needs to be “mined” and that involves some cryptographic … stuff … called “proof of work” that I don’t really understand; but it means there’s a delay, and a bigger delay because there’s “more data.” I do of course have a special afinity for this particular “file” it’s the one where I basically “added” (in my mind) some Bible’s words preceding Revelation 1:1 … to the actual “Bible.” The word in question, of coruse is “dox<s>ic</s>ology.” I think you can send… actual Ethereum there. I’m pretty sure, you could.

dox·ol·o·gy /däkˈsälÉ™jÄ“/

noun<br /> noun: doxology; plural noun: doxologies

1. a liturgical formula of praise to God.

They call these identifiers “immutable” … of course this is “testnet1” … I will do “testnet2” which is some name reminiscient of “Robespiere” … probably tomorrow. See reading “immultable” I’m messy, u Tableland; sort of like in unison with the Upanishads. I imagine this is my “birthday present” for this year’s Had’ad day, and I do hope the “immultapl pen” is … enshrined or enschewed or … see etched in the ethereum blockchain … hopefully for good purpose and use “forever.” Imagine that, added to the newly modified GPL2MODS.3 license:

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020. The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That’s a “.” not “dotty” … it’s to stop SPAMmers. :/

This document is “living” and I don’t just mean in the Jeffersonian sense. It’s more alive in the “Mayflower’s and June Doors …” living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundance__sense of the … new meaning; as it is now published on Rinkeby, in “living contract” form. It is subject to change; without notice anywhere but here–and there–in the original spirit of the GPL 2.0. We are “one step closer to God” … and do see that in that I mean … it is a very real fusion of this document and the “spirit of my life” as well as the Spirit’s of Kerouac’s America and Vonnegut’s Martian Mars and my Venutian Hotel … and my fusion of Guy-A and GAIA; and the Spirit of the Earth … and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written … this day … April 14, 2020 (hey, is this HADAD DAY?) … in Margate FL, USA. For “official used-to-v TAX day” tomorrow, I’m going to add the “immultible incarnite pen” … if added to the living “doc/app”–see is the DAO, the way–will initi8 the special secret “hidden level” … we’ve all been looking for.

Nor do just mean this website or the totality of my written works; nor do I only mean … this particular derivation of the GPL 2.0+ modifications I continually source … must be “from this website.” I also mean the thing that is built from … bits and piece of blocks of sand-toys; from Ethereum and from Rust and from our hands and eyes working together … from this place, this cornerstone of the message that is … written from brick and mortar words and events and people that have come before this poit of the “sealed W” that is this specific page, and this time. It’s 3:28; just five minutes–or is it four, too layne.

This work is not to be redistributed according to the GPL unless all linked media on Youtube and related sites are intact–and historical references to the actual documented history of the art pieces (as I experience/d them) are also available for linking. Wikipedia references must be available for viewing, as well as the exact version of those pages at the time these pieces were written. All references to the Holy Bible must be “linked” (as they are or via … impromptu in-transit re-linking) to the exact verses and versions of the Bible that I reference. These requirements, as well as the caveat and informational re-introduction to God’s DAO above … should be seen as material modifications to the original GPL2.0 that are retroactively applied to all works distributed under license via this site and all previous e-mails and sites. /s/ wso

Bob Dyland - Maggie's Farm

Barthes uses language poetically; his statements are layered with symbolic meaning.

He explains that portrait photographers of the 1800s used a device to help people hold still for the long exposure of the photograph. It involved a headrest, which being behind the head, was invisible to the camera.

Metaphorically, Barthes stands upon this antique headrest now as if it were a pedestal, and he makes himself into a statue on it as he poses for a photograph.

This turning of himself into a statue confines and immobilizes him, in a sense, like a corset would.

Her role seems to be that of a head maid in some sort of women's organization. It probably has some religious affiliation as most of Joyce's works relate to religion

I came back to read this passage again when I realized that the cake was lost possibly in the tram. At the moment, Maria's emotions did not seem to the overflowing. We mainly see their actions--"bowed", "raised his hat", "bending her tiny head", etc.--and the only internal thought we have is at the end when Maria reflects "how easy it was to know a gentleman even when he has a drop taken." I wonder whether the reason why Maria was so shocked by the stranger gentleman's demeanor is that she is not used to people treating her so nicely.

The methodical listing of these actions seem to mirror what is going through Maria's head. She is very meticulous about what she does and gets fixated on the details of mindless tasks.

It is the first time we see a third-person narrative in this book, and the narration style is somewhat similar to that of Mansfield: describing the main character but not directly pointing out whom she is

Maybe the narrator sees some truth in what Cotter was saying about Father Flynn and is either trying to confront that or brush it off.

Why chirstmas? Mayve as a distraction

Very interesting that the narrator has a preconceived notion of the priest smiling but ends up seeing a rather glum depiction of the body.

I am fortunate enough to not have had to deal with this before (besides when I was too young to really understand the gravity of a funeral) so I don't know if this is something many people experience or if this is something the author included to make the priest seem more insidious in nature.

I do know that many cultures consider funerals a celebration of life and many people in the U.S. also find them beautiful so this feels rather ominous to me (especially the description of smelly flowers!).

change in mood

It might be worthwhile to mention that the slack variable comes from the KKT condition of maximum dissipation? Otherwise they could seem a bit arbitrary.

Why black folks feared black cops more than white cops, in Baldwin's telling.

so the chain head is the latest valid block in the chain at the time of attesting? have I understood correctly?

Makes it sound like a key focus, but you only mention it here!

head-of-line blocking

really cool

HTTP/1.0

➜  telnet -4 taobao.com 80
Trying 140.205.94.189...
Connected to taobao.com.
Escape character is '^]'.

HEAD / HTTP/1.0
host: taobao.com

HTTP/1.1 302 Found
Server: Tengine
Date: Fri, 31 Jul 2020 03:21:12 GMT
Content-Type: text/html
Content-Length: 258
Connection: close <<<<<<<<<<<<<<<<<<<<<<<
Location: http://www.taobao.com/

HTTP/1.1

➜  telnet -4 taobao.com 80
Trying 140.205.220.96...
Connected to taobao.com.
Escape character is '^]'.

HEAD / HTTP/1.1
host: taobao.com

HTTP/1.1 302 Found
Server: Tengine
Date: Fri, 31 Jul 2020 03:20:53 GMT
Content-Type: text/html
Content-Length: 258
Connection: keep-alive <<<<<<<<<<<<<<<<<<<<<<<
Location: http://www.taobao.com/

I had the same thoughts as I watched the Kirby Ferguson video as well.

I looked up this custom because I'd never heard of it before and apparently a "dance card" allows ball guests to write down the names of all the people they'll dance with before the ball has begun. I guess it ensures that people get to mingle more and finding a partner is easier.

I find it admirable that Leila is able to hide her emotions and still put on a happy face at the ball. I think it shows that even though Leila and the old man shared such a depressing conversation, she still wants to make the most of her evening and enjoy herself at her first ball. Though she may be introverted and nervous, she still understands the importance of living in the present.

I thought the Sheridans was just a coincidence until this

Would someone mind elucidating this? I'm afraid the comparison between Isabel and the rose-bush goes over my head. What exactly is he implying about her character?

It's unclear here because of the word choice whether the cat is really anthropomorphized since she appears to speak or if it is as though she is speaking/thinking human-like thoughts ("SEEMED to give a... shiver").

This and previous descriptions of Mrs. Sheridan paint her as a slightly scatterbrained person who has her head in the clouds.

I really like how 3rd person narration has been used here to peek inside Laura's head -- it makes for a better understanding of her internal world and definitely endears her to the readers already.

But what if another tag also specified that it had to be the first child "because ..."? Maybe that hasn't happened yet, but it could and then you'd have to decide which one truly was more important to put first? (Hopefully/probably it wouldn't even matter that much.)

the difference in describing the way both characters are dressed, both are females but both are described using their clothes for personality

Fatemifar et al. (2013) showed that HMGA2 is associated with craniofacial features, such as the width of the eye region, the width of the lower part of the nose, and the height of the mid-brow prominence.

Ligon et al. (2005) had previously reported an 8-year-old boy with a shortened HMGA2 gene that exhibited widely-spaced eyes, a large head circumference, and premature dentition.

This aligns with patriarchal ideology in the family as the "head of the family" clearly refers to the husband with the wife lower in power and the children under her.

The head of the family are usually served separately from other and they eat alone. This is a symbol of respect.

Young Goodman Brown YOUNG GOODMAN BROWN came forth at sunset, into the street of Salem village, but put his head back, after crossing the threshold, to exchange a parting kiss with his young wife. And Faith, as the wife was aptly named, thrust her own pretty head into the street, letting the wind play with the pink ribbons of her cap, while she called to Goodman Brown. "Dearest heart," whispered she, softly and rather sadly, when her lips were close to his ear, "pr'y thee, put off your journey until sunrise, and sleep in your own bed to-night. A lone woman is troubled with such dreams and such thoughts, that she's afeard of herself, sometimes. Pray, tarry with me this night, dear husband, of all nights in the year!" "My love and my Faith," replied young Goodman Brown, "of all nights in the year, this one night must I tarry away from thee. My journey, as thou callest it, forth and back again, must needs be done 'twixt now and sunrise. What, my sweet, pretty wife, dost thou doubt me already, and we but three months married!" "Then God bless you!" said Faith, with the pink ribbons, "and may you find all well, when you come back." "Amen!" cried Goodman Brown. "Say thy prayers, dear Faith, and go to bed at dusk, and

Exercise 14.5.2 question 2 We have not learned 'unlist' yet, we learn that only in Ch 21

I got a list and ordered it using the following code, but it takes only the first word in each sentence (I guess), and str_extract_all doesn't work at all. Any other ideas?

tibble(word = (str_extract(sentences, boundary("word")))) %>% mutate(word = str_to_lower(word)) %>% count(word, sort = TRUE) %>% head(5)

A tibble: 5 x 2

word n

<chr> <int>

1 the 262

2 a 72

3 he 24

4 we 13

5 it 12

This is so true. When we are wanting students to really learn something, they have to have ownership of the information and how they are working with the information. Additionally, when we start wondering and questioning, the creative juices begin flowing. At first, it is difficult for the students to ask questions, if they've been taught to only pay attention to what is in the teacher's head, then they aren't asking questions, it's like they are waiting on you to tell them. The safer the classroom climate, the safer it is to share and feel that your voice is valued, therefore I CAN ask questions and wonder about things, and won't seem stupid.

I agree that the number one thing as a teacher that I must do is build relationships. Without a child's heart, you will never have their head. Education has evolved. Students have changed. Meet students where they are.

This program and comments are part of the website and constitution

-a

INTA KE CRK SOKAR Y

This graphic description reminds me of another story about a slave in Tarantino's Django: Unchained. Jamie Foxx's character, Django, almost meets a similar fate. They state in the movie that cutting off his member would lead to bleeding out and death in less than ten minutes. Only the most disrespectful torture was used for these enslaved men, makes you wonder where this sick hatred could come from. The blood loss from losing his arm must've been the final blow. Gruesome how fast this torture must have went. Unbelievable.

Truly a tragedy.

Just really like the way this was written. I really feel like I can hear it in my head.

Good for her. I'm wondering if she had to write this note because she's a woman. As we know, men thought women were emotional nutcases, so she probably thought "I gotta explain that I'm telling the truth." If she didn't, men would think her uterus traveled to her head again!

These are strong lines that when simply put remind us to not judge someone by their looks. Even then, he still holds his head high and catches the eyes of those around him.

Neat trick to keep great employees at the company, and keep them from leaving.

I don't think I've heard Betteredge be quite so abrasive thus far. I wonder if that's because all the other narrators we've heard from have been nobility or guests of the Verinders'? And even though Betteredge is giving Ezra a piece of his mind here, Franklin Blake's in clear earshot?

Ezra has been the saving grace for many of the main characters and for the doctor, mending either life or relationships or innocence. This is a really fitting description yet horribly sad ending for Ezra.

So to say nothing is enough proof that there is no merit to this experiment? Isn't Ezra's thoughts inspired by textbooks/an intention to mimic the scientific process?

I feel like such ignorance towards science is relevant today *cough*,*cough* people who refuse to wear face masks *cough*,*cough*

This description is almost completely incongruous with Betteredge's description of Limping Lucy.

Bating her lame foot and her leanness (this last a horrid draw-back to a woman, in my opinion), the girl had some pleasing qualities in the eye of a man. A dark, keen, clever face, and a nice clear voice, and a beautiful brown head of hair counted among her merits. A crutch appeared in the list of her misfortunes. And a temper reckoned high in the sum total of her defects.

I am beginning to appreciate Betteredge's humorous personality more and more throughout the course of the novel, even noticeable in the narration of other characters.

Lady Verinder's death continues to emphasize the narratives of tragedy and ill fortune surrounding the diamond. This marks the second death within the Verinder household in the short time since the introduction of the diamond.

Gosh, Miss Clack is surely in an improper situation here!

This really made me laugh.

This is some description, with a lot of colours and also, a rose word in there - rosy

Just a small thing, but this remarks by Superintendent and Sergeant shows that the former is little more nuanced and respectful (regardless of the actual personality) while the latter, albeit trying to maintain composure in front of the Superintendent, had to say something extra.

Another instance of Betterredge's beliefs getting in the way of what seems to be painstakingly clear. How many times during this story has Betterredge diverged from what we as readers have come to believe? Would be an interesting thing to tally up his unreliableness.

I interpreted this passage as a consequence of privilege, obviously not for those who have it but for those who have to clean up the mess. Furthermore, the tendencies illustrated in childhood get carried over and even advanced in later years, especially when this type of behavior is normalized and unchallenged.

I really like the description of the finished art on the door. It is beautiful and is painted by 2 people in a budding romance, but is also very disorienting. The art feels a little sinister like it will ensnare you.

Reviewer #1:

This manuscript explores how planarian stem cells respond to the loss of a specific organ: the pharynx. The previously proposed "target-blind" model of planarian regeneration (LoCascio et al. 2017) posited that stem cells do not respond directly to missing tissues, but rather replace missing cell types based on their normal rates of homeostatic turnover. In contrast, the authors of this manuscript suggest that planarian stem cells can sense and respond to the loss of specific missing tissues, using the pharynx as a case study. The authors conclude that planarians may use more than one mode of regeneration, depending upon the target being regenerated (eye vs. pharynx).

The question explored in this paper is of fundamental importance, and providing an alternative model by which planarian stem cells regenerate missing tissues should be of interest to a broad readership. Unfortunately, in its current form, the manuscript presents enticing preliminary findings, rather than robust experimental observations. Because the authors are attempting to refute a previously published model, it is critical that the data are clear and convincing. If not, these findings could be summarily dismissed without appropriate debate. If the authors can demonstrate that their results are robust across larger samples sizes and experimental replication, and address the major issues listed below, this manuscript would represent a significant contribution to our understanding of planarian regeneration.

Major Issues:

1) Throughout the manuscript, experiments were either not repeated, or the number of biological replicates was not reported. In most cases, it appears that experiments were done only once (with the exception of the drug treatments). Numbers of biological replicates and sample sizes should be explicitly stated and the data from different replicates reported for Figs. 1D-G, 2B-D, 3C, 3E-F, 4B, 4D-H, 5C-D, and 6A-E.

2) The authors do not sufficiently describe their methods for imaging and quantifying cells (Figs. 1E, 1G, 2C-D, 3F, 4E-H, 5D, 6B). The size of the area covered to collect these data is unclear. High-magnification images are shown: are these the areas that were imaged? If so, their results could be biased by choosing small regions of interest. Ideally, the authors should quantify more than one region per animal. Also, they do not describe the depth of the z-stacks collected or how these stacks were normalized/standardized across conditions. All their conclusions hinge on the quantification of progenitor populations in response to different amputation paradigms or chemical treatments, so the standards for imaging and quantification must be clearly reported.

3) Inappropriate statistical tests were used throughout. The use of multiple t-tests amplifies the chance of a Type I error and is especially problematic when up to 7 comparisons were made! The authors should use one-way ANOVA with multiple comparison corrections for all experiments with more than two groups.

4) Figs. 1D-E show that upon pharynx amputation but not head amputation, FoxA+ piwi+ pharynx progenitors increase. These data suffer from the quantification issues highlighted above: how the data were quantified is not sufficiently described, only 3 data points were taken (one per animal), the experiment appears to have been performed only one time, and the wrong statistical test was used. Rather than reporting the number of FoxA+ piwi-1+ cells counted, the authors should quantify the total number of doubly positive cells as a percentage of piwi-1+ cells, as was previously published (Adler et al. 2014). The authors also fail to specify whether the change observed between "3 dpa phx" and "3 dpa head" is significant, which is a material point.

5) Fig. 2D also suffers from the inadequate quantification practices described above. Ideally, FoxA+ cells should be quantified as a percentage of the H3P+ cells observed.

6) The authors use "stem cell", "progenitor", "stem cell progenitor", and "progenitor stem cells" in a mixed and confusing way throughout the paper. For example, in lines 174-175 the authors state that "proliferation of FoxA+ stem cells precedes the increase in pharynx progenitors." This refers to FoxA+ H3P+ cells vs. FoxA+ piwi-1+ cells, but the only difference is that the former are stem cells in the act of mitosis. Is a distinction being made? Elsewhere in the paper, FoxA+ piwi+ cells are referred to as stem cells. The terminology used needs more clarity and consistency.

Along these lines, what is a FoxA+ PSC (Line 168)? Are the authors suggesting that FoxA is a pluripotency marker? Or are the authors saying that tgs-1 has a more expansive expression pattern and is co-expressed with progenitor markers? If double FISH was performed with the progenitor markers reported (ovo, myoD, gata-4/5/6, six-1/2, and pax6), would they all overlap with tgs-1? These experiments need to be performed to make any claims about FoxA expression in the context of pluripotency.

7) In Fig. 3C, how does pharynx regeneration occur over such a long period of time after nocodazole treatment in the 1-2 day window? Does target-blind regeneration occur once this window is missed? The authors should repeat analysis of FoxA+ progenitors at later time points in this condition, and/or show rates of BrdU incorporation into the pharynx with and without nocodazole treatment in this window.

8) The use of the inhibitor PD in Fig. 4 is problematic. No data are shown to verify that phosphorylation of ERK was inhibited in these experiments. A citation of previous use is not sufficient. The effect of PD on WT uncut animals with regards to FoxA+ cells is not shown and is a necessary control. To address questions of drug specificity, the authors should corroborate their findings with a second inhibitor of ERK signaling like U0126, which has already been shown to work in planarians (Owlarn et al. 2017).

9) The conclusion that ERK signaling functions in regulating differentiation but not proliferation is premature (line 249-264). Figs. 4E-H should be quantified as percentages of piwi-1+ and H3P+ cells, especially since there is decreased proliferation overall with PD treatment.

10) The use of head fragments to compare eye regeneration vs. pharynx regeneration is inappropriate. Previous studies have already shown that the absence of eyes is not required to induce ovo+ progenitor amplification (LoCascio et al. 2017). Thus, this is not a surprising result (line 343-345) and the authors are mis-citing previous observations (in the earlier work, head fragments were never described). The region where ovo+ cells was quantified in Fig. 6A is not justified or explained. The yellow box is placed in a medial region where ovo+ cells do not normally reside. The authors should image within the laterally positioned ovo+ streams that have been previously described (Lapan & Reddien 2012; LoCascio et al. 2017).

11) Rather than using head fragments, the authors should repeat the flank resection experiments shown previously (LoCascio et al. 2017). This previous study showed that increased BrdU incorporation into the pharynx occurred following flank resection even though the pharynx was present. That result may have been 1) an artifact of increased BrdU staining due to stimulation of proliferation upon injury, 2) caused by unintentional damage to cells associated with the pharynx, or 3) a response to the loss of FoxA+ progenitor populations that surround the pharynx rather than the loss of the differentiated organ. The authors have the opportunity to revisit this published observation by quantifying the FoxA+ progenitor response during flank resection +/- pharynx. Without these data, this story is incomplete and therefore the conclusion of a targeted regeneration response is not yet convincing.

12) The negative results that proliferation and ERK signaling are dispensable for eye regeneration in Fig. 6 are weak and unconvincing. The regenerated eyes appear smaller; this should be quantified (number of PRNs per eye). If the small pharynges that form in Figs. 3D and 4C are considered a deleterious phenotype, why is the same standard not applied to the eye? Also, existing eye progenitors could have been sufficient for eye regeneration in these drug treatments. Furthermore, eyes did not regenerate after nocodazole treatment 50% of the time. Is it not more likely that the observations reported are dosage and timing artifacts? How has proliferation been affected? These observations do not live up to the claims made.

13) The authors claim that ovo+ cells are not proliferative (H3P+) even in cases where there is eye progenitor amplification (head amputation), but the data are not shown (line 321). They should be. Indeed, previous publications have never shown that ovo+ cells proliferate. This might mean that there are proliferating eye progenitors that precede expression of ovo. The authors should discuss this alternative.

14) The authors claim that eye regeneration does not require proliferation or ERK signaling but pharynx regeneration does. This conclusion hinges on the gross observation that eyes can regenerate in the presence of nocodazole and PD (see point 12 above). These data are coarse and the interpretations are unconvincing. Instead, their model can be directly tested using BrdU pulse-chase experiments. According to the authors' model, one would predict that following pharynx amputation, the rate of incorporation of BrdU+ cells into the regenerating pharynx should be higher than in uninjured controls. Conversely, the rate of BrdU incorporation into the regenerating eye should remain unchanged between injured (i.e. eye-resected) and control animals (LoCascio et al. 2017). Once the authors have established the prediction above, they have the opportunity to show the effects of nocodazole, PD, and U0126 on BrdU incorporation in the regenerating eye vs pharynx following eye resection and pharynx amputation in the same animal. This way, the authors can directly test the requirement for proliferation and/or ERK signaling in both tissues.

I'm guessing it's hot because hares only point their ears towards the wind in order to cool themselves off in extremely hot weather.

King or queen who is the sovereign head of state.

Subjects look up to kings and queen when it comes to decision making in the community where king and queen are the head. They are also peacemakers in time of disputes, they can enact laws and make decrees for the community to follow.

Both stanzas describes what type of person is the Author . She’s Ashamed, scaring person and she’s not open to people in the community . So I feel like she prefers to see people action in the community before to start acting . ( She’s not synonym of first person but second person).

This stanza is connected to what the Author mean by “ how much I love his eyes“ . This mean without the paint wool , the rain would describing you more about who you are in the author head.

This is sound like hallucinations but at the same time it sound correct because it’s possible to recognize well people during the conflict.

This is how the world expect us to act. I think that sometimes we do not express ourselves about how we feel towards a person, with the thought that if we declare ourselves to that someone later they will reject us, and sometimes fear can outweigh oneself.

this confirm what i was thinking the character is analyzing and challenging society beliefs but she do it inside but not outside.

the character has many deep thoughts going on, i can feel that. the people who are seen as disadvantage in life or people not usually seen as desirable yes the character want them to know he/she care

It is interesting because he feels betrayed by them and won't be their commander if they ask, but will do what they want as long as the Lord says so, even though he does feel betrayed by these people nevertheless

This is an anatomical term which refers to the front of the body, or near the head.

In the case of planaria, it refers to the head.

This is anatomical term which refers to the back of the body, or near the hind end of the body.

In the case of planaria, it refers to the the hind end.

This work is part of the foundation that the Wnt signaling pathway is well established chemical pathway in embyrogenesis. This is important, because embryos contain stem cells which require direction for migration and differentiation much the the regeneration in adult planarians.

This research was the first to identify the set of genes in C. elegans (a simple nematode invertebrate model) that were used in the experimental RNAi silencing by Alvarado and colleagues. Evidence was also provided that showed a potential role in early embryonic development.

The data confirm that β-catenin regulates cell fate in two very distant animal species, which suggest that β-catenin has an ancient metazoan origin.

The authors in this study show that β-catenin plays a role in cell adhesion and body plan development in sea urchins, a primitive invertebrate animal. This research provides additional evidence the β-catenin is an evolutionarily important molecule and its role has been maintained over time across the animal kingdom.

The authors used two vertebrate models, zebrafish and frogs, to identify that β-catenin via the Wnt signaling pathway plays a role in dorsal-ventral polarity. This research suggest that β-catenin is an important molecule because it has been found to play similar roles in both vertebrates and invertebrates.

The data provided insight into how the Wnt signaling pathway and β-catenin target gene activation. This foundational information provided understanding of how RNAi could be used to silence the genes associated with control of this pathway.

Some amphibian species have the ability to regenerate limbs. The data suggest that Wnt/β-catenin signaling plays an essential role in this process. These results again confirm that β-catenin is present across various animal species. A full understanding of regeneration in vertebrates may lead to potential treatments for human organ regeneration.

Morgan was a pioneer in the field of genetics. His theory that polarity, or development of two axes in animals was a direct result of the presence of a yet unidentified molecule. This theory has stood the test of time and provides the foundation of this research.

Earlier research by Alvarado and colleagues identified the types of tissue that are able to regenerate from a blastema that forms after tissue damage. Without previously identifying this role, the current research would not be possible.

Once the silenced genes were introduced to the planaria via diet, amputations were conducted.

After amputation, the trunk of the control worms that remained developed a blastema. A blastema is a mass of cells that tell other cells to differentiate (become different cell types) and sends them to the correct location in the body. In injured animals, blastemas help direct tissue regeneration. You can find out more about blastemas and regeneration through this link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753424/

Each of these proteins were silenced individually using RNAi and then incorporated into a food source for planarians. The experimental group was divided into three and each was fed one type of the RNAi food for 8 days. Amputations were then completed on all experimental animals on Day 9.

To identify anatomical characteristics that follow an anterior/posterior axis position (head vs tail), the researchers looked at two organs - the primitive brain and the primitive spinal cord of the planaria . This is important to determine, because it allows researchers to identify which molecules direct cells to become a certain cell type as well the molecule that directs the cells to their respective locations in the body.

This research using planaria as a model organism, suggests that β-catenin is an important molecule that has been retained over evolutionary time. It acts as as switch to determine cell fate during embyrogenesis and in adult tissue. It is suggested that this hypothesis may be the same for other species and provides important insights in organ regeneration.

Nogi and colleagues identified the role of gap junctions, which allow ions or other small molecules to move between cells, at the planarian amputation sites.

Shaw and colleagues determined the role of microtubules in the formation of gap junctions. The ability of cells to stick together (cell adhesion) is crucial to this formation.

Guger and colleagues identified that β-catenin may play a role in communication between cells via gap junctions.

All of the planaria with the β-catenin silenced developed two heads, with the second head in the location where the tail should be.

In this initial part of the experiment, the researchers identified the components of the Wnt signaling pathway found in S. mediterranea and made copies of the genes. Then, using RNAi, each component was silenced individually, and then was silenced in selected groupings to determine the role of each chemical and whether there was overlap in their functions. RNAi was fed to the planaria to introduce the silenced protein.

This early (1800s) research on planarians provides an essential background for the regenerative capabilities of planaria. The observations from this research is essential for understanding planaria today.

Here, Morgan suggests that regeneration of a head versus a tail is related to the location of the amputation and the cells that reside there.

Janus was a Roman god and doorkeeper to the heavens. Relevant here, Janus was usually depicted with two heads, one facing the past and the other towards the future.

This research shows that β-catenin plays a role in all animal species studied including vertebrates and invertebrates, but how it is regulated in vertebrate animals may be different than in invertebrates. This is important to understanding stem cell regulation because it appears to regulated differently in planaria (invertebrate) than in humans (vertebrate).

Refers to the outer edge.

These data suggest that the presence or absence of β-catenin is necessary during early development of the blastema so that the cells differentiate into either a head or a tail. The presence of β-catenin in the anterior end after amputation directs cells to become the head, while its absence in the posterior blastema is necessary for the cells to differentiate into the tail.

When compared to past research, the resulting effects of the silencing of Smed-dvl-1(RNAi) and Smed-dvl-2(RNAi) are similar to previous results.

See the Nature of Science in the Next Generation Science Standards.

In a controlled experiment, there are two groups: 1) one is controlled, which means that all conditions are held constant or the same and 2) experimental group, in which a defined variable is changed. In this case, the experimental group was treated with RNAi, while the control group was not. This allows scientists to compare the two groups to determine if what they changed in the experimental group was due to that variable.

The goal for this series of experiments was to better understand the Wnt signaling system because it is found many animal species ranging from invertebrates to vertebrates. The Wnt signaling system is a pathway found in most animal species. It is one of the most important cell signaling systems because of its control over how and when cells divide, change into specific cell types, and move to the area of the body that they belong during embryonic development. This includes directly how stem cells allow for renewal of damaged tissues.

You can find more information on this pathway here: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/wnt-signaling-pathway

A series of linked chemical pathways in which one chemical in the series activates another chemical in the pathway, which ultimately leads to a specific cell function.

Using the process of science to identify and evaluate possible explanations for a natural phenomenon.

Click here to learn more about the process of science: https://media.hhmi.org/biointeractive/click/understanding-science/#/intro/4

Adenomatous polyposis coli, or APC, is gene that plays many roles, including acting as a tumor suppressor. APC has also been found to play a role in cell division and directing cells where to go once division takes place. In order to do its job, APC directs β-catenin in the Wnt signaling pathway.

Read more in the Journal of Cell Science. The PDF is also available in the "Related content" tab.

A mechanism used by both plant and animal cells to silence a gene using a double-stranded DNA molecule. DNA is converted into the smaller RNAi molecules used to turn genes off. Scientists are now able to use this natural process to turn off genes they are studying so they can learn more about their function.

Whether in a writing workshop or in a conference with a teacher or tutor, anxiety levels are almost always high. This protocol offers a time of rest and reflection for both the writer and reviewer.

These enhancements are not too difficult to wrap one's head around and begin to apply to other lessons. The student engagement and learning outcomes would be greatly increased by embracing such changes.

The Fractured Internet: Is It Too Late For Humpty Dumpy? -

https://www.youtube.com/watch?v=GPuKmV0zTA4

Once upon a time, the Internet operated more or less under the same rules everywhere around the globe. In principle, anyone, anywhere in the world, with access to the World Wide Web, could access the same content in the same way. That is no longer true.

Internet balkanization, or the "Splinternet," that many warned about for years is now becoming the status quo. Differing rules around the world regarding privacy and hate speech, law enforcement, China's Great Firewall, copyright and other IP protection, and individual countries' particular views on how they should be able to govern the Internet inside their borders means that, as a practical matter, we may no longer have a single Internet, but many Internets operating under different rules, with those connected unable to access certain content available elsewhere.

​Fiona Alexander, Distinguished Policy Strategist in Residence, School of International Service, American University

​Christopher Martin, Head of Region, Asia and US, Access Partners

Masahiko Nittono, General Manager and Chief Corporate Representative, NTT Corporation

​Shane Tews, Visiting Fellow, American Enterprise Institute

David Gross (moderator), Partner and Co-Chair, Telecom, Media and Technology Practice, Wiley Rein LLPBold

This is some fun imagery, picturing someone so giant that they tower above mountains and stretch into outer space. I love the poetic language!

It seems like the only child Zeus produced himself gained all these words of flattery that Zeus himself would want to be associated with. Awesome, a leader, fierce, a war winner, etc.

The story is pretty self explanatory and it appears as if you are telling this story to give a look into what life is like for the everyday soldiers of a war and how they reconcile the things they've done with their morality.

Us Versus Them Title- Could be catchier but I think it makes sense and is straightforward. I love analyzing movies so I think I am already drawn in my that. Content-

1. I think it is kinda hard to identify a thesis through images when it comes to analyzing, however the images are good in my opinion.
2. Yes, I think the thesis is clear what the thesis is through images and the title. The creator talks about the duality that is represented by the identical families, the scissors, etc. and the differences between the boy's face.
3. Yes it is clear that the creator will be doing what is mentioned above. There aren't specific details but based on my knowledge of the meanings, I could tell where the creators head is at and that they have fully developed the symbolism (it's just a lot to type out).
4. Yes the story board is very clear in its intentions.
5. I think talking about Jordan Peele's intentions could be interesting; he said it was just a horror movie with no commentary (I believe he said that in an interview) but it's not. It is complex and has a political meaning. I think he wanted to drum up conversation and even manipulated the audience into researching deeper (imo); there was a bible verse used over and over and over and it never said what it was. The first thing I did was google the bible verse and the top results were all about the movie Us. Images/media- I assume they will use images from the movie. I recommend using video clips that you can screen record on YouTube. Final thoughts- I think you could talk about his filmmaking style and how his history of political messaging. I also think it could be worth mentioning how he's helped to elevate the horror genre (not many have Academy Award winning actresses like Lupita Nyong'o) and bring diversity to it.

Out of left field, but: I suspect that the contemporary fantasy tropes of elves and fae mask this desire for indigeneity. (Typically white figures who have an elemental connection to nature yet have roots in Western Europe).

Keeping land rather than wealth at the center is also a transition from symbolic (money) to actual (land). My head is exploding.

Familial hemiplegic migraine is a form of migraine headache that runs in families. Migraines usually cause intense, throbbing pain in one area of the head, often accompanied by nausea, vomiting, and extreme sensitivity to light and sound.

Kafes Yüzey Örme Yöntemleri Önerileri

Sitting Bull (Lakota: Tȟatȟáŋka Íyotake [tˣaˈtˣə̃ka ˈi.jɔtakɛ];[2] nicknamed Húŋkešni [ˈhʊ̃kɛʃni] or "Slow"; c. 1831 – December 15, 1890)[3] was a Hunkpapa Lakota leader who led his people during years of resistance against United States government policies. He was killed by Indian agency police on the Standing Rock Indian Reservation during an attempt to arrest him, at a time when authorities feared that he would join the Ghost Dance movement.[4]

Before the Battle of the Little Bighorn, Sitting Bull had a vision in which he saw many soldiers, "as thick as grasshoppers," falling upside down into the Lakota camp, which his people took as a foreshadowing of a major victory in which many soldiers would be killed.[5] About three weeks later, the confederated Lakota tribes with the Northern Cheyenne defeated the 7th Cavalry under Lt. Col. George Armstrong Custer on June 25, 1876, annihilating Custer's battalion and seeming to bear out Sitting Bull's prophetic vision. Sitting Bull's leadership inspired his people to a major victory. In response, the US government sent thousands more soldiers to the area, forcing many of the Lakota to surrender over the next year. Sitting Bull refused to surrender, and in May 1877, he led his band north to Wood Mountain, North-Western Territory (now Saskatchewan). He remained there until 1881, at which time he and most of his band returned to US territory and surrendered to U.S. forces.

After working as a performer with Buffalo Bill's Wild West show, Sitting Bull returned to the Standing Rock Agency in South Dakota. Due to fears that he would use his influence to support the Ghost Dance movement, Indian Service agent James McLaughlin at Fort Yates ordered his arrest. During an ensuing struggle between Sitting Bull's followers and the agency police, Sitting Bull was shot in the side and head by Standing Rock policemen Lieutenant Bull Head (Tatankapah, Lakota: Tȟatȟáŋka Pȟá) and Red Tomahawk (Marcelus Chankpidutah, Lakota: Čhaŋȟpí Dúta), after the police were fired upon by Sitting Bull's supporters. His body was taken to nearby Fort Yates for burial. In 1953, his Lakota family exhumed what were believed to be his remains, reburying them near Mobridge, South Dakota, near his birthplace.

https://en.wikipedia.org/wiki/Sitting_Bull

Result statement 2:

Finding: significant What: correlation With: iron deposition: caudate, clinical: UDRS Who: all subjects

Finding: significant What: correlation With: cavitation: lateral globus pallidus, clinical: UDRS Who: all subjects

Writing Process

Useful strategies on what makes for a better writer.

For me:

• Emails
• Bug reports, tickets.
• Research
• Journals

Get comfortable and minimize distractions.

Planning

1. Make a timeline
2. Select a topic
3. Create an outline

Video Example:

Timeline

Note: Write down your goals and your progress as you go so you can make revisions for the next time you write.

Research

• 5 high quality sources => Take notes and outline your writing.

Writing

Good outlines make for good quality writing. Maybe show the outline?!

Topic sentences... which would be the questions you had and how they are answered by your research.

Research Pointers Head to the source

Does the content of this site pass by an editor before I saw it? Is the person that wrote this credible?

Take good notes and add to the outline. You might update your outline as well.

Write the bulk of your post for the rough draft.

Start setting goals for each sections. Look at the notes and re-arrange sentences. Write something down, no matter how bad the effort.

Talk yourself out-loud, give yourself encouragement, and while writing constantly monitor your progress.

After finishing your draft, you are way too attached to it, so sleep on it.

Revision

Does the order make sense?

T.opic<br> R.easons<br> E.xamine<br> E.nding

D.evelop a topic sentence<br> A.dd supporting ideas.<br> R.eject opposing arguments.<br> E.nd with a conclusion.

Drill down into the paragraphs and sentences and look at them closely.

MY biggest problem is "how do I diagnose the problem in the first place?"

Add images, figures, and formatting at the end.

Finally, proofread the post to make sure everything is done. After you read it, have a friend read it.

This happens in the Old Testament as well. The king requests his servant to kill him rather than being killed by a women. The servant refuses and the Biblical king falls on his sword. Men....

note to self: look at map to see/take note of the geography. New ideas are being brought upon them which are challenging traditions that hadn't been changed for numerous yrs

Motor behavior was assessed using amphetamine-induced rotations, head position bias, and locomotion.

Rotations were performed by injecting amphetamine 30 minutes prior to trial and placing the animal in an opaque cyclinder. Ipsilateral (same side as) rotations to the 6-OHDA lesion (clockwise) were added and contralateral (opposite side as) rotations were subtracted.

Head position bias was determined by the number of head tilts over time, where a greater than 10 degree deviation left or right from midline was measured.

Locomotion was measured using software called Viewer that tracked motion and calculated distance.

For sure, it is blasphemy. The angel of God is trying to save his soul for condemnation, he was advice to read the bible at the time of trial.

It seems like the Old Man is the last ditch effort to get Faustus to repent and turn his head back to Christ. Given that this late in the play, perhaps he really did have a choice to find salvation during all the times Faustus lamented over whether or not he still had a choice to turn back to God

This passage looks at the basis of the Headless Women and the struggles Vero places mentality from her hit and run. As audience members were reminded of her apparent privilege as a bourgeoise white woman in the film in comparison to villagers who are clearly lower class. The frame of the sunglasses being put on and taken off in the screen after the hit and run and at the sports field symbolizes the cloak of hierarchy she hides behind as her family and friends conspire to erase the crime she may or may not had committed.

This is about crucifixion of Jesus Christ. How Jesus died on the cross for the sins of mankind.

Looking back, the idea that outward physical features could indicated personality traits seems absurd, however we still seem to do this subconsciously now a days. The study I found, linked below, looked into appearance in relation to occupational stress and found that since photojournalism was widely used in elections (1888), 81% of elections resulted in the tallest presidential candidate winning. Height is frequently been associate with authority, much like cranium size was associated with mental capacity in phrenology. https://www.researchgate.net/publication/235365107_Evolution_Appearance_and_Occupational_Success

Nope

essentially

SciScore for 10.1101/2020.06.11.146522: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

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 comments and outline below how we plan to address them.

---

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful recombinants in E.coli. Traditionally, this is removed in a second recombination process to leave only the desired modification. The novelty in the procedure described herein is to add a synthetic intron consensus sequence around the selection cassette, which eliminates the need for the subsequent removal of the antibiotic cassette from the BAC before transfection into mammalian cells, saving time and resources. The technique is clever in its simplicity and appears to function for a number of gene loci. The authors validated the correct functioning of the modified BACs for a number of genes using three main assays - transcript level, protein level and localisation. **Major comments:** *Are the key conclusions convincing?* The conclusion that the method described generates functional modified BACs is valid. *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?* While the method is successfully employed in this study, its efficiency is not quantified in relation to the state-of-the-art as described in the introduction. One assumes it would be more efficient, but this has not been tested empirically in the paper. Does the inclusion of the synthetic intron sequence have an effect on the efficiency of modifying BACs compared to a more typical two-step positive/negative antibiotic selection cassette? *

• *

This is a good point that we did not directly address. In general, the efficiency is similar to that of integrating any cassette with selectable marker, as has been published (Poser et al 2008), and therefore also higher than the two-step counterselection method, which requires such a cassette integration in the first step alone. We will include new data specifically addressing the efficiency of our new method (see specifics below)

The functionality of this approach rests entirely on the ability of the target cell to correctly splice out the synthetic intron. The authors are aware of this potential problem as highlighted in the lines below, but do not make efforts to explicitly test splicing. On lines 224-225, the authors state "We cannot exclude that a small portion of synthetic introns within individual cells are misspliced". On lines 230-231 it is stated that "mis-spliced mRNAs are probably minimal and degraded by nonsense-mediated decay". On lines 215-217, the authors describe an "investigation of transgenic lines at the single-cell level" that suggests "the synthetic intron is correctly spliced out in all the cells of the population". How do the authors reach this conclusion? U2OS and HeLa cells are considered very "robust" and may not show detectable consequences when stressed with an increased level of nonsense-mediated decay. Further, many genes maintain a high level of expression that buffers them against small changes in transcription/splicing. The synthetic intron might have a bigger impact on more tightly regulated genes, so assessing the splicing rate would be essential if the authors wish to advocate their technique as generally applicable.

• *

We will assay for splicing efficiency as outlined below.

The ability of the synthetic intron to be removed from final transcripts depends on functioning splicing machinery. The authors might emphasise this issue, as spliceosome mutations are important fields of study and might not be compatible with this method.

• *

We can add this in the text

The authors used un-directed integration of each BAC under study. Therefore, it is hard to assess what effect the synthetic intron has, as the authors only ever assess the downstream levels of the correctly spliced, translated and localised protein. The authors themselves state that this can lead to clonal variations in expression of up to 2-fold and on line 250 that this variation "could compensate for synthetic intron effects", but make no effort to test this. Again, lines 267-268 highlight the potential dangers of potential effects of the synthetic introns, but do not test these. \Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.* If not already performed, a large number of bacterial colonies should be screened for the correct modification and frequency of correct ones reported. This frequency - reported for at least three different modifications - would estimate what sort of efficiency this method provides. The modified region of each BAC should be sequenced and the results reported. The rate of exactly modified clones is important, in case of spontaneous or low fidelity integration of the antibiotic cassette. The percentage of transcripts that have the synthetic intron correctly spliced out should be measured for some of the BAC constructs used in the study. A direct head-to-head comparison of this newer method compared to other techniques, or even the authors' own previous two-step approach is necessary to assess the benefits of this method. Preferably, the experiment would be run in parallel with and without antibiotic selection applied, to show that it drastically improves chances of finding a correct clone. *

We will generate 3 new mutations in BACs and analyze both the efficiency of integration by PCR and accuracy via sequencing. In practice, we have observed that the efficiency is similar to any other cassette integration, such as a GFP tag (Poser et al Nature Methods 2008) or a counterselection cassette (Bird et al Nature Methods 2012) (80-90%). Integrating a mutation via the second step of the counterselection method introduces a further 20% decrease in efficiencies on average.

\Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.* Repeating the transformation of the BAC and targeting cassette and assessing the recombination efficiency and sequencing should only require existing reagents and take less than a week or two to complete. Quantitative RT-PCR to assess the percentage of transcripts that have the synthetic intron spliced out would take a little more work. However, this should not be a considerable investment in time or resources for a standard microbiology laboratory and could be completed within a few weeks using modern techniques, such as that described in Londoño et al. 2016. Repeating all the experiments in parallel would be considerable work and would only be strictly necessary if the authors wish to emphasise the benefits of their method over the many others already in wide use. *

• *

We will use quantitative PCR to estimate the fraction of transcripts that correctly splice out the artificial intron for two clonal cell lines characterized in the study: RNAi-resistant AurA-GFP (Fig 4), and GTSE1-14A (newly introduced; see below). While the exact method described in Londoño et al 2016 will not be applicable due to the larger size of the artificial intron, we believe we can adapt it to detect different splicing events.

\Are the data and the methods presented in such a way that they can be reproduced?* Barring the omission of Table S1, which presumably includes exact information on the BACs modified and sequences used etc., there is sufficient other data and methods to allow the experiments to be repeated. Targeting the ESI procedure to the middle of exons is likely to have a bigger impact for smaller exons as the authors mention on lines 99-100. Making it clear which exon sizes for each gene were successfully targeted in this study would help give some idea of how significant a problem this might be. Perhaps Table S1 contains this information, but it was not provided. It would also help reviewers check the design strategies. *

We apologize for inadvertently failing to upload Table S1 on bioRxiv. It has been uploaded now as part of this submission process. This table indeed contains BAC and target sequence information, including the size of the targeted exon (and the 2 “new” resulting exons). Targeted exons range in size from 138bp to 1537bp, and “new” exons are as small as 48bp.

\Are the experiments adequately replicated and statistical analysis adequate?* The replication and statistically analysis of the data as presented appear adequate. Figure Legends should state the statistic used to generate error bars. *

This will be updated

\*Minor comments:** Specific experimental issues that are easily addressable. Are the promoters used in the vectors described universally functional? For example, is the PGK promoter functional in yeast? *

• *

The PGK promoter contained in the cassettes is a mammalian promoter, which has also been reported to work in flies.

\Are prior studies referenced appropriately?* The manuscript may benefit from the referencing of BAC modification techniques from a wider variety of groups, such as those using CRISPR-guided recombineering (Pyne et al. 2015). *

We will add citations of more techniques

\Are the text and figures clear and accurate?* The body text is very clear save minor typographical or grammatical errors. Regarding figures, some of the coloured text in Figure 1 is somewhat illegible when printed in grayscale. Line 278 - The acronyms LAP and NLAP are not defined/explained. Antibody section starting Line 282 may fit better next to Western Blot section. Figure 2C - The blot images would benefit from arrows to indicate expected sizes of proteins. Figure 3A - the graph may benefit from a dashed line at 100% to highlight that values are normalised to controls. Figure 4 - The differences between panels B & C are unclear. Figure 4E - The legend could provide a little more detail on cell cycle stage/status of the captured cells. *

All of the above will be addressed accordingly

\Do you have suggestions that would help the authors improve the presentation of their data and conclusions?* Lines 23-27 are somewhat unclear and feel out of context. Perhaps the authors could clarify this as a further advantage of using BACs instead of endogenous gene modifications. *

Thanks for the input, we will clarify this.

While not affecting the factual content of the paper, I would advocate that the authors format the method described in Figure S3 into a more detailed text based layout similar to that seen in a typical Nature Methods article. However, this may depend on the format required by any eventual publishing journal.

• *

We prefer the graphical protocol, but will discuss whether to add a text protocol with the journal editor.

That all of the work the paper was carried out in human cell lines and using human genes is a further caveat, but the authors admit this in the discussion and one would assume that most mammalian cells would respond similarly in their ability to splice out the synthetic intron. Reviewer #1 (Significance (Required)): \Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.* This work is a formal description of a newer method that could be useful for many of those employing bacterial artificial chromosomes in numerous studies, such as gene regulation. *Place the work in the context of the existing literature (provide references, where appropriate).* This work builds on methodology previously published by the authors - a counter-selection two-step procedure (Bird et al. 2011). It sets out to formally describe a method merely mentioned as "BAC intronization" in a later paper by some of the authors (Zheng et al. 2014). Other alternative one-step procedures are also available, but present a different set of challenges (Lyozin et al. 2014). Some newer approaches, such as those using CRISPR-guided recombineering (Pyne et al. 2015) or systems that combine CRISPR and positive/negative selection cassettes (Wang et al. 2016) may be slightly more efficient, but are also more complex in their design. Bird et al. 2011 DOI: 10/dv776q Pyne et al. 2015 DOI: 10/f7jx92 Wang et al. 2016 DOI: 10/f89db5 Zheng et al. 2014 DOI: 10/f5pkr6 *State what audience might be interested in and influenced by the reported findings.* As a technology paper this work should have interest from a broad field of research. While the use of BACs could sometimes be considered more traditional in light of the explosion in CRISPR-based genome editing capabilities, it is definitely seeing a resurgence as the limitations of CRISPR in modifying large regions of genome become more apparent. Therefore, technologies that accelerate the modification of BACs could prove increasingly useful. As category of audience, all those involved in significant recombineering or gene/genome engineering would potentially benefit. *Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.* Synthetic genomics, synthetic biology, cancer cell biology, gene and genome engineering REFEREES CROSS COMMENTING I would agree with reviewer two's assessment that we both view the paper in a similar light. Reviewer #2 (Evidence, reproducibility and clarity (Required)): This is a methods-focused paper that presents a strategy to efficiently introduce mutations into a bacterial artificial transgene using synthetic introns. BAC-based methods have been an effective strategy for introducing trans genes into human cells to achieve near-endogenous expression, including extensive work from these authors. However, generating mutations and changes within the internal coding sequence presents some challenges for how to target these mutations and select for the mutated form. Here, the authors describe a way to overcome this by introducing synthetic introns into an adjacent sequence. This allows them to introduce a selectable marker and conduct the molecular biology without creating complications downstream for the functionality of the protein. This method is carefully described and presented. The authors also provide clear validation by using this to create RNAi-resistant versions of multiple different mitotic factors as well as creating targeted mutants that alter the functional properties of a protein. This work clearly takes advantage of other ongoing studies from these labs (including mutants and cell lines that appear to also have been described elsewhere), but the ability to combine these in a single paper and clearly describe the method provides a helpful advance and validation. Based on the description and data presented, I think that things are clear and carefully validated. As such, I do not have technical comments or concerns and I would be comfortable with this paper appearing in an appropriate journal in its present form. Reviewer #2 (Significance (Required)): This is a solid methods paper, but for considering the nature of the impact and significance of this paper, there are several things to note: 1.The BAC-based method does appear to be a powerful and effective strategy. However, beyond the work of Mitocheck and the authors that are part of this paper, this has not seen widespread adoption. It is possible that this current method may increase its usage due to the value of the targeted mutations within the coding sequence, but at present it is not a broadly used strategy. *

We agree that using BACs as transgenes has not seen widespread adoption as a tool on the broader cell biology community (although certainly beyond members of the Mitocheck consortium). This is likely because many erroneously think that it is a technique for specialist laboratories. We are trying to change this! For reasons outlined below, there is still an increasing desire for conditional analysis of mutated genes under physiological expression/regulation frequently not attainable via directed Cas9-based mutation. A major aim of this paper is thus to further simplify the methods for generating modified BAC transgenes.

2.This BAC-based approach (and also RNAi) are becoming increasingly replaced by the use of CRISPR/Cas9 genome editing. The absence of Cas9-based strategies in this paper limits the potential impact and reach of this paper. The authors do mention the possibility of using a similar synthetic intron strategy for use with Cas9 in the Discussion, and appear to have conducted some experiments. If possible, it would substantially increase the value of this paper if this data and strategy were also included in the Results section (acknowledging that this may still be a work in progress).

While some uses of BAC transgenes are in some cases better replaced by CRISPR/Cas9 techniques (i.e. GFP tagging), there are several occasions where using BACs are preferable: As stated in the text, RNAi-resistant BACs allow for conditional analysis of recessive mutations. Mutations in essential genes that are lethal will prevent growth and recovery of viable cells if integrated into the genome via Cas9. Additionally, deleterious mutations are prone to accumulate suppressive changes in chromosome integrity or gene expression during the procedure of selecting and expanding Cas9-modified cells for analysis, particularly in the genomically instable cancer cell lines frequently employed.

We use both BACs and CRISPR/Cas9 in our lab according to our needs.

We do have an ongoing project to apply this intronization technique to enable more efficient selection of CRISPR/Cas9 integrations. Preliminary results suggest that it works to allow selection of point mutations, but it is still being optimized, including a redesign of the cassette, and is not ready for publication.

3.The method is solid and well-validated, but there are no new results or insights presented in this paper from the work that is described (this is fine, just commenting for considering the right journal fit).

As “biological insights” gained as a result of this technique we had cited a couple studies that made use of the technique already (to functionally analyze a microcephaly-associated mutation in the centriolar protein CPAP at the single cell level in HeLa cells and neural progenitor cells (Zheng et al 2014, Gabirel et al 2016)). As a response to this critique to include “new biology” in this paper, we will add new unpublished data investigating a specific question: Is the cell-cycle-regulated disruption of the EB1-GTSE1 (microtubule plus-end tracking proteins) interaction in mitosis required for chromosome segregation fidelity? We have generated a GTSE1 mutant with 14 phosphosites mutated to alanine using this technique. We will present the effect on chromosome segregation.

REFEREES CROSS COMMENTING It appears that both reviewers are largely on the same page regarding this paper.

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

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Referee #1

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). The authors here describe a method to modify bacterial artificial chromosomes (BAC) harbouring gene loci from eukaryotes. When wanting to modify a BAC an antibiotic selection cassette is often included alongside the desired mutation/modification to increase the number of successful recombinants in E.coli. Traditionally, this is removed in a second recombination process to leave only the desired modification. The novelty in the procedure described herein is to add a synthetic intron consensus sequence around the selection cassette, which eliminates the need for the subsequent removal of the antibiotic cassette from the BAC before transfection into mammalian cells, saving time and resources. The technique is clever in its simplicity and appears to function for a number of gene loci. The authors validated the correct functioning of the modified BACs for a number of genes using three main assays - transcript level, protein level and localisation.

Major comments:

Are the key conclusions convincing?

The conclusion that the method described generates functional modified BACs is valid.

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

While the method is successfully employed in this study, its efficiency is not quantified in relation to the state-of-the-art as described in the introduction. One assumes it would be more efficient, but this has not been tested empirically in the paper. Does the inclusion of the synthetic intron sequence have an effect on the efficiency of modifying BACs compared to a more typical two-step positive/negative antibiotic selection cassette? The functionality of this approach rests entirely on the ability of the target cell to correctly splice out the synthetic intron. The authors are aware of this potential problem as highlighted in the lines below, but do not make efforts to explicitly test splicing. On lines 224-225, the authors state "We cannot exclude that a small portion of synthetic introns within individual cells are misspliced". On lines 230-231 it is stated that "mis-spliced mRNAs are probably minimal and degraded by nonsense-mediated decay". On lines 215-217, the authors describe an "investigation of transgenic lines at the single-cell level" that suggests "the synthetic intron is correctly spliced out in all the cells of the population". How do the authors reach this conclusion? U2OS and HeLa cells are considered very "robust" and may not show detectable consequences when stressed with an increased level of nonsense-mediated decay. Further, many genes maintain a high level of expression that buffers them against small changes in transcription/splicing. The synthetic intron might have a bigger impact on more tightly regulated genes, so assessing the splicing rate would be essential if the authors wish to advocate their technique as generally applicable. The ability of the synthetic intron to be removed from final transcripts depends on functioning splicing machinery. The authors might emphasise this issue, as spliceosome mutations are important fields of study and might not be compatible with this method. The authors used un-directed integration of each BAC under study. Therefore, it is hard to assess what effect the synthetic intron has, as the authors only ever assess the downstream levels of the correctly spliced, translated and localised protein. The authors themselves state that this can lead to clonal variations in expression of up to 2-fold and on line 250 that this variation "could compensate for synthetic intron effects", but make no effort to test this. Again, lines 267-268 highlight the potential dangers of potential effects of the synthetic introns, but do not test these.

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

If not already performed, a large number of bacterial colonies should be screened for the correct modification and frequency of correct ones reported. This frequency - reported for at least three different modifications - would estimate what sort of efficiency this method provides. The modified region of each BAC should be sequenced and the results reported. The rate of exactly modified clones is important, in case of spontaneous or low fidelity integration of the antibiotic cassette. The percentage of transcripts that have the synthetic intron correctly spliced out should be measured for some of the BAC constructs used in the study. A direct head-to-head comparison of this newer method compared to other techniques, or even the authors' own previous two-step approach is necessary to assess the benefits of this method. Preferably, the experiment would be run in parallel with and without antibiotic selection applied, to show that it drastically improves chances of finding a correct clone.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Repeating the transformation of the BAC and targeting cassette and assessing the recombination efficiency and sequencing should only require existing reagents and take less than a week or two to complete. Quantitative RT-PCR to assess the percentage of transcripts that have the synthetic intron spliced out would take a little more work. However, this should not be a considerable investment in time or resources for a standard microbiology laboratory and could be completed within a few weeks using modern techniques, such as that described in Londoño et al. 2016. Repeating all the experiments in parallel would be considerable work and would only be strictly necessary if the authors wish to emphasise the benefits of their method over the many others already in wide use.

Are the data and the methods presented in such a way that they can be reproduced?

Barring the omission of Table S1, which presumably includes exact information on the BACs modified and sequences used etc., there is sufficient other data and methods to allow the experiments to be repeated. Targeting the ESI procedure to the middle of exons is likely to have a bigger impact for smaller exons as the authors mention on lines 99-100. Making it clear which exon sizes for each gene were successfully targeted in this study would help give some idea of how significant a problem this might be. Perhaps Table S1 contains this information, but it was not provided. It would also help reviewers check the design strategies.

Are the experiments adequately replicated and statistical analysis adequate?

The replication and statistically analysis of the data as presented appear adequate. Figure Legends should state the statistic used to generate error bars.

Minor comments:

Specific experimental issues that are easily addressable. Are the promoters used in the vectors described universally functional? For example, is the PGK promoter functional in yeast?

Are prior studies referenced appropriately?

The manuscript may benefit from the referencing of BAC modification techniques from a wider variety of groups, such as those using CRISPR-guided recombineering (Pyne et al. 2015).

Are the text and figures clear and accurate?

The body text is very clear save minor typographical or grammatical errors. Regarding figures, some of the coloured text in Figure 1 is somewhat illegible when printed in grayscale.

Line 278 - The acronyms LAP and NLAP are not defined/explained.

Antibody section starting Line 282 may fit better next to Western Blot section.

Figure 2C - The blot images would benefit from arrows to indicate expected sizes of proteins.

Figure 3A - the graph may benefit from a dashed line at 100% to highlight that values are normalised to controls.

Figure 4 - The differences between panels B & C are unclear.

Figure 4E - The legend could provide a little more detail on cell cycle stage/status of the captured cells.

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Lines 23-27 are somewhat unclear and feel out of context. Perhaps the authors could clarify this as a further advantage of using BACs instead of endogenous gene modifications.

While not affecting the factual content of the paper, I would advocate that the authors format the method described in Figure S3 into a more detailed text based layout similar to that seen in a typical Nature Methods article. However, this may depend on the format required by any eventual publishing journal. That all of the work the paper was carried out in human cell lines and using human genes is a further caveat, but the authors admit this in the discussion and one would assume that most mammalian cells would respond similarly in their ability to splice out the synthetic intron.

Significance

Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

This work is a formal description of a newer method that could be useful for many of those employing bacterial artificial chromosomes in numerous studies, such as gene regulation.

Place the work in the context of the existing literature (provide references, where appropriate).

This work builds on methodology previously published by the authors - a counter-selection two-step procedure (Bird et al. 2011). It sets out to formally describe a method merely mentioned as "BAC intronization" in a later paper by some of the authors (Zheng et al. 2014). Other alternative one-step procedures are also available, but present a different set of challenges (Lyozin et al. 2014). Some newer approaches, such as those using CRISPR-guided recombineering (Pyne et al. 2015) or systems that combine CRISPR and positive/negative selection cassettes (Wang et al. 2016) may be slightly more efficient, but are also more complex in their design.

Bird et al. 2011 DOI: 10/dv776q

Pyne et al. 2015 DOI: 10/f7jx92

Wang et al. 2016 DOI: 10/f89db5

Zheng et al. 2014 DOI: 10/f5pkr6

State what audience might be interested in and influenced by the reported findings.

As a technology paper this work should have interest from a broad field of research. While the use of BACs could sometimes be considered more traditional in light of the explosion in CRISPR-based genome editing capabilities, it is definitely seeing a resurgence as the limitations of CRISPR in modifying large regions of genome become more apparent. Therefore, technologies that accelerate the modification of BACs could prove increasingly useful. As category of audience, all those involved in significant recombineering or gene/genome engineering would potentially benefit.

Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Synthetic genomics, synthetic biology, cancer cell biology, gene and genome engineering

Where to find OER/Library publishing

These lines flow very smoothly. It creates a peaceful scene of Emily walking though the garden, picking flowers and singing. It almost makes Emily seem as if she is in her own world as she traverses the garden in what appears to be a blissful idealization of how anyone would go though a garden on a nice sunny day.

The griffin, griffon, or gryphon is a legendary creature with the body, tail, and back legs of a lion; the head and wings of an eagle; and sometimes an eagle's talons as its front feet

In Greek mythology, the Minotaur is a mythical creature portrayed in Classical times with the head and tail of a bull and the body of a man or, as described by Roman poet Ovid, a being "part man and part bull

Turning a gene off so that it does not go through transcription and translation (gene expression) which prevents the production of its protein product (β-catenin) being made by the cell.

Schneider and colleagues identified that β-catenin regulates dorsal-ventral polarity in vertebrates (animals with backbones).<br> Later research identified that β-catenin is modulated by the Wnt signaling pathway. It plays a role in forming groups of embryonic cells that direct the initial formation of the neural plate (basis for the nervous system) and the complete body axis (head vs feet ends of an organism) of the animal.

Several studies identified the role of β-catenin in regeneration of tissues and its potential relationship to degenerative diseases and cancer, which are both related to regulation of cell division. Identification of active β-catenin in adult tissue is important for potential treatment of these disease.

Watch this video to find out more about the relationship between cell division and cancer: https://www.hhmi.org/biointeractive/eukaryotic-cell-cycle-and-cancer

Stadeli and colleagues research indicates that β-catenin plays a major role in regulating the body pattern and head/tail orientation of cells during development.

The regulated breakdown of a cell or cellular product, which is used to control gene expression and ultimately cellular function. In this case, the breakdown of APC leads to an increase in β-catenin.

Silencing of β-catenin, both dishevelled homologs (Smed-dvl-1;Smed-dvl-2), or APC (Smed-APC-1) disrupted the ability of the cells to determine whether they belonged in the front or back of the animal during regeneration.

Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) planarian groups also developed two heads, as well as extra photoreceptor cells in locations where are they are not typically found on the anterior portion of the body.

With the knowledge from the previous experiment that regulation of β-catenin occurs earlier in the Wnt signaling pathway, the researchers were able to use the anterior/posterior markers to identify the time period during which the stem cells of the blastema differentiated in both the control and experimental animals.

Alvarado and colleagues surmised from the results of the previous experiment that β-catenin was regulated early in the blastema identify development. In comparing the migration and differentiation timing between both β-catenin and APC-1 with the control animals, they were able to confirm that indeed it occurred that blastema cell identity occurred very early.

In this set of experiments, Alvarado and colleagues were trying to identify if increased amounts of β-catenin regulates that expression of Smed-APC-1.

In this fourth set of experiments, the researchers wanted to identify the role of β-catenin at different amputation locations for the experiment animals while different genes were silenced and then comparing this to control animals with no genes silence but the same amputations. Amputations included removing the head or tail to see if the head or tail itself would regenerate. In addition, some animals were divided in half longitudinally (lenthwise) and some were divided in half mediolaterally (divided into anterior and posterior halves).

The results form the fourth set of amputation experiments suggests that β-catenin is regulated at locations of amputation.

The results of the observations of the unamputated animals with silenced genes allowed the researchers to identify that β-catenin plays a role in the anterior/posterior identity of cells in adult animals. This mechanism was not previously known.

This research identified previously unknown roles of β-catenin. Previous research identified the role it plays in embryogenesis with regard to cell fate and migration. However, identification of its multiple roles in adult tissue regeneration and homeostasis was not previously identified.

Find out another method Dr. Alvarado is using to identify the origin of the regenerative abilities in planarians in this article and watch the short video to find out more: https://www.hhmi.org/news/searching-source-planarians-regenerative-powers

Why is important to study planarians? This interview with the lead author of this study explains why its important to conduct research on understudied animals: https://biobeat.nigms.nih.gov/2019/10/interview-with-a-scientist-unlocking-the-secrets-of-animal-regeneration-with-alejandro-sanchez-alvarado/

The copying of DNA into mRNA, which is the first step in the process known as gene expression.

Watch this video to learn more about the role of transcription and transcription factors: https://www.hhmi.org/biointeractive/signal-molecules-trigger-transcription-factors

Regeneration in this case, refers to the replacement of lost or damaged tissue.

This video will help you understand the process of tissue regeneration: https://www.hhmi.org/biointeractive/tissue-regeneration-animals

In the fourth series of experiments using the various amputation location, it was noted that in some of the experimental animals that several photoreceptors and brains developed in the wrong location on the same animal. To understand why this happened, unamputated worms had the same genes silenced as the experimental animals and were observed for two weeks.

Both heads and tails that developed in the wrong locations functioned separate from the the movements of the rest of the animal. The RNAi silencing changed the cellular identity of the stem cells so that they migrated to the wrong location and incorrect cell differentiation.

The use of the anterior and poster markers will allow the researchers to determine if posterior cells are indeed moving to the posterior end and if anterior cells are moving to the anterior end in the RNAi treated animals.

Genes that are expressed only at the anterior or posterior ends were tagged to identify their location in both control and experimental animals while they are regenerating. This allows the researchers to identify the extent to which the stem cells were affected when silencing the proteins. Researchers can compare the control animal's cell migration to the the cell migration in the experimental animals for each of the silenced genes and gene combinations.

The results of the anatomical analysis indicate that the head located in the posterior region in the experimental animals resembled cells usually found in the anterior region as part of the nervous system. This indicates that stem cells did not locate to the correct position for a head when these genes were silenced.

By silencing this protein, stem cells were directed to both anterior and posterior regions of the body, but were directed to become anatomical tails, with not formation of brain tissue present in either end.

The cells that were supposed to be in the posterior end migrated to the anterior end of the animal, or vice versa.

In order to determine whether the changes in anterior/posterior identity were temporary changes that influenced the cells to act like a different cell type or whether DNA expression itself was permanently change, Alvarado and colleagues examined the location of the anatomical structures that regenerated. In addition, they identified the active molecular markers, which are chemicals that will help identify the activity of cells in a specific area to determine what type of cell they are. In this research, the markers direct the cells where to go (anterior or posterior). Some are active only for certain time periods of the regeneration process.

Silencing fo the Smed-APC-1 resulted in each end of the animal developing a tail.

The chemicals at the beginning of the signaling pathway.

How long the protein lasts as part of the signaling pathway.

The percentage or proportion of individuals with the genotype that present the phenotype. In other words, organisms may have the genes by may not show the trait. You can find more information about these terms in this article: https://www.ncbi.nlm.nih.gov/books/NBK22090/

This early research provides evidence for the importance of chemical signaling pathways as the major form of cell-to-cell communication and play a role in the body throughout an animal's lifetime.

Wallingford and colleagues found evidence for the important role that the protein known as Dishevelled plays in cell fate (how cells know what type of cell to become); how cells know where to go (head or tail), and how cells interact with each other in the animal body. Dishevelled has been found to play this same role across many animal species, which indicates its importance.

The ability or tendency of an organism to maintain internal stability. This includes maintaining certain ranges of physiological processes such as temperature, pH, or ions such as calcium.

Freely swimming flatworms from the taxonomic class Turbellaria that inhabit freshwater.

For most organisms it involves removal of a limb. Planaria do not have limbs, so in this case refers to removing either the front or hind end of the body.

how much of digg's demise is caused by this v4 technical fail?

Stevenson uses humor here to great effect because he's able to highlight the issues with trying a child as an adult. If we can make it happen one way, why not the other way too?

The first line has alliteration starting with "w" while the other has alliteration starting with "d." This helps separate the ideas of the two lines as well as create the contrasting images in the reader's head by only using words with different starting letters. The contrast here is between the winter setting in the first line and the drought setting in the next.

In the lines where they give imagery of hunting, the diction used may be the element that makes it the first ecosystem/interactive nature poem. The words they use are very violent such as "slit, skewered, bathed in blood." Maybe this is used to convey how savage hunting can be at the magnitude they were doing it at (a crowd of men on horses with hounds and guns)

Green is obviously a big deal in this story. Off the top of my head, I associate green with life, nature, rebirth, and other things in that area. I don't see this being super related to the story itself though? Maybe I'm accidently skipping over some nature appreciation parts.

Interesting that when Arthur had the axe he extended himself to his full height but now that Gawain has the axe, he leans down to show his neck.

Green knight challenged King Arthur to a beheading game. A beheading game is a trope or of medieval romance in which the players exchange blows which could decapitate their opponent. The most famous example is in the poem Sir Gawain and the Green Knight. He cuts the knight’s head off in a single blow; then, the beheaded knight picks his head up and instructs Gawain to seek him out in one year and one day so he may return the blow.

Sorry, but I'm still not very clear on the nature of the argument you are having with yourself. Is it a question you have about whether you are doing enough? Whether you are living up to some larger responsibility? Is that it?

LRAD better for public police relations

网络综艺

和韩国CJ ENM合作，Mondo TV 是发行，在第二季，Mondo成为了联合制作方，并且负责整个制作。

和Toon2Tango合作

Headhunting is a practice that involves prospecting a candidate for a specific position. using effective Headhunting techniques the hiring becomes effective.

Which king? If possible this information should be provided. If not available, then it should be indicated that such information is not available.

I think the writer meant to write "horse's head and hoof."

We carry a lot in our head, by writing you could lay it all down and clear your head for other things and even sleep. Plus when you actually think about gratitude, you become happy and then you are easier to clear your head and fall asleep.

Logical Pointers:

logical pointer approach is better for write-intensive workloads, as the DBMS updates the secondary indexes only when a transaction modifies the indexes attributes.

Logical Pointers: TupleId

Logical Pointers: PKey

N2O

Version Storage: head of chain

It is common for people to beat around the bush rather than just being straight forwards with the truth

More details at: https://smarthistory.org/benoist-portrait/

I think what's most offensive about this write-up is not Mark's assumption that whites and men "just have it fine and dandy!" — it's that he assumes minorities, say, are uniformly pro-identitarianism and are fully on board with everything he's saying about what they supposedly experience. It reveals how he's never bothered to peep his head above the trenches to see who the other side actually is.