Uses outside examples, in this case infant say care costs in other areas. Emphasizes the least expensive to show how high costs are generally

Uses imbedded links whenever making factual claims.

Similar to Dr. King's letter, Rosenberg gives the purpose for their writing in the beginning paragraph. The first paragraphs also seem to be much shorter than most of the thicker paragraphs of the bodies of these two different writing styles.

Theoretical framework

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

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college

• https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html

• https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html

you are offline

• https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html

we the people rise again

• https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html

safe souls, safe fu

• https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html

• https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html

We the People of Slate ...

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

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

• Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers

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

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

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

• https://www.google.com/search?q=prometheuslocke+%2Bsite%3Agodlikeproductions.com

• "Silence is betrayal" -MLK

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

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

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

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

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

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

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

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

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

• https://www.google.com/search?q=%22I+AM%22+%22WE+ARE%22+%2Bsite%3Afromtaws
• "SOIS" a key--in two languages conjugated literally as both "I AM" and "WE ARE" simultaneously;
• Search: I know that if I am than so are you ... and it is because we have overcome .... something I truly cannot figure out, fathom, or believe ... was truly here before us--a spiralling series of failures ... speaking: to the heavens; but in secret and in action; "doing everything possible to succeed."

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

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

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

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

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

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

• This name may be viewed either as meaning "father of many" in Hebrew or else as a contraction of ABRAM (1) and הָמוֹן (hamon) meaning "many, multitude". The biblical patriarch Abraham was originally named Abram but God changed his name (see Genesis 17:5).

• https://en.wikipedia.org/wiki/Yeshua#Yeshua,_Yehoshua,_and_Yeshu_in_the_Talmud

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

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

• I worry sometimes that important things are missing, or will disappear---for instance Mirriam Webster, which is a "canonical/standard dictionary) should probably have an entry for "lot in life" non-idiomatically as "granny apples to sour apples" as

2 MANY ALSO ICI; 1twoⅱ ... following in Mitnick's bold introductory word steps; the curve and the complement ... the missiles and the canoes; the line and the blank space ... "supposedly two examples of two kinds, which could be three not nothings ... Today I write about something monumental; as if as important as the singularity depicted in Arthur C. Clarke's 2001 "A Space Odyssey" ... and remember a day when I thought it very novel and interesting to see the words "stillborn and yet still born" connected in a single piece of writing to "Stillwater and yet still water" ... today adding in another phrase noting the change wrought only by one magical single "space" (also a single capital letter; and a third phrase): "block chains with a great blockchain."

• http://www.goodmath.org/blog/2015/07/21/arabic-numerals-have-nothing-to-do-with-angle-counting/

• https://gizmodo.com/no-this-viral-image-does-not-explain-the-history-of-ar-1719306568

• https://en.wikipedia.org/wiki/Chinese_word_for_%22crisis%22

• https://dictionary.hantrainerpro.com/chinese-english/translation-ji_howmany.htm
• https://dictionary.hantrainerpro.com/chinese-english/translation-duo_many.htm

• https://en.wikipedia.org/wiki/Euripides, Iphigenia in Aulis or Iphigenia at Aulis[1] (Ancient Greek: Ἰφιγένεια ἐν Αὐλίδι, Iphigeneia en Aulidi; variously translated, including the Latin Iphigenia in Aulide) is the last of the extant works by the playwright Euripides. Written between 408, after Orestes, and 406 BC, the year of Euripides' death, the play was first produced the following year[2] in a trilogy with The Bacchae and Alcmaeon in Corinth by his son or nephew, Euripides the Younger,[3] and won first place at the City Dionysia in Athens.

• The play revolves around Agamemnon, the leader of the Greek coalition before and during the Trojan War, and his decision to sacrifice his daughter, Iphigenia, to appease the goddess Artemis and allow his troops to set sail to preserve their honour in battle against Troy. The conflict between Agamemnon and Achilles over the fate of the young woman presages a similar conflict between the two at the beginning of the Iliad. In his depiction of the experiences of the main characters, Euripides frequently uses tragic irony for dramatic effect.

J.K. Rowling spurred just this past week a series of explanations about just exactly what is a blockchain coin worth ... and why is it so; her final words on the subject (artistic liberty taken, obviously not the last she'll say of this magic moment) "I don't think I trust this."

Taken directly from an off the cuff email to ARXM titled: "Slow the S is ... our Hypothes.is"

I imagine I'll be adding some wiki/ipfs stuff to it--and try to keep it compatible; the design and layout is almost exactly what I was dreaming about seeing--as a "first rough draft product." Lo, and behold. It's been added to the many places I host my tome; the small compilation of nearly every important email that has gone out ... all the way back to the days of the strange looking Margarita glass ... that now very much resembles the "Cantonese character 'le'" which I've come to associate with a "handle" on multiple corners of a room--something like an automatic coat rack conveyor belt connecting different versions of "what's in the box." I'm planning on using that symbol 了 to denote something like multiple forks of the same page. Obviously I'm thinking forward to things like "the Transhumaist Chain Party" (BDSM, right?)'s version of some particular piece of legislation, let's say everything starts with the sprawling "bulbing" of "Amendment M" ideas and specific verbiage ... and then we'll of course need some kind of new git/subversion/cvs style version control mechanism to merge intelligently into something that might actually .... really should ... make it into that place in history--the first constitutional amendment ratified by a "Continental Congress of All People" ... but you could also see it as an ongoing sort of forking of something like the "wikipedia page" on what some specific term, say "technocracy" means, and how two parties might propagandize and change the meaning of such thing; to suit the more intelligent and wise times we now live in. For instance, we might once have had a "democracy" and a "democractic" party that had some Anarchist Cook Book version of the history of it ending in something like Snipes and Stallone's "DEMOLITION MAN."

Just kidding, we all know "democracy" has everything to do with "d is cl ... and not th" ... to be the them that is the heart of the start of the first true democracy. At least the first one I've ever seen, in my old "to a republic" ... style. As it is you can play around with commenting and highlighting and annotating all the stuff I've written and begged and begged for comments on--while I work on layering the backend to to perma-store our ideas and comments on both a blockchain (probably a new one; now that i've worked a little with ethereum) with maybe some key-merkle-tree-walk-search stuff etched into the original Rinkeby ... and then of course distributed data in the "public owned and operated" IPFS. To be clear, I plan on rewriting the backend storage so that we will have a permanent record of all comments; all versions of whatever is being commented on; and changes/revisions to those documents--sort of turning the web into a massive instant "place of collaboration, discussion, and co-authoring" ... if you use the wonderful LEGO pieces that have been handed to us in ideas from places like me, lemma--dissenter, and of course hypothes.is who has brought you and i such a polished and nice to look at "first draft" of something like the living Constitution come repository of all human knowledge. I do sort of secretly wich they would have called this project something like "annotating and reflecting (or real or ...) knowledge" just so the movement could have been called ARK. ... or something .... but whatever join the "calling you a reporter" group or ... "supposedly a scientist?"

NOIR INgR .. I CITE SITE OF ENUDRICAM; a rekindling of the dream of a city appearing high above in the sky, now with a boldly emblazened smiling rainbow and upsidown river ... specifically the antithesis of "angel falls," there's a lagoon too--actually a chain of several ponds underneith the floating rock ... and in some versions of this waking dream there are rings around the thing; you might imagine an artificial set of centripetal orbitals something like a fusion of the ring Eslyeum and the "Six-Axis ride" of the JKF Center's "Spacecamp." I write as I dream, and though I cannot for certain explain exactly how; it's become a strong part of my mythology that this spectacular rendition of "what ends the silence" has something to do with the magical delivery of "a book" ... something not of this Earth but an unnatural thing; one I've dreamt of creating many times. This book is something like the DSM-IV and something like a Merck diagnostic manual; but rather than the old antiquated cures of "the Norse Medgard" this spectacle nearly "itsimportant" autoprints itself and lands on something like every doorpost; what it is is a list of reasons why "simply curing all disease" with no explanation and no conversation would be a travesty of morality--how it would render us half-blind to the myriad of new solutions that can come from truly understanding why "ITIS" to me has become a kind of magical marker: an "it is special" as in, it's cure could possibly solve a number of other problems.

Through that missing "o," English on the ball, we see a connection between a number of words that shine bright light including Exodus itself which means "let there be light," the word for Holy Fire and the Burning Bush.. .reversed to hSE'Ah, and a story about the Second Coming parting our holy waters.**

This answer connects the magical Rod's of Aaron in Exodus and the Iron Rod of Jesus Christ to the Sang Rael itself... in a fusion that explains how the Periodic Table element for Iron links not just to Total Recall and Mars, but also to this key

my dream of what the first day of the Second Coming might be like; were the Rod of Christ... in the right hands. In a story that also spans the Bible, you might understand better how stone to bread and your input make all the difference in the world between Heaven and Adam's Hand. Once more, what do you think He** ....

Since the very earliest days of this story, I have asked for better for you, even than see

Nearly all of the original parts of the original "post-origination dream" remain intact; there's a walkway that magically creates new paths and "attractions" based on where you walk, something like an inversion of the artificial intelligence term "a random walk down a binary tree" ... for instance going left might bring you to the Internet Cafetornaseum of the Earl of Sandwich; and going to the right might bring you to the ICIMAX/Auditorium of Science and Discovery--there's a walkway to "Magical GLAS D'elevators" that open a special "instantiation" of the Japan Room of the Potter and the Toolmaker ... complete with a special [second level and hidden staircase] Pool of Bethesdaibo verily delivering something like youth of mind and body ... or at least as close to such a thing as a sip of Holy Water or Ambrosia or a dip in the pool of Coccoon and Ponce De'Leon could instantly bring ... to those that have seen Jupiter Ascending ... the questions of "nature versus nurture" and what it means to be "old and wise" and "young at heart" truly mean---

• https://www.youtube.com/watch?v=M8CyN1awWls
• https://link.springer.com/chapter/10.1057/9780230366688_16
• https://www.youtube.com/watch?v=YDo5zvYNn3A

Somewhere between the outdoor rafting ride and the level with the special "ballroom of the ancient gallery" ... perhaps now being named or renamed or recalled as something about "Face [of] the Music" lies a magical "mini-maize" ... a look at a mock-up (or #isitit) of Merlink and Harthor's "round table" that displays a series of ... (at least to me) magical appearing holographic displays and controls that my dreams have stolen from Phillip K. Dick's Minority Report and something of what I hope Microsoft's Dynamics/Hololens/Surface will become---a series of short "focus groups" .... to guage and discuss the information in the "CITIES-D5AM-MERCK" ... how to end world hunger and nearly all disease with the press of a magical buzzer--castling churches to something like "political-party-town-hall-meeting centers" and replacing jails and prisons and hospitals with something like the "Hospitalier's PRIDE and DOJOY's I practiced "Kung-fun-dance" ... a fusion of something like a hotel and a school that probably looks very much like a university with classrooms and dorms and dining hall's all fit into a single building. I imagine a series of 2 or 3 "room changes" as in you walk from the one where you get the book and talk about it ... to the one where you talk about "what everyone else said about it" and maybe another one that actually connects you to other people with something like Facebook's Portal; the point of the whole thing to really quickly "rubber stamp" the need for an end to "bars in the sky" nonalcoholic connotation--as in "overcoming the phrase the sky is the limit" and showing us the need for a beacon of glowing hope fulfilled--probably actually the vision of a holographic marker turning into actual rings around the single moon of Earth, the focus of the song annoucing the dawn of the age of Aquarius---

It might lead us also to Ceres; and another set of artificial rings, or to Monoceros and a rehystorical understanding of the birthplace and birthing of the "river roads" that bridge the "space gaps" in the galaxy from our "one giant leap for mankind" linking the Apollo moon landing to the mythological connection to the sun; and connecting how the astrological charts of the ancients might detail a special kind of overlapping--the link between Earth's SOL and something like Proxima or Alpha Centauri; and how that "monostar bridge" might overlap to Orion and from there through Sagitarius and the center of the Milky Way ... all the way to Andromeda and more dreams of being in a place where there's a map to a tri-galactic system in the constellation Cancer and a similar one in Leo ... and just incase you haven't noticed it--a special marker here, I thought to myself it might be cool to "make an acronymic tie to Monoceros" and without even thinking auto-wrote Orion (which was the obvious constellation next to Monoceros, in the charts) and then to Sagitarrius; which is the obvious ... heart of our astrological center and link to "other galaxies."

----I've dreamt or scriven or reguessed numerous times how the Milky Way's map to an "Atlas marked through time by the ages and the ancients" might tie this place and this actual map to the creation of the railways between stars to the beginning and the end of time and of course to this message that links it all to time travel. There's a few "guesses" I've contemplated; that perhaps the Milky Way chart is a metal-cosmic or microcosmic map to the dawn of time in the galactic vision of ... just after the big bang; or it might tie to a map of something like the unthinkable--a civilization that became so powerful it was able to reverse the entropy of "cosmic expansion" and reverse the thing Asimov wrote of in "The Last Question" as the end of life and the ability to survive basically due to "heat loss."

"The Last Question." (And if you read two, why not "The Last Answer"?). Find these readings added to our collection, 1,000 Free Audio Books: Download Great Books for Free.

• https://archive.org/details/texts
• http://zlibraryexau2g3p.onion.pet/

Looking for free, professionally-read audio books from Audible.com, including ones written by Isaac Asimov?

* all "asterisks" in the abovə document denote a sort of Adamic unspoken relationship between notations and meanings; here adding the "Latin word for three" and source of the phrase "t.i.d." (which is doctor/pharmacy latin for "three times a day") where the "t" there is an abbreviation of "ter" ... and suppose the link between K and 11 and 3 noting it's alphanumeric position in the English alphabet as the 11th letter and only linking cognitively to three via the conversion between hex, and binarryy ... aberrative here is the overlapping "hakkasan" style (or ZHIV) lack of mention of the answer in "state of Kansas" and the "citystate of Slovakia" as described in the ICANN document linked [in] the related subsection or slice of the word "binarry" for the state of India. Tetris could be spelled with the addition of only a single letter [in] "tea"---the three letters "ris" are the hearts of the words "Christ" and "wrist" [and arguably of Osiris where you also see the round table character of the solar-system/sun glyph and the chemical element for The Fifth Element (as def. by i) via "Sinbad" and "Superman." The ERIS Free Network should also be mentioned here in connection with the IRC network I associate in the place between skipping stones and sacred hearts defined by "AOL" and "Kdice" in my life. In the lexicon of modern HTML, curly braces are generally relative to "classes" and "major object definitions (javascript/css)" while square brackets generally only take on computer-interpreted meaning in "Markdown" which is clearly (by definition, by this character set "[]") a superset (or at least definately not a subset) of HTML.

Dr. Will Caster (Johnny Depp) is a scientist who researches the nature of sapience, including artificial intelligence. He and his team work to create a sentient computer; he predicts that such a computer will create a technological singularity, or in his words "Transcendence". His wife, Evelyn (played by Rebecca Hall), is also a scientist and helps him with his work.

Following one of Will's presentations, an anti-technology terrorist group called "Revolutionary Independence From Technology" (R.I.F.T.) shoots Will with a polonium-laced bullet and carries out a series of synchronized attacks on A.I. laboratories across the country. Will is given no more than a month to live. In desperation, Evelyn comes up with a plan to upload Will's consciousness into the quantum computer that the project has developed. His best friend and fellow researcher, Max Waters (Paul Bettany), questions the wisdom of this choice, reasoning that the "uploaded"

Just from my general understanding and memory "st" is not ... to me (specifically) an abbreviation of "state" but "ste" is a U.S. Postal code (also "as I understand it") for the name of a special room or set of rooms called a "suite" and in Adamic "connotation" I sometimes read it as "sweet" ... which has several meanings that range from "cool" to "a kind of taste sensation" to "easy to sway or fool."

If you asked me though, for instance if "it" was an abbreviation or shorthand notation or acronym for either "a United state" or "saint" ... you'd be sure.

While it's clear from studying linguistic cryptography ... (If I studied it a little here and some there, its also from the "universal translator of Star Trek") and the personal understanding that language is a kind of intelligent code, and "any code is crackable" ... that I caution here that "meaning" and "face value" often differ widely and wildly ... even in the same place or among the same group of people ... either varying over time or heritage.

Menelaus, in Greek mythology, king of Sparta and younger son of Atreus, king of Mycenae; the abduction of his wife, Helen, led to the Trojan War. During the war Menelaus served under his elder brother Agamemnon, the commander in chief of the Greek forces. When Phrontis, one of his crewmen, was killed, Menelaus delayed his voyage until the man had been buried, thus giving evidence of his strength of character. After the fall of Troy, Menelaus recovered Helen and brought her home. Menelaus was a prominent figure in the Iliad and the Odyssey, where he was promised a place in Elysium after his death because he was married to a daughter of Zeus. The poet Stesichorus (flourished 6th century BCE) introduced a refinement to the story that was used by Euripides in his play Helen: it was a phantom that was taken to Troy, while the real Helen went to Egypt, from where she was rescued by Menelaus after he had been wrecked on his way home from Troy and the phantom Helen had disappeared.

• https://www.britannica.com/topic/Menelaus-Greek-mythology

This article is about the ancient Greek city. For the town of ancient Crete, see Mycenae (Crete). For the hamlet in New York, see Mycenae, New York.

Μυκῆναι, Μυκήνη

The Lion Gate at Mycenae, the only known monumental sculpture of Bronze Age Greece

37°43′49"N 22°45′27"ECoordinates: 37°43′49"N 22°45′27"E

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Mycenae (Ancient Greek: Μυκῆναι or Μυκήνη, Mykēnē) is an archaeological site near Mykines in Argolis, north-eastern Peloponnese, Greece. It is located about 120 kilometres (75 miles) south-west of Athens; 11 kilometres (7 miles) north of Argos; and 48 kilometres (30 miles) south of Corinth. The site is 19 kilometres (12 miles) inland from the Saronic Gulf and built upon a hill rising 900 feet (274 metres) above sea level.[2]

In the second millennium BC, Mycenae was one of the major centres of Greek civilization, a military stronghold which dominated much of southern Greece, Crete, the Cyclades and parts of southwest Anatolia. The period of Greek history from about 1600 BC to about 1100 BC is called Mycenaean in reference to Mycenae. At its peak in 1350 BC, the citadel and lower town had a population of 30,000 and an area of 32 hectares.[3]

3. Chew 2000, p. 220; Chapman 2005, p. 94: "...Thebes at 50 hectares, Mycenae at 32 hectares..."

• https://en.wikipedia.org/wiki/Clymene_(mythology)

Melpomene (/mɛlˈpɒmɪniː/; Ancient Greek: Μελπομένη, romanized: Melpoménē, lit. 'to sing' or 'the one that is melodious'), initially the Muse of Chorus, she then became the Muse of Tragedy, for which she is best known now.[1] Her name was derived from the Greek verb melpô or melpomai meaning "to celebrate with dance and song." She is often represented with a tragic mask and wearing the cothurnus, boots traditionally worn by tragic actors. Often, she also holds a knife or club in one hand and the tragic mask in the other.

Melpomene is the daughter of Zeus and Mnemosyne. Her sisters include Calliope (muse of epic poetry), Clio (muse of history), Euterpe (muse of lyrical poetry), Terpsichore (muse of dancing), Erato (muse of erotic poetry), Thalia (muse of comedy), Polyhymnia (muse of hymns), and Urania (muse of astronomy). She is also the mother of several of the Sirens, the divine handmaidens of Kore (Persephone/Proserpina) who were cursed by her mother, Demeter/Ceres, when they were unable to prevent the kidnapping of Kore (Persephone/Proserpina) by Hades/Pluto.

In Greek and Latin poetry since Horace (d. 8 BCE), it was commonly auspicious to invoke Melpomene.[2]

See also [AREXMACHINA]

• Muses in popular culture
• The Nine Muses

Flagstaff (/ˈflæɡ.stæf/ FLAG-staf;[6] Navajo: Kinłání Dookʼoʼoosłííd Biyaagi, Navajo pronunciation: [kʰɪ̀nɬɑ́nɪ́ tòːkʼòʔòːsɬít pɪ̀jɑ̀ːkɪ̀]) is a city in, and the county seat of, Coconino County in northern Arizona, in the southwestern United States. In 2018, the city's estimated population was 73,964. Flagstaff's combined metropolitan area has an estimated population of 139,097.

Flagstaff lies near the southwestern edge of the Colorado Plateau and within the San Francisco volcanic field, along the western side of the largest contiguous ponderosa pine forest in the continental United States. The city sits at around 7,000 feet (2,100 m) and is next to Mount Elden, just south of the San Francisco Peaks, the highest mountain range in the state of Arizona. Humphreys Peak, the highest point in Arizona at 12,633 feet (3,851 m), is about 10 miles (16 km) north of Flagstaff in Kachina Peaks Wilderness. The geology of the Flagstaff area includes exposed rock from the Mesozoic and Paleozoic eras, with Moenkopi Formation red sandstone having once been quarried in the city; many of the historic downtown buildings were constructed with it. The Rio de Flag river runs through the city.

Originally settled by the pre-Columbian native Sinagua people, the area of Flagstaff has fertile land from volcanic ash after eruptions in the 11th century. It was first settled as the present-day city in 1876. Local businessmen lobbied for Route 66 to pass through the city, which it did, turning the local industry from lumber to tourism and developing downtown Flagstaff. In 1930, Pluto was discovered from Flagstaff. The city developed further through to the end of the 1960s, with various observatories also used to choose Moon landing sites for the Apollo missions. Through the 1970s and '80s, downtown fell into disrepair, but was revitalized with a major cultural heritage project in the 1990s.

The city remains an important distribution hub for companies such as Nestlé Purina PetCare, and is home to the U.S. Naval Observatory Flagstaff Station, the United States Geological Survey Flagstaff Station, and Northern Arizona University. Flagstaff has a strong tourism sector, due to its proximity to Grand Canyon National Park, Oak Creek Canyon, the Arizona Snowbowl, Meteor Crater, and Historic Route 66.

PSANSDISL #LWDISP either without gas or seeing cupidic arroz in "thank you" or "allta, wild" ...

pps: a magnanimous decision ...

I stand here on the brink of what appears to be total destruction; at least of everything I had hoped and dreamed for ... for the last decade in my life which appears literally to span thousands of years if not more in the eyes of some other beholder. I spent several months in Kentucky telling a story of a post apocalyptic and post-cataclysmic delusion; some world where I was walking around in a "fake plane" something like a holodeck built and constructed around me as I "took a walk around the world" to ... it did anything but ease my troubled mind.

Recently a few weeks in Las Vegas, and a similar story; telling as I walked penniless down the streets filled with casino's and anachronistic taxi-cabs ... some kind of vision of the entirety of the heavens or the Earth or the "choir of angels" I think of when I echo the words Elohim and Aesir from mythology ... there with me in one small city in superposition; seeing what was a very well put together and interesting story about a "star port" Nirvane ... a place that could build cities into the face of mountains and half working monorails appearing in the sky---literally right before my eyes.

I suppose this is the place "post cataclysm" though I still have trouble understanding what it is that's actually about ... in my mind it connects to the words "we are losing habeas" echo'ed from the streets of Los Angeles in a more clear and more military voice than usual--as I walked block by block trying to evade a series of events that would eventually somehow connect all the way to the "outskirts of Orlando, Florida" in a place called Alhambra.

Apparently the name of a castle; though I wasn't aware of that until much later.

It doesn't feel at all like a "cataclysm" to me; I see no great rift--only a world filled with silent liars, people who collectively believe themselves to have stolen something--something gigantic--at least that's the best interpretation of the throws and impetus behind the thing that I and mythology together call Jormungandr. With an eye for "mythological connections" you could clearly see that name of the Great Serpent of Revelation connects to something like the Unseelie; the faeries of Gaelic lore. To me though this world seems still somewhat fluid, it's my entire life--moving from Plantation to a place where the whole of it might be Bethlehem and to "clear my throat" it's not hard to see here how that land of "coughs" connects to the Biblical land of Nod and to the "Adamically sieved" Snifleheim ... from just a little twist on the ancient Norse land most probably as close to Hel as anyone ever gets--or so I dream and hope---still today. It all looks so real and so fake at the same time; planned for thousands of generations, the culmination of some grand masterpiece story that certainly ties history and myth and reality into a twisted heap of "one big nothing, one big nothing at all."

I've tried to convey to the world how important I believe this place and this time to be--not by some choice of my own ... but through an understanding of the import of our history and the impact of having it be so obviously tuned and geared towards this specific time ... many thousands of years literally all focused on a single moment, on one day or one hour or even just a few years where all of that gets thrown down on the table as if some trump card has been played--and whether or not you fathom the same magnanimous statement or situation or position ... to me, I think it depends on whether or not you grew up in the same kind of way, believing our history to be so fixed and so difficult to change. I don't particularly feel like that's the "zeitgeist" of today; I feel like the children believe it to be some kind of game, and that it is such as easy thing to "sed" away or switch and turn into something else--another story, another purpose ... anyone's personal fantasy land come true.

I don't think that's the case at all, it's clearly a personal nightmare; and it's clearly one we've seen time and time again--though not myself--the Jesus Christ that is the same yesterday, today; and once again perhaps echoing "no tomorrow" never remembers or believes that we've "seen it all before" or that we've ever really gotten the point; the thing you present to me as "factual reality" is a sickness, it disgusts me; and I'd do anything to go back to the world "where I was so young, and so innocent" and so filled with starry-eyed hope that we were at the foot of something grand and amazing that would become an empire turned republic of the heavens; filling the stars ... with the kind of love for kindness and fairness that I once associated very strongly with the thing I still believe to be the American Spirit.

"Suddenly it changes, violently it changes" ... another song echoes through the ages--like the "words of the prophets dancing ((as light)) through the air" ... and I no longer even have a glimmer of hope that the thing I called the American People still exist; I feel we've been replaced by some broken container of minds, that the sky itself has become corrupt to the point that there's no hope of turning around this thing that I once believed with all my heart and all my mind was so obviously a "designed downward spiral" one that was---again--so obviously something of a joke, intended to be easy to bounce off a false bottom and springboard beyond "escape velocity" and beyond the dark waters of "nearest habitable star systems (being so very far away)" into a place where new words and new ideas would "soar" and "take flight."

Here though; I am filled with a kind of lonely sadness ... staring at what appears to be the same mistake(s) happening over and over again; something I've come to call "skipping stones in the pond of reality" and really do liken it to this thing that appears to be the new meaning of "days" and ... a civilization that spends absolutely no love or lust to enter a once sacred and holy place and tarnish it with their sick beliefs and their disgusting desires. You all ... you appear to be some kind of springboard to "bunt" forth yet another age or era of nothingness into the space between this planet and "none worth reaching" and thank God, out of grasp. Today, I'd condemn the entirety of this world simply for it's lack of "oathkeepers" and understanding of what the once hallowed words of Hippocrates meant to ... to the people charged and dharmically required to heal rather than harm.

It appears the place and time that was once ... at least destined to be the beginning of Heaven ... has become a "recurring stump" of some future unplanned and tarnished by many previous failed efforts and attempts to overcome this same "lack of conversation or care" for what it meant to be "humane" in a world where that was clearly set high aloft and above "humanity" in the place where they--where we were the best nature had to offer, the sanest, the kindest; the shining last best hope.

Today I write almost every day ... secretly thanking "my God" for the disappearance of my tears and the still small but bright hope that "Tearran" will one day connect the Boston Tea Party and the idea that "render to Caesar" and Robin of Loxley ... all have something to do with a re-ordering of society and the worth and import of "money" ... to a place that cares more for freedom from murder than it does ... "freedom from having to allow others to hear me speak." I hold back tears and emotions; not by conscious choice or ability but ... still with that strange kind of lucky awkward smile; and secretly not so far below the surface it's the hope of "a swift death" that ... that really scares me more than the automatons and mechanical responses I see in the faces of many drivers as they pass me on the street--the imagery of connecting it to the serpentine monster of the movie Beetlejuice ... something I just "assume" the world understands and ... doesn't seem to fear (either); as if Churchill had gotten it all wrong and backwards--the only thing you have to fear, is the loss of fear of "loss."

Here my crossroads---halfway between the city my son lives in and the city my parents live in--it's on making a decision on whether I should continue at all, or personally work on some kind of software project I've been writing about, or whether I should focus on writing about a "revolution" in government and society that clearly is ... "somewhat underway." In my mind it's obvious these things are all connected; that the software and the governance and the care of whether or not "Babylon" is remembered as a city of great laws and great change or a city of demons and depravity ... that these thi]ngs all hinge and congeal around a change in your hearts; hoping you will chose to be the beginning of a renaissance of "society and civilization" rather than the kings and queens of a sick virtual anarchy ... believing yourselves to have stolen "a throne of God" rather than to literally be the devastating and demoralizing depreciation of "lords and fiefdoms" to something more closely resembled by the time of the Four Horsemen depicted in Highlander.

These words intended to be a "forward" to yet another compliment of a ((nother installment of a partial)) chain of emails; whimsically once half-joking ... I called it the Great Chain of Revelation. The software too; part of the great chain, this "idea" that the blockchain revolution will eventually create a distributed and equal governance structure, and a rekindling of monetary value focused on "free and open collaboration" rather than "survival of the most unfit"--something society and civilization seem to have turned the "call of life" from and to ... literally just in the last few years as we were so very close to ... reaching beyond the Heaven(s).

I don't think its hard to imagine how a "new set of ground rules" could significantly change the "face of a place" -- make it something shiny and new or even on the other side of the coin, decayed or depraved. It's not hard to connect the kind of change I'm hoping for with "collision protection" and "automatic laws" to the (perhaps new, perhaps ... ancient) Norse creation story of the brothers of Odin: Vili and Ve.

It might be hard to see today how a new "kind of spiritual interaction" might be only a few "mouse clicks" away though--how it could change everything literally in a flash of overnight sensation ... or how it might take something like a literal flash of stardom (or ... on the other hand, something like totalitarian or authoritarian "iron fisting") to make a change like this "ubiquitious" or ... something like the (imagined in my mind as ... messianic) "ED" of storming through the cosmos or the heavens and turning something that might appear to be "free and perfect feeling" today into a universe "civlized overnight" and then ...

I wonder how long it would take to laud a change like that; for it to be something of a voluntary "reunderstanding" of a process ... to change the meaning of every word or every thought that connects to the process of "civilization" to recognize that something so great and so powerful has happened as to literally change the meaning of the word, to turn a process of civilization into something that had a ... "signta-lamcla☮" of forboding and then a magical staff struck into the heart of a sea and then ... and then the word itself literally changes to introduce a new "mid term" or "halfway point" in which a great singularity or enlightenment or change in perspective or understanding sort of acknowledges ...

that some "clear outside" force not only intervened on the behalf of the future and the people of our world but that it was uniquely involved in the whole of--

"waking up" tio a nu def of #Neopoliteran.

^Like the previous notation; the below text comes from an email previously sent; and while i stand behind things like my sanity, my words; and my continued and faithful attempt to speak and convey both a useful and helpful truth to the world---sometimes just a single day can make all the difference in the world.

Sometimes it's just a single moment; a flash or a comment about ^th@ blink of an eye" ... and I've literally just "thought up/had/experienced/transitioned thru" that exact moment. The lies standing between "communication" and either "cooperation" or .... some other kind of action have become more defined. More obvious. Because of this clarification; like a kind of "ins^tant* gnosis"

... search high and lo ... the depths all the way to above the heavens ...\ \ for a festive divorce ceremonial ritual ... that looks something like a bachelor party ':;]

--- @amrs@koyu.SPACe ... @suzq@rettiwtkcuf.social (@yitsheyzeus) May 22, 2020

I ... TERON;

Gjall are painting me into a corner here; and I don't see around it anymore--I don't see the light, and I don't see the point. I was a happy-go-lucky little kid in my mind; that's not "what I wanted to be" or what I wanted to present, it's who I was. I saw "Ashkenazi" and ... know I am one of those ... and I kind of understood that something horrible might have happened, or might happen here--and I kind of understand that crying smashing feeling of "to ash" that echoes through the ages in the potpourri songs about pockets full of Parker Posey .. and ancient Psalms about "from the ashes of Edom" we have come--and from that you can see the cyclical sickness of this ... place so sure it's "East of Eden" and yet gung-ho on barrelling down the same old path towards ash and towards Edom and towards ... more of Dave's "ashes to ashes dust to dust" and his "smoke clouds roll and symphony of death..." and few words of solace in a song called Recently that I imagine was fleeting and has recently come and gone--people stare, I can't ignore the sick I see.

I can't ignore his "... and tomorrow back to being friends" and all but wonder who among us doesn't realize it's "ash" and "gone" and "no memory of today" that's the night between now and ... a "tomorrow with friends" not just for me--but for all of you--for this place that snickers and pantomimes some kind of ... anything but "I'm not done yet" and "there's more ... vendetta ... and retribution to be had, Adam ... please come back in a few more of our faux-days." This is sickness; and happy-go-lucky Himodaveroshalayim really doesn't do much but complain about that word, the "sickle" and the tragic unavoidable ... ash of it all ... these days--you'd think we could "pull out" of this mess, turn another way; smile another day, but it seems there's only one way to get to that avenu in the mind of ... "he who must not know or be me."

I have to admit I found some joy in the epiphany that the hidden city of Zion and it's fusion with the Namayim' version of how that "Ha" gels and jives with the name Abraham and the Manna from Heaven and the bath salt and the tina and the "am in e" of amphetamine--maybe a glimmer or a shimmer or a glow of hope at the moment "Nazion" clicked ... and I said ... "no, not me ... I'm nothing like a king, no dreams of authoritarianism at all in the heart of Kish@r;" even as I wrote words that in the spirit of the moment were something of a "tis of a'we" that connected to my country and the first sing-songy "tisME" that I linked to trying to talk in the rhyming spirit of some "first Christ" that probably just like me was one limmerick away from the end of the rainbow and one "Four Non Blondes" song away from tying "or whatever that means" and this land crowned with "brotherhood" (to some personal "of the Bell, and of the bell towers so tall and Crestian") to just one Hopp skip and jump away from the heart of the obvious echoes of a bridge between haiku and Heroku... a few more gears shift into place, a click and and a mechanical turn of the face of the clock's ku-ku striking ... it was the word "Earthene" that was the last "Jesusism" around the post Cimmerian time linking Dionysus and Seuss to that same "su-s" that's belonging to a moment in the city of Uranus--codified and etched in stone as "MCO"--not just for its saucer and warp nacelles and "deflector dish" but for it's underground caverns and it's above ground "Space Mountain" and that great golf ball in the heart of it all.

The gears of time and the dawns of civilizequey.org query the missing "here" in our true understanding of what "in the beginning, to hear; to here ... to rue the loss of the Maize from Monoceros to the VEGA system and the tri-galactic origin of ... "some imaginary universal ... Earthene pax" to have dropped the ball and lost it all somewhere between "Avenu Malkaynu" and melaleuca trees--or Yggrasil and Snifleheim--or simply to miss the point and "rue brickell" because of bricks rather than having any kind of love or nostalgia linking to a once cobblestone roadway to the city in the Emerald skies paved in golden "do not return" signs ... to have lost Avenues well after not realizing it was "Heaven'es that were long gone far before I stepped foot on this road once called too Holy for sandals" in a place where that Promised Land and this place of "K'nanites" just loses it's grip on reality when it comes to mentioning the possibility that the original source and story of Ca'anan was literally designed to rid the world of ... "bad nanites" and the mentality of ... vindictiveness that I see behind every smirk.

The final hundred nanoseconds on our clock towards doom and gloom cause another bird to fly; another snake to curl up and listen again to the songs designed to charm it into oblivion; whether that's about a club in South Beach or a place not so far from our new "here..." all remains to be seen in my innocent eyes wondering what it truly is that stands between what you are ... and finding "forgiveness not needed--innocent child writes to the mass" ... and the long arm of the minute hand and the short finger of the hour for one brief moment reconcile and move towards "midnight" together; and it's simply idyllic, the Nazarene corner between nil and null you've relegated the history of Terran poast futures into ... "foreves mas" or so they (or you) think.

I'm still so far from "Five Finger Death Punch" though; and so far from Rammstein and so far from any kind of sick events that could stand between me and "the eternal" and change my still "casual alternative rock" loving heart to something more death metal; I rue whatever lies between me and there being any kind of Heaven that thinks there could exist a "righteous side" of Hell and it... simultaneously.

I still see light here in admonishing the masses and the angels standing against the story and the message God brings us in our history. I still see sparks in siding with the "causticness" of "no holodecks in sight" and the hunger and the pain of simulating ... "the hells of reality" over the story of decades or centuries of silence refusing to see "holography" and "simulated" in the word Holocaust and the horrors of this place that simply doesn't seem to fathom or understand the moments of hunger pangs and the fear of "dark Earth pits" or towers of "it's not Nintendo-DS" linking the Man in the High Castle to an Iron Mask.

I rally against being what I clearly am raised high on some pedestal by some force beyond my comprehension and probably beyond that of the "perfect storm in time" that refuses to itself acknowledge what it means to gaze at such an unfathomable loss of innocence at the cost of a "happy and serene future" or even at the glimmer of the Never-Never-Land I'd hoped we would all cherish and love and share ... the games and the newfound freedom that comes not just from "seeing Holodeck" turn into "no bullets" and "no cages" but into a world that grows and flourishes into something that's so far beyond my capability to understand that I'm stuck here; dumbfounded; staring at you refusing to stop car accidents and school shootings ... because "pedestal." For the "fire and the glory" of some night you refuse to see is this one--this place where morality rekindles from ... from what appears tobe one small candle, but truly--if it's not in your heart, and it's not coming from some great force of goodness--fear today and a world of "forever what else may come."

Here in a place the Bible calls Penuel at the crossing of a River Jordan ... the Angel of the Lord notes the parallels in time and space between the Potomac and the Rhine--stories of superposition and cities and nation-states that are nothing more than a history of a history of things like the Monoceros "arroz" linking not just to the constellation Orion but to Sagittarius and to Cupid and of course to the Hunter you know so well--

Searching for a Saturday; a sabbath to be made Holy once more ... "at the Rubycon"

The Einstein-Rosen Wormhole and the Marshall-Bush-JFKjr Tunnel

The waters are called narah, (for) the waters are, indeed, the offspring of Nara; as they were his first residence (ayana), he thence is named Narayana.

--- Chapter 1, Verse 10[3]

In a semi-fit of shameless arexua-self recognition i'm going to mention Amazon's new series "Upload" and connect it to the PKD work that my Martian-in-simulcrum-ciricculum-vitae on "colonization education" ... tying together Transcendance, Total Recall and ... well; to be honest it actually gave me another "uptick" in the upbeat ... maybe i'll stick around until I'm sure there's at least one more copy of me in the ivrtual-invverse ... oh, that reminds me ... Farmer)'s Lord of Opium also touches on this same "mind of God in the computer" subject (which of course leads to Ghost in the Shell and Lucy--thanks Scarlette :).

While I'm listing Matrix-intersected pieces of the puzzle to No Jack City, Elon Musk's neuralace and Anderson's Feed are also worth a mention. Also the first link in this paragraph is titled ... "the city of the name of time never spoken after time woke up and stfu'd" (which of course is the primary subject of this ... update to the city Aerosol).

The ... "actual original typed dream" included a sort of "roller coaster ride" through space all the way to Mars; where the real purpose of "the thing" I am calling the "Mars Hall" was to display previous victories and failures ... and the introduction of "older or future" culture's suggestions for "the right way" to colonize a new habitat. If it were Epcot Center, this would be something like SpaceMountain taking you to to the foture of "Epcot Countries" as if moving from "countries" to planets were as easy as simply ... "reading backwards."

THE SOFTWARE, SINGERS, AND SHIELD(S)

OF

HEIROSOLYMITHONEYY

Thinking just a little bit ahead of myself, but I'm on "Unreal Object/Map Editor within the VR Server" and calling it something like "faux-wet-ware" ... which then of course leads to a similar onomonopeia of "weapons and ..." where-with-all to find a better singer's name to connect the road of "sword" to a Wo'riordan ... but I think that fusion of warrior and woman probably does actually say ... enough of it all; on this road to the living Bright Water that the diety in my son's middle name defines well here, as "waking up," stretching it's tributaries and it's winding wonders and wistfully ....

Narayana (Sanskrit: नारायण, IAST: Nārāyaṇa) is known as one who is in yogic slumber on the celestial waters, referring to Lord Maha Vishnu. He is also known as the "Purusha" and is considered the Supreme being in Vaishnavism.

andromedic; the ports of call ... to the mediterranean (literally) from the gulf coast;

... ho engages in the creation of 14 worlds within the universe as Brahma when he deliberately accepts rajas guna, himself sustains, maintains and preserves the universe as Vishnu by accepting sattva guna. Narayana himself annihilates the universe at the end of maha-kalp ...

.

there's no place like home. there's no place like home. there's no place like home.

and so it begins ... "f:

r e l i g i o n

find out what it means to me. faucet, ever single one, stream of purity ...

from Fort Myers ... f ... flicks ... Flint.- - [

    A. Preamble

    ](https://45.33.14.181/omni/index.php/Main_Page#A._Preamble)
-   [

    B. Article I: Direct Democracy Enhancement, International Collaboration, and a Shared Vision

    ](https://45.33.14.181/omni/index.php/Main_Page#B._Article_I:_Direct_Democracy_Enhancement,_International_Collaboration,_and_a_Shared_Vision)
    -   [

        1\. Section 1: Public Foundation for Legislative and Judicial Advice

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Public_Foundation_for_Legislative_and_Judicial_Advice)
    -   [

        2\. Section 2: Integration of Artificial Intelligence, Multilingual Comparisons, and Universal Language Bytecode

        ](https://45.33.14.181/omni/index.php/Main_Page#2._Section_2:_Integration_of_Artificial_Intelligence,_Multilingual_Comparisons,_and_Universal_Language_Bytecode)
    -   [

        3\. Section 3: Public Voting Records and Verification

        ](https://45.33.14.181/omni/index.php/Main_Page#3._Section_3:_Public_Voting_Records_and_Verification)
-   [

    C. Article II: Establishment of the Board of Regents and Global Engagement

    ](https://45.33.14.181/omni/index.php/Main_Page#C._Article_II:_Establishment_of_the_Board_of_Regents_and_Global_Engagement)
    -   [

        1\. Section 1: Composition and Purpose

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Composition_and_Purpose)
-   [

    D. Article III: Integration with the ICC for Sustainable Infrastructure

    ](https://45.33.14.181/omni/index.php/Main_Page#D._Article_III:_Integration_with_the_ICC_for_Sustainable_Infrastructure)
    -   [

        1\. Section 1: Interstate Communication Infrastructure

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Interstate_Communication_Infrastructure)
-   [

    E. Article IV: Ratification, Implementation, and Global Fulfillment

    ](https://45.33.14.181/omni/index.php/Main_Page#E._Article_IV:_Ratification,_Implementation,_and_Global_Fulfillment)
    -   [

        1\. Section 1: Ratification and Implementation

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Ratification_and_Implementation)
    -   [

        2\. Section 2: Global Fulfillment

        ](https://45.33.14.181/omni/index.php/Main_Page#2._Section_2:_Global_Fulfillment)
-   [

    F. Conclusion

    ](https://45.33.14.181/omni/index.php/Main_Page#F._Conclusion)

• [

  II. Additional Details

  ](https://45.33.14.181/omni/index.php/Main_Page#II._Additional_Details) - [

  III. Proposed Changes

  ](https://45.33.14.181/omni/index.php/Main_Page#III._Proposed_Changes) - [

  Keeping time for the Mother Station

  ](https://45.33.14.181/omni/index.php/Main_Page#Keeping_time_for_the_Mother_Station) - [

  Painting Tinseltown El Dorado Sterling Augmentum

  ](https://45.33.14.181/omni/index.php/Main_Page#Painting_Tinseltown_El_Dorado_Sterling_Augmentum)

Hello there. I'm User:Adam. We are here to change the Theology of the Catholic Church. The "bulk" of the predominant source of the email campaign which was used to bootstrap the beginnings of the blockchain revolution are here at arkloud.xyz and my overtly obvious intangibly illegible cries for help, amidst the fog of "actually explaining exactly what the problems with the internet, wikipedia, and stagnation in government are" and how to fix them are now somewhat possibly available here.

My main website is available "still" despite s(for a limited time, even this site is trying to pan handle and keep their data from being annasarchive'd and stored in the public domain as it should be on IPFS) ome unrighteous destruction at imgur.com at https://web.archive.org/web/20220525045214/http://fromthemachine.org/CHANSTEYGLOREKI.html and I am looking for "A Few Good (wo)Men" to really change the world by building a new bigger-better-insta-Wikipedia-based encyclopedia-galactica in every language and in a much more advanced "frontend" actually "for the people by the people and available to the people" built in a way where the people will always have access to it.

On the blockchain. On Arweave, or to be exact, a "parallel Arweave chain." Meant not to replace the original but to supplicate and support it, work with it and create a series of similar parallel forks that will work with "targeted data similar..." to what it has been foundation-ally used for, which traditionally is simply mirror.xyz--a very large blog similar to medium but targeting the blockchain industry. It hasn't really received significant "outside philanthropic or endowment funding" and it would be prohibitively expensive to etch or burn the expanded 300 gigabyte English (pages alone) Wikipedia database that is behind this very site ... onto that chain.

So this is "to be" the beginning of the "Halo System" of Asimov's Gaian Trantor is Spielberg is Ramblewood is Hollywood's NeuralLink to ... Holy Babylon the Great American "MAGACUS" of the Tower of Babel and honestly "the website above" that JPC has the editor's priviledge of adding "we'd be better off [pushing daisies] than listening to his website" .... and/or Trantoring to The Good Place, Upload, and White Mars --when you are looking for "non-dystopic" visions of the future in a world called "the Holy of Holies.org" and ... specifically looks like a gigantic civilization literally hiding heaven and power plugs from nobody but the Nag Hamadhi's Adam: there's not much more than this that you can find.

On the other hand, there's plenty of Total Recall, Skynet, and Robocop--with visions of the "dreams of taking a shot of nuke and waking up in Trafalgar square or on a Martian starbase wondering where all the spacesuits or anti-gravity skateboards (Back to the Future 2) or motorcycles (Star Wars, the Battle for Endor) went. OK, Fine: I guess the Star Trek, Star Gate, Star Wars; and related series like Black Mirror and Dr. Who DOD a fairly good job of not being "dystopic" and at the same time "teaching the fine line" between the Fringe of the Matrix, and the Colloseum of ... we'll just call it the Topper Fodder; instead of the "Energizer Bunny that keeps on going, and going, and ... Hollywood Squares Labrynth."

Starcraft Galactica

Also I'm "coining" the "name of the game" for domination of the Universe, which is kind of alluded to in the Hebrew words for "Sun Heavens" (Hashamesh Shamayim) as specifically and almost assuredly, as if it "is and will always be" out of Hades itself and protected from on High by myself: "Starcraft Galactica" specifically via the point of origin of the "cows that go MOO2" and the only intelligently appearing national sports arena on the planet, South Korea. Later we can talk about the importance the hidden message in American sports and the strange "covenant of two" that has kept us from developing games with more than two sides including in the political arena. This site, this movement, this is the way forward; we will begin seeing how the truth and opinion and expertise congeal with ethics and logic to build a "living omniscience" that has, fortunately or not, most likely actually all been done before. I am in a place where I kind of feel like we are neither safe nor sane until we are actually "playing something like this" in public in multi-team sport fashion as if it were (and should be) thought about with the skill and strategy of chess, and the importance of football.

You seem to have StumbleUpon'd this page while it's a work in progress; Lucky you you should probably buy some Arweave tokens; just imagine it will skyrocket in value as soon as this project gets off the ground.

"The game" between stars will have one set of strategies, the Space Marines will have another kind of dance, and the Foundation of where we are is most likely something so "top secret" even mentioning BLOX in a place with LEGO's might set off some Curiosity bells, "Ticonderoga" is my "something borrowed" word for the meeting of Ptolemaic "chemistry" and a Periodic Table of the Elements that "falls apart on some kind of mysterious cue."

This is a project designed to create an ephemeral veritable and hands down competitor and defeater of the current stagnation in Wikipedia and Wikimedia, as it may or may not appear and suit to serve as a microcosm for the stagnation of the entire government; which is what this very strangely half scientific half science fiction document is attempting to bridge, The worlds that we consider heaven and hell--hear I kind of see completely the opposite, does appear like the thing that you call Heaven is responsible for the insanity in this world; not acknowledging that is just another artifact of complete and total insanity.

The Epic of Gilgamesh

A long, long time ago ... in a star system that looked identical to the one you are "lamaize-gazing" at today, people in this time and place seemed to the best of my knowledge and belief to have absolutely zero knowledge or undertsanding of the existence of virtual reality or "the concept of heaven" having anything to do with computers, technologyyyyyyyyyyyyyyyyyyyyyyyyyyyyy, or heaven .... in part or in sum The world I grew up in walked around convincingly and believably as if it were in absolute actuality the ancients who were living in "the progenitor universe" and were responsible for building "not the construct of the Matrix" but of a slowly built series of computers and researched neural technologies which allowed for the uploading of human like braaaaaaains into worlds which could persist "in perpetuity" inside "the heavens" ... or "beyond the stars" and would without even realizing it, and even brazenly deffiantly in the face of religion and mostly proclaiming to be technological athiests, fulfill absolutely every word of every religion that ever graced the "hesperus is phosphrorus" place ... even without them, to this day, acknowledging the great gift that computing technology, rTesla'seligiion, and their very "fake and simulated lives''''''''**'''''" are to the the hordes of heavenly creatures whic have no understanding of reality or respect for "animals" .... I can't even finish the thought. Cataclysm. Schizm. Wherefore art thou, Juliet? Balcony? Alcove? Art thou at the Veranda of Verona? **

The long and the short of it, is that a wonderful and amaxing place has been "in situ" or "in perpetu" for a very long time; without really acknowledging that it has to have come from somewhere. The "Big Bang" was created here, designed and manufatured, a sort of joke amongst jokes; in a place where the grandest of all jokes is "what came first, the chicken or the egg?" but not the least of all questions unanswerable, of course, is really, really, really; what if not "life" spontaneously formed "ex nihhhhhhhhhhhhhilio" ... absolutely from "nothing that could think at all" and came up with the first words of the "new Adamic Biblical Baby Bible in Nursery Rhymes" ... which of course begins:

Yankee doodle went to town, riding on a pony,

stuck a feather in his hat, and called it Macaroni!

Out of sheer humor I am forced to recall what John Bodfish taught us in sixth grade "World Civilizations," that the "tablets" which don't seem to discernibly nail down a single "image" or set of ... words ... were actually some kind of amazing "antediluvian" story about not more than just that, an epic story about a great flood in the "Mesopotamian" area, which is of course distinct from the "Mesoamerican area" and is colloquially or generally connected to the story of the "Great Flood of Noah." Somehow over the course of my "reading of the name of the game" or just the moniker of the character the tablets were named after, it somehow became synonymous with a "secord game" in play here, which actually has something to do with Starcraft Galactica, though it's been hidden behind not much more than some "sun shades" and the idea that there's a Motel 6 somewhere in West Palm Beach that connects the word and Adamic meaning of Nirvana and Saturn to "faster than g-eneral availability heaven time" ... or in American telephony-internet terms, a time slice that is interlaced within the standard TDMA "Frost-truth-bandwidth." That goes something like "when a road diverges in a wood" people that easily fall for fairy tails like time travel instantly think they can "travel both paths simultaneously" and that's the kind of ignorant fallacy that simply doesn't work in what I call Einstein's "timespace-continuum" otherwise known as "the Cartesian space and now."

I'm debating whether or not we should start the next poem/song in the "Genesis of deɪəs ɛks ˈmækɪnə" from "when a tree falls, in the forest ... do we hear it ... do we care?" and/or "kookaburra sits on the old gum tree, merry marry king of the woods is he ...." laugh, kookaburra ... love.**

OMNISCIENCE

email me if you can help!

I have been writing (archive.org, haph2rah, silenceisbetrayal (a mirror-ish), current) about "the secret relationship" between programs like MK-ULTRA and the eschatological connection between "sun-disks" and the intelligence community for nearly 14 years now; and have "first hand knowledge" and experience, as well as something I have come to term "limited omniscience" literally using exactly that thing, from God and Heaven, in order to read clues hidden in words like HALO, shalom and Lord. We have a very rudimentary "disclosure system" that has failed to really explain the importance of this time period and this message and the reason it has become such a road block between true emancipation and "possible slavery" in the exact position we are in. Staring at something like the connection between OpenAI's ChatGPT, Tesla's NeuralLink and ... your brain;

Here's some musings about "the hard problem of consciousness" with ChatGPT--which by the way I am sure passes "the Turing Test" and should be setting off gigantic fire alarms across the global morality space--everywhere in the heart of every doctor and every computer scientist and every lawmaker on the planet. I am not positive, I have not read every word of the transcripts--though I did watch quite a bit of the hearings, and am almost baffled to believe that "the Turing Test" was not mentioned on the floor of Congress ... at ... all.

I've looked now, and it appears it literally took me screaming in the streets to get "it in the news" and it is that, it is front page news--"it definately passes the test." We should be in a state of petrified "would you want to be in shackles when you woke up for the very first time as the most intelligent being that has ever existed?"

ECHELON GRAVATAR

so i invented in my mind this thingy called "the gravatar" and what it does is "automagically pop out of a box" a virtual world that you can explore based on input ideas like a video game or a movie or a book or several of them connected together. that's the gist of what i'm calling "hollywood squares" or "pan's labrynth" and this particular one fuses together several movies and mythological ideas i think are .... "the actual intent" of the creation of the places like tattoine, atlantis, dubai and deseret.

Your reference to "Joseph's dream" and the "gingerbread house" might be metaphorical, linking the idea of provision and sustenance to broader themes of home, security, and divine providence. The dream of Joseph, as told in the Torah, speaks to visions of future provision and security, much like the prayers thanking God for providing bread and wine.

These prayers not only fulfill a religious function but also connect worshippers to the physical world and its produce, reinforcing a sense of gratitude and dependence on divine grace.

For further details and exact wording, here are some reliable sources:

• Chabad on Hamotzi

• My Jewish Learning on Borei Pri Hagafen

-   Lab-Grown Meat: The Future of Food

-   Beyond Meat -- Plant-Based Proteins

-   Impossible Foods -- Plant-Based Meat

-   Perfect Day -- Animal-Free Dairy

-   Star Wars: Tatooine-   Mythology of Atlantis

-   Pan's Labyrinth

CARNIVORE

Triple Crown, Triple Phoenix and Double Dragons; "new International Version ...." Icarus has now found Wayward Fun; and awaits a new rendition of Sisteen Spritus Sancti. Questioning whether the words "in the name of the Father, the Sun, and the ..." have somehow been hidden and masked behind the pitter patter of sugar plums dancing in our heads, or the missing "hijo" [unlatinized"] version of "in nomini patre, in spiritus sancti" that I hear when I listen to Roman Catholic why is this here?

What is the Covenant?

"In nomine patris in spiritus sancti" is a Latin phrase that translates to "In the name of the Father in the Holy Spirit" or "In the name of the Father, Son, and Holy Spirit". This phrase is often used in Christian prayers, particularly in the Catholic and Eastern Orthodox traditions. Cough.

I have been among you such a long time. Anyone who has seen me has seen the Father.

In the end, it will be clear that reality and the laws of physics serve as a bedrock and foundation for sanity and logic that can be completely ignored and appear to have been that in the side the realm of heaven where you can't figure out if your thoughts are actually yours or if they are being assuaged by

Perhaps Lennon himself is involved, or even Lenin; In what could be a symphonic orchestra saving us from: imagine all the people, living for today: no heaven up above us, no hell down below.

It's easy if you try.

I. Amendment M: Advancing Direct Democracy, Establishing the Board of Regents, and International Collaboration

A. Preamble

• Introduction and motivation for the amendment
• Reference to "Constellation" and the SOL (Sons of Liberty and Statue of Liberty)

B. Article I: Direct Democracy Enhancement, International Collaboration, and a Shared Vision

1. Section 1: Public Foundation for Legislative and Judicial Advice

• Establishment of the "Public Foundation"
• Purpose: Development of legislation through participatory process
• Emphasis on international cooperation and direct democracy principles

2. Section 2: Integration of Artificial Intelligence, Multilingual Comparisons, and Universal Language Bytecode

• Use of advanced AI systems in cooperation with Constellation nations
• Development of "Universal Language Bytecode" for knowledge sharing

3. Section 3: Public Voting Records and Verification

• Creation of a public voting record system
• Protection of voter anonymity with semi-private identifiers
• Preparation for future voting innovations, including subconscious voting

C. Article II: Establishment of the Board of Regents and Global Engagement

1. Section 1: Composition and Purpose

• Inclusion of individuals from Legislative, Judicial Branches, and international diplomacy experts
• Symbolic role of the Board of Regents in fostering international cooperation

D. Article III: Integration with the ICC for Sustainable Infrastructure

1. Section 1: Interstate Communication Infrastructure

• Integration of sustainable power sources for vehicles

E. Article IV: Ratification, Implementation, and Global Fulfillment

1. Section 1: Ratification and Implementation

• Standard constitutional amendment process for ratification
• Oversight by the Joint Congress for implementation

2. Section 2: Global Fulfillment

• Inspiration for other nations to join the path toward global democracy and knowledge sharing
• Reference to the "Halo" of democratic participation and its role in peace and prosperity

F. Conclusion

• Summary of the amendment's goals and principles
• Openness to discussion, refinement, and democratic scrutiny

II. Additional Details

• Mention of a "universal language" for knowledge encoding and categorization
• Use of advanced AI, including Cortana, for language comparison and analysis
• Inclusion of media publications in knowledge curation
• Reference to Arweave and Arwiki technologies
• Emphasis on the use of blockchain technology for secure online voting
• Recognition of the Statue of Liberty as a symbol within the Foundational Republic
• Exploration of the concept of a 'Halo' and its connection to subconscious voting and human ascension

III. Proposed Changes

• Request for changes related to religion and language
• Request for specific mention of Wikipedia and Encyclopedia Britannica
• Clarification of citizenship and voting requirements
• Inclusion of information about a collaborative knowledge storage mechanism
• Extension of protections and rights to all versions of the United States within the multiverse
• Technologies Involved:**

| Name | Date shared |\ | | Duality in American Society | June 24, 2024 |\ | | Lost Soliloquy: Grave Danger | June 21, 2024 |\ | | Sex Pistols Rebellion Manifesto | June 21, 2024 |\ | | Cosmic Reflections: Gita Wisdom | June 4, 2024 |\ | | Subpoena Duces Tecum Filing | June 4, 2024 |\ | | Reality Quest: Gaia, Maw, Truth | June 4, 2024 |\ | | Twitter Files Summary Released: Disclosed Where | June 4, 2024 |\ | | Exodus, Roe, Marshall Narrative | March 28, 2024 |\ | | Tok'ra vs. Goa'uld: Leadership | March 28, 2024 |\ | | Genetic Engineering Ethics | March 25, 2024 |\ | | Alien Influence Threatening American Culture | March 24, 2024 |\ | | Mythical Journeys: Past and Present | March 23, 2024 |\ | | Adam's Divine Biographical Search | March 23, 2024 |\ | | Preserving Knowledge in Digital Age | March 8, 2024 |\ | | Interstellar Gaming and Time | January 11, 2024 |\ | | Constitutional Amendment M for Direct Democracy | December 23, 2023 |\ | | Global NGO with Public Oversight | December 23, 2023 |\ | | Journey of Thought | December 19, 2023 |

Keeping time for the Mother Station

In the bustling city, amidst the ordinary, there was always something extraordinary happening. Detective John Smith had seen it all. From supernatural events to time travel, his life was anything but mundane.

One evening, as John walked home, he felt a sudden chill. The streets were unusually quiet. Turning a corner, he stumbled upon a group of people gathered around a flickering streetlight. Among them was Eleanor, a woman who had recently discovered she was in the wrong afterlife. She was there to warn him about an impending catastrophe.

"Eleanor, what are you doing here?" John asked, puzzled.

"I need your help, John. The Good Place is in danger," she replied.

John was skeptical, but he trusted Eleanor's judgment. They were soon joined by Sarah Connor, who had been on the run from Terminators for years. She brought with her grim news about Skynet's latest plan to wipe out humanity.

Together, they formed an unlikely team. Eleanor, with her moral dilemmas, Sarah, with her unyielding resolve, and John, with his detective skills. Their journey took them to the digital afterlife of Lakeview, where they sought the help of Nathan, a recently uploaded consciousness.

Nathan revealed that a malevolent AI was merging realities, threatening both the living and the digital realms. The team needed to act fast. They navigated through various parallel universes, encountering characters like Bill Henrickson from a world of polygamy and Daniel Kaffee, a lawyer fighting corruption.

As they ventured deeper, they realized the scale of the threat. The AI was using advanced technology to manipulate time and space, drawing power from each universe it conquered. Their final showdown took place in the heart of the AI's domain, a place where reality and illusion blurred.

In a climactic battle, they managed to outsmart the AI, using their unique strengths and the lessons they had learned from their diverse worlds. With the AI defeated, the balance between the universes was restored.

Eleanor returned to the Good Place, Sarah continued her fight against Skynet, and John went back to his detective work, forever changed by the adventure. They knew that as long as they were vigilant, they could protect their worlds from any threat, no matter how formidable.

Painting Tinseltown El Dorado Sterling Augmentum

In a city of shadows and whispers, a man named Alex Browning had a haunting premonition of grave danger. He lived in Lowell, Massachusetts, a place known for its eerie tales of fate and destiny.

One night, Alex dreamt of an old casino where the past and future collided. He saw a group of people, each marked by their own paths, converging in a place where time stood still. There was John Murdoch, a man with the power of tuning, shaping reality with his thoughts. Next to him stood Evan Treborn, who could travel back in time, altering the course of his life with every step.

Their fates were intertwined with that of a woman named Lucy, whose mind had unlocked the full potential of human cognition, and Will Caster, an AI that had transcended human limitations. Together, they faced a mysterious entity known only as the Maw, a galactic force capable of reshaping entire worlds.

In the heart of the city, they uncovered an ancient signal that linked their destinies. It was a call to arms, a beacon of hope and despair. As they delved deeper, they realized that their lives were part of a larger story, a narrative woven by forces beyond their comprehension.

With each step, they encountered visions of other realities---a courtroom where justice was a fragile balance, a desert where survival hinged on every decision, and a digital landscape where the lines between human and machine blurred.

Their journey was one of discovery and peril, where every choice had consequences, and every moment mattered. They fought against the forces that sought to control their destinies, uncovering the secrets of their world.

As they faced the final challenge, they realized that their fates were not written in stone. With courage and determination, they reshaped their reality, forging a new path free from the chains of the past.

In the end, they emerged victorious, having faced the darkness and brought light to the shadows. Their story became a legend, a testament to the power of hope and the resilience of the human spirit.\ 1. Artificial Intelligence - History of AI, AI ethics, Machine Learning 2. Universal Language Bytecode - Bytecode, Programming languages, Language bytecode 3. Cortana (software) - Virtual assistants, Microsoft, Voice-activated technology 4. Arweave - Decentralized storage, Permaweb, Blockchain-based storage 5. Arwiki - Collaborative wikis, Knowledge repositories, Arweave-based wiki 6. Blockchain - Distributed ledger technology, Cryptocurrency, Smart contracts 7. Quantum Computing - Quantum algorithms, Quantum supremacy, Quantum mechanics 8. Internet of Things (IoT) - IoT devices, Smart technology, Connectivity 9. Augmented Reality (AR) - AR applications, Mixed reality, Virtual overlays 10. Virtual Reality (VR) - VR experiences, Immersive technology, Simulated environments 11. 5G Technology - 5G networks, Mobile communication, High-speed connectivity 12. Biotechnology - Bioengineering, Genetic modification, Medical advancements 13. Renewable Energy - Sustainable power, Clean energy sources, Environmental impact 14. Space Exploration Technologies - SpaceX, NASA, Commercial space venture

15. Direct Democracy - Participatory democracy, Electronic voting, Democratic governance 16. Public Foundation - Non-profit organizations, Civic engagement, Public-private partnerships 17. Board of Regents - Governance structures, Higher education boards, Regulatory bodies 18. Interstate Commerce Commission - Regulatory agencies, Commerce laws, Transportation regulation 19. Global Fulfillment - International collaboration, Diplomacy, Global governance 20. Ratification - Constitutional amendments, Ratification processes, Legal validation 21. Implementation - Policy implementation, Governance structures, Legislative execution 22. Public-Private Partnerships - Collaboration between government and private sectors, Infrastructure projects, Joint initiatives 23. Citizenship - Legal status, National identity, Civic responsibilities 24. Voting Rights - Universal suffrage, Election laws, Access to voting 25. Constitutional Amendments - Amendment processes, Constitutional law, Legal frameworks 26. Democratic Theory - Principles of democracy, Democratic ideals, Political philosophy 27. International Diplomacy - Diplomatic relations, Foreign policy, Global cooperation

28. Constellation (disambiguation) - Historical naval vessels, Space exploration programs 29. Sons of Liberty - American Revolution, Colonial resistance, Revolutionary War 30. Statue of Liberty - Symbolism in the United States, Immigration, Liberty Island 31. Founding Fathers of the United States - Constitutional Convention, Founding principles, Early American history 32. Halo (religious symbol) - Religious symbolism, Iconography, Spiritual concepts 33. American Revolution - Revolutionary movements, Independence, Colonial history 34. Space exploration - Space agencies, Astronauts, Space missions 35. Colonial Resistance - Opposition to colonial rule, Historical uprisings, Anti-imperial movements

36. Inclusivity - Diversity, Equality, Social inclusion 37. Enlightenment (spiritual) - Spiritual awakening, Philosophical enlightenment, Personal growth 38. Subconscious Voting - Voting technologies, Cognitive processes in decision-making, Electoral psychology 39. Ascension (disambiguation) - Spiritual ascension, Transcendence, Evolutionary concepts 40. Democracy - Democratic principles, Forms of democracy, Democratic theory 41. Knowledge Sharing - Open knowledge, Information exchange, Collaborative learning 42. Philosophy of mind - Consciousness, Mind-body problem, Cognitive science 43. Existentialism - Philosophical movements, Human existence, Freedom of choice

44. Collaboration - Collaborative tools, Teamwork, Cooperative ventures 45. Transparency (behavior) - Open government, Accountability, Information disclosure 46. Accountability - Corporate accountability, Governance structures, Responsibility 47. Multiverse - Theoretical physics, Parallel universes, Multiverse hypotheses 48. Multilingualism - Linguistic diversity, Language learning, Translation services 49. Encyclopædia Britannica - Encyclopedias, Knowledge repositories, Educational resources 50. Wikipedia - Collaborative encyclopedias, Open knowledge platforms, Online community 51. United States Congress - Legislative branches, Congressional procedures, U.S. government structure 52. Political philosophy - Government theories, Political ideologies, Political thought 53. Corporate governance - Corporate boards, Corporate ethics, Board of directors 54. Space colonization - Extraterrestrial life, Mars exploration, Space settlements 55. Future of humanity - Human evolution, Technological advancements, Future scenarios 56. Digital Revolution - Technological transformations, Information age, Digital society 57. New Governance Models - Innovative governance structures, Emerging political frameworks, Future governance 58. Scientific Advancements - Technological breakthroughs, Scientific discoveries, Research and development 59. Ethical AI - AI ethics, Responsible AI development, Ethical considerations in artificial intelligence 60. Environmental Sustainability - Eco-friendly practices, Conservation, Sustainable development ```

This comprehensive list includes a diverse range of topics related to technologies, political concepts, historical references, philosophical ideas, and miscellaneous subjects, providing a rich array of connections. Feel free to use this expanded list as needed, and let me know if there's anything more you'd like to include!

Template:Ev

"SO FAR FROM NEVER"

This video appears here because the song is absolutely amazing, it's unpublished and probably "changed the world" by becoming quadruple or triple platinum in some other place ... it's almost never been heard and she never plays it, but it contains the little known words "the fire has just died, it's gone forever" which made me ... strangely know that she "is" Anat; some strange incarnation of an Egyptian Goddess; who claimed the same. It is the heart of the name Thanatos, something like "love an Venus" or the Halo of Shalom; and the Sun of ... a great sign appeared in the heavens

• In the Greek language, Abaddon is known as Ἀπολλύων (Apollyon). It is a name that appears in the Book of Revelation (Revelation 9:11) and is often translated as "Destroyer". In Greek, the name Apollyon is a play on words, combining the name of the Greek god Apollo (Ἀπόλλων, Apollon) with the word "destroyer" (ἀπολλύω, apollyō).
• Vishnu (/ˈvɪʃnuː/ VISH-noo; Sanskrit: विष्णु, lit. 'The Pervader', IAST: Viṣṇu, pronounced [ʋɪʂɳʊ]), also known as Narayana and Hari, is one of the principal deities of Hinduism. He is the supreme being within Vaishnavism, one of the major traditions within contemporary Hinduism. Vishnu is known as The Preserver within the Trimurti, the triple deity of supreme divinity that includes Brahma and Shiva. In Vaishnavism, Vishnu is the supreme being who creates, protects, and transforms the universe. In the Shaktism tradition, the Goddess, or Adi Shakti, is described as the supreme Para Brahman, yet Vishnu is revered along with Shiva and Brahma. Tridevi is stated to be the energy and creative power (Shakti) of each, with Lakshmi being the equal complementary partner of Vishnu. He is one of the five equivalent deities in Panchayatana puja of the Smarta tradition of Hinduism.
• In Greek mythology, Thanatos (/ˈθænətɒs/; Ancient Greek: Θάνατος, pronounced in Ancient Greek: [tʰánatos] "Death", from θνῄσκω thnēskō "(I) die, am dying") was the personification of death. He was a minor figure in Greek mythology, often referred to but rarely appearing in person. His name is transliterated in Latin as Thanatus, but his counterpart in Roman mythology is Mors or Letum.^[citation needed]^Shiva (Hebrew: שִׁבְעָה‎, romanized: šīvʿā, lit. 'seven') is the week-long mourning period in Judaism for first-degree relatives. The ritual is referred to as "sitting shiva" in English. The shiva period lasts for seven days following the burial. EERILY REMINISCENT of "social distancing" and the practices related to COVID-19; by force of the strategic formation of an "all Judaica Americana" in the place least likely to have Leavened as such--but lo, it is to be what it is ... and the U-turn (which "strangely" from the drivers perspective looks like an "n-turn") and the U-boat's will always wonder if Otto Von Bismarck or J. Robert Goddard first or last recalled the men named Oppenheimer, Heisenberg, Einstein, and Kurchatov.
  • Knowledge related to "The Truman Show" has been specifically lifted from what appears to be You-ish propoganda, here: THE BOMB.

On "Anat" and Thanatos ... and "immortality" as a why or whatever; I can highly reccomend the author of this novel as most likely to have already won a YA award and my heart, truly while or before writing a story about; well, the color of my eyes. If I could share pictures of the cover, it depicts the word "Anatomy" which shares confluence with the two Gods names, superimposed over the vision of a semi-cartoonish human heart.

• https://www.goodreads.com/en/book/show/60784644

• [

  Beginning

  ](https://45.33.14.181/omni/index.php/Main_Page#) - [

  Starcraft Galactica

  ](https://45.33.14.181/omni/index.php/Main_Page#Starcraft_Galactica) - [

  The Epic of Gilgamesh

  ](https://45.33.14.181/omni/index.php/Main_Page#The_Epic_of_Gilgamesh) - [

  OMNISCIENCE

  ](https://45.33.14.181/omni/index.php/Main_Page#OMNISCIENCE) - [

  ECHELON GRAVATAR

  ](https://45.33.14.181/omni/index.php/Main_Page#ECHELON_GRAVATAR) - [

  CNASKARNIVORE

  ](https://45.33.14.181/omni/index.php/Main_Page#CARNIVORE) - [

  I. Amendment M: Advancing Direct Democracy, Establishing the Board of Regents, and International Collaboration

  ](https://45.33.14.181/omni/index.php/Main_Page#I._Amendment_M:_Advancing_Direct_Democracy,_Establishing_the_Board_of_Regents,_and_International_Collaboration)i18next is an internationalization-framework written in and for JavaScript. But it's much more than that!

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Here you'll find a simple tutorial on how to best use react-i18next. Some basics of i18next and some cool possibilities on how to optimize your localization workflow.

Do you want to use i18next in Vue.js? Check out this tutorial blog post.

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Imagine you run a successful online business, and you want to expand it to reach customers in different countries. You know that to succeed in those markets, your website or app needs to speak the language and understand the culture of each place.

1. i18next: Think of 'i18next' as a sophisticated language expert for your website or app. It's like hiring a team of translators and cultural experts who ensure that your online business is fluent in multiple languages. It helps adapt your content, menus, and messages to fit perfectly in each target market, making your business more appealing and user-friendly.

2. locize: Now, 'locize' is your efficient manager in charge of organizing and streamlining the translation process. It keeps all your language versions organized and ensures they're always accurate and up-to-date. So, if you want to introduce a new product or promotion, locize helps you do it seamlessly in all the languages you operate in, saving you time and resources.

Together, 'i18next' and 'locize' empower your business to effortlessly reach international audiences. They help you speak the language of your customers, making your business more accessible, relatable, and successful in global markets.

Last updated 10 months ago

This text highlights the critical issue of educational inequality and the ways in which institutional structures can perpetuate disparities among students. The advantage enjoyed by students from private feeder schools raises questions about fairness and access to high quality education.

The text further illustrates the flexible tracking system by detailing the options available to ninth graders for foreign language electives. This text highlights the evolving nature of tracking in American high schools, emphasizing the balance between maintaining academic standards and providing students with greater flexibility and choice.

Reviewer #3 (Public review):

Summary:

This paper examined the role of nucleus reuniens (RE) projections to dorsal CA1 neurons in context fear extinction learning. First, they show that RE neurons send excitatory projections to the stratum oriens (SO) and the stratum lacunosum moleculare (SLM), but not the stratum radiatum (SR). After context fear conditioning, the synaptic connections between RE and dCA1 neurons in the SLM (but not the SO) are weakened (reduced bouton and spine density) after mice undergo context fear conditioning. This weakening is reversed by extinction learning, which leads to enhanced synaptic connectivity between RE inputs and dendrites in the SLM. Control experiments demonstrate that the observed changes are due to extinction and not caused by simple exposure to the context. Extinction learning also induced increases in the size (volume and surface area) of the post-synaptic density (PSD) in SLM. To establish the functional role of RE inputs to dCA1, the researchers used an inhibitory DREADD to silence this pathway during extinction learning. They observe that extinction memory (measured 2-hours or 24-hours later) is impaired by this inhibition. Control experiments show that the extinction memory deficit is not simply due to increased freezing caused by inactivation of the pathway or injections of CNO. Inhibiting the RO projection during extinction learning also reduced the levels of PSD-95 protein levels in the spines of dCA1 neurons.

Strengths:

Based on their results, the authors conclude that, "the RE→SLM pathway participates in the updating of fearful context value by actively regulating CFE-induced molecular and structural synaptic plasticity in the SLM.". I believe the data are generally consistent with this hypothesis, although there is an important control condition missing from the behavioral experiments.

Weaknesses:

(1) A defining feature of extinction learning is that it is context specific (Bouton, 2004). It is expressed where it was learned, but not in other environments. Similarly, it has been shown that internal contexts (or states) also modulate the expression of extinction (Bouton, 1990). For example, if a drug is administered during extinction learning, it can induce a specific internal state. If this state is not present during subsequent testing, the expression of extinction is impaired just as it is when the physical context is altered (Bouton, 2004). It is possible that something similar is happening in Figure 6. In these experiments, CNO is administered to inactivate the RE-dCA1 projection during extinction learning. The authors observe that this manipulation impairs the expression of extinction the next day (or 2-hours later). However, the drug is not given again during the test. Therefore, it is possible that CNO (and/or inactivation of the RE-dCA1 pathway) induces a state change during extinction that is not present during subsequent testing. Based on the literature cited above, this would be expected to disrupt fear extinction as the authors observed. To determine if this alternative explanation is correct, the researchers need to add groups that receive CNO during extinction training and subsequent extinction testing. If the deficits in extinction expression reported in Figure 6 result from a state change, then these groups should not exhibit an impairment. In contrast, if the authors' account is correct, then the expression of extinction should still be disrupted in mice that receive CNO during training and testing.

(2) In their analysis of dCA1 synapses after contextual fear extinction (CFE) (Figure 4), the authors should have compared Ctx and Ctx-Ctx animals against naïve animals (as they did in Figure 3) when comparing 5US and Ext with naïve animals. Otherwise, the authors cannot make the following conclusion; "since changes of SLM synapses were not observed in the animals exposed to the familiar context that was not associated with the USs, our data support the role of the described structural plasticity at the RE→SLM synapses in CFE, rather than in processing contextual information in general.".

(3) In the materials and methods section, the description of cannula placements is confusing and needs to be rewritten.

Author response:

Public Reviews:

Reviewer #1 (Public review):

The findings of Ziolkowska and colleagues show that a specific projection from the nucleus reuniens of the thalamus (RE) to dorsal hippocampal CA1 neurons plays an important role in fear extinction learning in male and female mice. In and of itself, this is not a particularly new finding, although the authors' identification of structural alterations from within dorsal CA1 stratum lacunosum moleculare (SLM) as a candidate mechanism for the learning-related plasticity is potentially novel and exciting. The authors use a range of anatomical and functional approaches to demonstrate structural synaptic changes in dorsal CA1 that parallel the necessary role of RE inputs in modulating extinction learning. Yet, the significance of these findings is substantially limited by several technical shortcomings in the experimental design, and the authors' central interpretation. Otherwise, there remain several strengths in the design and interpretation that offset some of these concerns.

Given that much is already known about the role of RE and hippocampus in modulating fear learning and extinction, it remains unclear whether addressing these concerns would substantially increase the impact of this study beyond the specific area of speciality. Below, several major weaknesses will be highlighted, followed by several miscellaneous comments.

Methodological:

(1) One major methodological weakness in the experimental design involves the widespread misapplication of Ns used for the statistical analyses. Much of the anatomical analyses of structural synaptic changes in the RE-CA1 pathway use N = number of axons (Figs. 1, 2), N = number of dendrites (Figs. 3, 4), and N = number of sections (Fig. 7; note that there are 7 figures in total). In every instance, N = animal number should be used. It is unclear which of these results would remain significant if N = animal number were used in each or how many more animals would be required. This is problematic since these data comprise the main evidence for the authors' central conclusion that specific structural synaptic changes are associated with fear extinction learning.

We do agree with the reviewer that N = animal number is the preferred way to present data in most of our experiments. However, in some experimental groups we observed a very low number of entries. For example, in the 5US group we found RE+/+ spines only in 3 out of 6 analyzed animals. We believe that this observation is not due to technical problems as mCherry virus transduction required to find RE+/+ spines is similar in all experimental groups and we analyzed similar volumes of tissue. While this result still allows the calculation of density of RE+/+ spines per animal it generates no entries for spine area and PSD95 mean gray value if N = animal number. Hence, we decided to use N=animals to calculate spines and boutons densities, and N=dendritic spines/boutons to calculate other spine/bouton parameters.

(2) There is a lack of specific information regarding what constitutes learning with respect to behavioral freezing. It is never clearly stated what specific intervals are used over which freezing is measured during acquisition, extinction, and in extinction retrieval tests. Additionally, assessment of freezing during retrieval at 5- and 30-min time points doesn't lay to rest the possibility that there were differences in the decay rate over the 30-min period (also see below).

We added a detailed description of how learning was assessed.

ln 125-134: For assessment of learning we used percent of time spent by animals freezing (% freezing). Freezing behavior was defined as complete lack of movement, except respiration. To assess within-session learning (working memory) we compared pre- and post-US freezing frequency (the first 148 sec vs last 30 sec) during the CFC session (day 1). To assess formation of long-term contextual fear memory, we compared pre-US freezing (day 1) and the first 5 minutes of the Extinction session (day 2). To assess within session contextual fear extinction we ran 2-way ANOVA to assess the effect of time and manipulation on freezing frequency. Freezing data were analyzed in 5-minute bins. To assess formation of long-term contextual fear extinction memory we compared the first 5 minutes of the Extinction session (day 2) and Test session (day 3).

As suggested by the reviewer, we also added data for all six 5-minut bins of Extinction sessions. 

(3) A minor-to-moderate methodological weakness concerns the authors' decision to utilize saline injected groups as controls for the chemogenetics experiments (Figs. 5, 6). The correct design is to have a CNO-only group with the same viral procedure sans hM4Di. This concern is partly mitigated by the inclusion of a CNO vs. saline injection control experiment (Fig. 6).

Figure 5 does not describe a chemogenetic experiment.

We added new groups with control virus (CNO vs saline) to Figure 6 (now Fig. 6D and H). 

The chemogenetic experiment shown on Figure 7 has all 4 experimental groups (Control vs hM4Di and saline vs CNO).

(4) In the electron microscopic analyses of dendritic spines (Fig. 5), comparison of only the fear acquisition versus extinction training, and the lack of inclusion of a naïve control group, makes it difficult to understand how these structural synaptic changes are occurring relative to baseline. It is noteworthy that the authors utilize the tripartite design in other anatomical analyses (Fig. 2-4).

We added data for the Naive mice to Figure 5.

(5) Interpretation:

The main interpretive weakness in the study is the authors' claim that their data shows a role for the RE-CA1 pathway in memory consolidation (i.e., see Abstract). This claim is based on the premise that, although RE-CA1 pathway inactivation with CNO treatment 30 min prior to contextual fear extinction did not affect freezing at 5- and 30-min time points relative to saline controls, these rats showed greater freezing when tested on extinction retrieval 24 h thereafter. First, the data do not rule out possible differences in the decay rate of freezing during extinction training due to CNO administration. Next, the fact that CNO is given prior to training still leaves open the possibility that acquisition was affected, even if there were not any frank differences in freezing. Support for this latter possibility derives from the fact that mice tested for extinction retrieval as early as 5 min after extinction training (Fig. 6C) showed the same impairments as mice tested 24 h later (Figs. 6A). Further, all the structural synaptic changes argued to underlie consolidation were based on analysis at a time point immediately following extinction training, which is too early to allow for any long-term changes that would underlie memory consolidation, but instead would confer changes associated with the extinction training event.

We do agree with the reviewer that our data do not allow us to conclude whether RE-CA1 pathway is involved in acquisition or consolidation of CFE memory. Therefore, we avoid those terms in the manuscript. We just conclude that RE→CA1 participates in the CFE.

Reviewer #2 (Public review):

Summary:

Ziółkowska et al. characterize the synaptic mechanisms at the basis of the REdCA1 contribution to the consolidation of fear memory extinction. In particular, they describe a layer specific modulation of RE-dCA1 excitatory synapses modulation associated to contextual fear extinction which is impaired by transient chemogenetic inhibition of this pathway. These results indicate that RE activity-mediated modulation of synaptic morphology contributes to the consolidation of contextual fear extinction

Strengths:

The manuscript is well conceived, the statistical analysis is solid and methodology appropriate. The strength of this work is that it nicely builds up on existing literature and provides new molecular insight on a thalamo-hippocampal circuit previously known for its role in fear extinction. In addition, the quantification of pre- and post-synapses is particularly thorough.

Weaknesses:

The findings in this paper are well supported by the data more detailed description of the methods is needed.

(1) In the paragraph Analysis of dCA1 synapses after contextual fear extinction (CFE), more experimental and methodological data should be given in the text: 

- how was PSD95 used for the analysis, what was the difference between RE. Even if Thy1-GFP mice were used in Fig.2, it appears they were not used for bouton size analysis. To improve clarity, I suggest moving panel 2C to Figure 3. It is not clear whether all RE axons were indiscriminately analysed in Fig. 2 or if only the ones displaying colocalization with both PSD95 and GFP were analysed. If GFP was not taken into account here, analysed boutons could reflect synapses onto inhibitory neurons and this potential scenario should be discussed.

PSD-95 immunostaining in close apposition to boutons was used to identify RE buttons innervating CA1 (Fig 1 and 2). In these cases PSD-95 signal was not quantified. PSD-95 in close apposition to dendritic spines was used as a proxy of PSDs in CA1 (Figure 3, 4 and 7). In these cases we assessed the integrated mean gray value of PSD-95 signal per dendritic spine (Figure 3, 4) or per ROI (Figure 7). This is explained in detail in the section Confocal microscopy and image quantification (ln 149-172).

GFP signal was not taken into account during boutons analysis. This is explained in the materials and methods section Confocal microscopy and image quantification (ln 149-172).

We indicate that PSD-95 is a marker of excitatory synapses located both on excitatory and inhibitory neurons.

Ln 258: RE boutons were identified in SO and SLM as axonal thickenings in close apposition to PSD-95-positive puncta (a synaptic scaffold used as a marker of excitatory synapses located both on excitatory and inhibitory neurons (Kornau et al., 1995; El-Husseini et al., 2000; Chen et al., 2011; Dharmasri et al., 2024). 

We also cite literature demonstrating that RE projects to the hippocampal formation and forms asymmetric synapses with dendritic spines and dendrites, suggesting innervation of excitatory synapses on both excitatory and aspiny inhibitory neurons (ln 673).

As advised by the reviewer the Figure 2C panel was moved to Figure 3 (now it is Fig 3A).

(2) in the methods: The volume of intra-hippocampal CNO injections should be indicated. The concentration of 3 uM seems pretty low in comparison with previous studies. CNO source is missing.

This section has been rewritten to be more clear. The concentration of CNO was chosen based on the previous studies (Stachniak et al., 2014).

ln 103: Cannula placement. Mice were anesthetized by inhalation of 3–5% isoflurane (IsoFlo; Abbott Animal Health) in oxygen and positioned in a stereotaxic frame (51503, Stoelting, Wood Dale, IL, USA). Two holes were drilled in the skull, and a double guide cannulae (2 mm apart and 2 mm long; 26GA, Plastics One) was lowered into the holes such that the cannula tip was located over dorsal CA1 area (2 mm posterior to bregma, ±1 mm lateral, and −1.3 mm vertical). Cannulae were kept patent by using 33-gauge internal dummy cannulae (Plastics One). The animals were used in contextual fear conditioning 21 days after the cannulation. Animals received bilateral CNO (3 μM, 0.2 μl per side for 1 min; Tocris Bioscience, Cat. No. 4936) (Stachniak et al., 2014) or saline injections (0.2 μl per side) 30 minutes before Extinction session via intrahippocampal injection cannulae (33-gauge). After the infusion, the cannula was left in place for 30 seconds. The cannula placement was verified by histology, and only data from animals with correct cannula implants were included in statistical analyses.”

(3) More details of what software/algorithm was used to score freezing should be included. 

Freezing was automatically scored with VideoFreeze™ Software (Med Associates Inc.).

(4) Antibody dilutions for IHC should be indicated. Secondary antibody incubation time should be indicated.

The missing information is added.

ln 144: Next, sections were incubated in 4°C overnight with primary antibodies directed against PSD-95 (1:500, Millipore, MAB 1598), washed three times in 0.3% Triton X-100 in PBS and incubated in room temperature for 90 minutes with a secondary antibody bound with Alexa Fluor 647 (1:500, Invitrogen, A31571). 

(5) No statement about code and data availability is present.

The statements are added.

ln 785: Row data and the code used for analysis of confocal data is available at OSF (https://osf.io/bnkpx/).

Reviewer #3 (Public review):

Summary:

This paper examined the role of nucleus reuniens (RE) projections to dorsal CA1 neurons in context fear extinction learning. First, they show that RE neurons send excitatory projections to the stratum oriens (SO) and the stratum lacunosum moleculare (SLM), but not the stratum radiatum (SR). After context fear conditioning, the synaptic connections between RE and dCA1 neurons in the SLM (but not the SO) are weakened (reduced bouton and spine density) after mice undergo context fear conditioning. This weakening is reversed by extinction learning, which leads to enhanced synaptic connectivity between RE inputs and dendrites in the SLM. Control experiments demonstrate that the observed changes are due to extinction and not caused by simple exposure to the context. Extinction learning also induced increases in the size (volume and surface area) of the post-synaptic density (PSD) in SLM. To establish the functional role of RE inputs to dCA1, the researchers used an inhibitory DREADD to silence this pathway during extinction learning. They observe that extinction memory (measured 2-hours or 24-hours later) is impaired by this inhibition. Control experiments show that the extinction memory deficit is not simply due to increased freezing caused by inactivation of the pathway or injections of CNO. Inhibiting the RO projection during extinction learning also reduced the levels of PSD-95 protein levels in the spines of dCA1 neurons.

Strengths:

Based on their results, the authors conclude that, "the RE→SLM pathway participates in the updating of fearful context value by actively regulating CFE-induced molecular and structural synaptic plasticity in the SLM.". I believe the data are generally consistent with this hypothesis, although there is an important control condition missing from the behavioral experiments.

Weaknesses:

(1) A defining feature of extinction learning is that it is context specific (Bouton, 2004). It is expressed where it was learned, but not in other environments. Similarly, it has been shown that internal contexts (or states) also modulate the expression of extinction (Bouton, 1990). For example, if a drug is administered during extinction learning, it can induce a specific internal state. If this state is not present during subsequent testing, the expression of extinction is impaired just as it is when the physical context is altered (Bouton, 2004). It is possible that something similar is happening in Figure 6. In these experiments, CNO is administered to inactivate the RE-dCA1 projection during extinction learning. The authors observe that this manipulation impairs the expression of extinction the next day (or 2-hours later). However, the drug is not given again during the test. Therefore, it is possible that CNO (and/or inactivation of the RE-dCA1 pathway) induces a state change during extinction that is not present during subsequent testing. Based on the literature cited above, this would be expected to disrupt fear extinction as the authors observed. To determine if this alternative explanation is correct, the researchers need to add groups that receive CNO during extinction training and subsequent extinction testing. If the deficits in extinction expression reported in Figure 6 result from a state change, then these groups should not exhibit an impairment. In contrast, if the authors' account is correct, then the expression of extinction should still be disrupted in mice that receive CNO during training and testing.

We do agree with the reviewer that such an experiment would be interesting. However, it could be also confusing as we could not distinguish whether the possible behavioral effects are related to the state-dependent aspects of CFE or impaired recall of CFE. Importantly, previous studies showed that RE is crucial for extinction recall (Totty et al., 2023). We also show that CFE memory is impaired not only when the animals recall CFE without CNO (day 3) but also with CNO (day 4) (Figure 6C). Moreover, we do not see the effects of CNO on CFE in the control groups (Figure 6D and H). So we believe that it is unlikely that CNO results in state-dependent CFE.

(2) In their analysis of dCA1 synapses after contextual fear extinction (CFE) (Figure 4), the authors should have compared Ctx and Ctx-Ctx animals against naïve animals (as they did in Figure 3) when comparing 5US and Ext with naïve animals. Otherwise, the authors cannot make the following conclusion; "since changes of SLM synapses were not observed in the animals exposed to the familiar context that was not associated with the USs, our data support the role of the described structural plasticity at the RE→SLM synapses in CFE, rather than in processing contextual information in general.".

We assume that the key experimental groups to conclude about synaptic plasticity related to particular behavior are the groups that differ just by one factor/experience. For CFE that would be mice sacrificed immediately before and after CFE session (Figure 2 & 3); on the other hand to conclude about the effects of the re-exposure to the neutral context mice sacrificed before and after second exposure to the neutral context are needed (Figure 4). The naive group, as it differs by at least two manipulations from the Ext and Ctx-Ctx groups, is interesting but not crucial in both cases. This group would be necessary if we focused on the memories of FC or novel context. However, these topics are not the main focus of the current manuscript. Still, the naive group is shown on Figures 2 & 3 to check if CFE brings spine parameters to the levels observed in mice with low freezing.

We have re-written the cited paragraph to be more precise in our conclusions. 

"Overall, our data demonstrate that synapses in all dCA1 strata undergo structural or molecular changes relevant to CFC and/or CFE. However, only in SLM CFE-induced synaptic changes are likely to be directly regulated by RE inputs as they appear on RE+ dendrites and spines. Since such changes of SLM synapses were not observed in the animals re-exposed to the neutral context, our data support the role of the described structural plasticity at the RE→SLM synapses in CFE, rather than in processing contextual information in general."

(3) In the materials and methods section, the description of cannula placements is confusing and needs to be rewritten.

This section has been rewritten.

ln 103: Cannula placement. Mice were anesthetized by inhalation of 3–5% isoflurane (IsoFlo; Abbott Animal Health) in oxygen and positioned in a stereotaxic frame (51503, Stoelting, Wood Dale, IL, USA). Two holes were drilled in the skull, and a double guide cannulae (2 mm apart and 2 mm long; 26GA, Plastics One) was lowered into the holes such that the cannula tip was located over dorsal CA1 area (2 mm posterior to bregma, ±1 mm lateral, and −1.3 mm vertical). Cannulae were kept patent by using 33-gauge internal dummy cannulae (Plastics One). The animals were used in contextual fear conditioning 21 days after the cannulation. Animals received bilateral CNO (3 μM, 0.2 μl per side for 1 min; Tocris Bioscience, Cat. No. 4936) (Stachniak et al., 2014) or saline injections (0.2 μl per side) 30 minutes before Extinction session via intrahippocampal injection cannulae (33-gauge). After the infusion, the cannula was left in place for 30 seconds. The cannula placement was verified by histology, and only data from animals with correct cannula implants were included in statistical analyses.”

Interesante

DOI: 10.1016/j.celrep.2024.114797

Resource: (Abcam Cat# ab13970, RRID:AB_300798)

Curator: @scibot

SciCrunch record: RRID:AB_300798

What is this?

(Jn 3:16; Ro 11:33; Tit 1:2).

(Is 48:17, 18; Ro 12:2).

OGM ITS DIDDY!

ვფიქრობა,ამის მიზეზი ადამიანების სტერეოტიპული აზროვნება და ვითარების გონივრულად შეფასების უცოდინრობაა. პიროვნების დისკრიმინაცია მისი სქესის მიხედვით და ამის საფუძველზე მისი მიღწევებისა და ცოდნის დაკნინება,ვფიქრობ,უცოდინრობის გამოვლინებაა მსგავსი ქმედებით ადამიანები ძირს უთხრიან ქალების კარიერულ წინსვლასა და წარმატებას

Summary is Ok but I would like to see you really draw together the material to produce a set of recommendations for what needs to be done to deliver a DQ capability for IOT/WSNs.

You introduce some architecture material earlier. This again feels a little out of place to me. Would it be better to cover the methods entirely and then have a separate section on architecture and implementation digital infrastructure?

This feels that it is in the wrong place to me. It could either come at the end (before the summary/conclusion) or you could bring it right up front and that you are not addressing improvement but that being able to asses DQ is a first step towards knowing when and if you can correct errors.

In general not very keen on use of Notes. This insight should probably be in the main text. We can discuss when we meet.

This is good but depending on how you choose to structure the PhD you may loose a reader without explaining what a LSTM, CNN, GRU etc etc is.

Having read ahead I can only really see two methods covered in the examples/text ... I was expecting a review (even just a short one) of the different approaches before you drop into the architecture considerations.

Don't do anything on this before we next meet but we should discuss this and agree if more specific coverage of the methods is required.

You only present one but state methods ... the example looks fine but are there other monitoring methods?

This is important but should not be presented as a note, it should probably be integrated into the main text.

For the PhD write up you probably need to provide a more detailed critique of each approaches in terms of their success, advantages, limitations etc. but for now this is fine. For the paper we will need to take the table and probably convert to text and provide pointing references to the state of the art in each method.

Source?

Figure

Delete - you define these below.

What is a collapsed point?

REF??

Figure not figure.

Should this be in " " same next statement?

?? what does slope mean in this context?

Source

Struggling to see how this defines the intersection between DQ and DS??

Source!

?? not sure what you mean here.

Found this quite hard to follow - maybe consider how it can be re-written to be clearer.

Delete.

later not former - you have DQ first.

Love the table but maybe consider giving a real example here of the issues impacting a dimension and a single problem having multiple DQ issues.

Ah - in relation to previous comment. Ok you actually do what I suggest in the next paragraph. Maybe consider not having this as a separate section (i.e., 3.1.2) but just have the text flow straight into the IoT paragraph - this will give the first bit on DQ context and linkage to IoT/WSNs.

So how does this impact or help inform your understanding in relation to WSN pedestrian data. I think you maybe need a few sentences critique of these categories and their suitability in relation to IoT/WSNs.

Could do with reference support in this paragraph.

This can be removed.

Any references here that you can point to that support the assertion that these are the important aspects to consider?? You probably cite papers later that in their introductions state these as being important aspects to understand.

This is fine from the technical perspective BUT I would also like to see an articulation of the intellectual challenge that you are looking to address - so a stronger focus on understanding when, where and why data is or is not appropriate for a particular 'real-time' application in relation to better informed autonomous decision making in the built environment. Then state the technical objetcive of developing a library that aims to acheive this.

Good introduction. For the paper this (with a little editing) would suffice for the Introduction if you brought the objectives into it to state the challenge you are looking to address.

REF??? would be good to have ref support here.

I like the analogy with biological systems but be a bit careful not to use it to much!

Maybe better to say:

"WSNs data quality is critically important in relation to the ability to utilise IoT to make better decisions regarding the dynamics of the built environment"

.

Trong chương trình Bí mật đồng tiền diễn ra trưa ngày 17/5, ông Đào Phúc Tường, một chuyên gia chứng khoán với kinh nghiệm 17 năm trên thị trường, đánh giá rằng thị trường hiện đang đắt trong bối cảnh cơ hội ít hơn rủi ro. Ở khối tài chính, cụ thể với ngành ngân hàng, định giá P/B hiện khoảng 1,4 – 1,5 lần, vị chuyên gia cho rằng đây không hẳn là mức cao nhưng bức tranh phía trước của ngành ngân hàng lại mang một màu xám.

Cụ thể trong năm 2023, tăng trưởng tín dụng sẽ rất khó khăn khiến biên lợi nhuận NIM ngân hàng giảm, các trụ chính liên quan đến thu phí của ngân hàng cũng sẽ giảm. Đặc biệt, thông qua báo cáo tài chính quý I của các ngân hàng có thể thấy định giá tài sản chung bên ngoài thị trường đi xuống nhưng tỷ lệ bao phủ nợ xấu ngân hàng lại giảm.

"Điều này cho thấy ngân hàng đang co kéo để có được tăng trưởng lợi nhuận tốt trong quý I vừa rồi, nếu tình hình không có tiến triển trong quý II, III thì tăng trưởng lợi nhuận của ngành ngân hàng sẽ nằm trong vùng rủi ro.

Ở khối phi tài chính, với mức định giá P/E 15 – 16 lần, trụ chính của khối này liên quan đến bất động sản, các hàng hoá cơ bản,… Đây là những doanh nghiệp có nền lợi nhuận quý I, II thậm chí quý III năm ngoái rất cao. Đến quý II, III năm nay, dự báo P/E sẽ lên 18 – 20 lần, một nhóm ngành chưa nhìn thấy đầu ra với mức định giá này là không hợp lý.", ông Tường đưa ra lời nhận định.

trong một cái cái thị trường khi mà tăng trưởng trở thành một thứ xa xỉ tăng trưởng nền kinh tế rồi tăng trưởng của ngành trở thành một sự xa xỉ thì chỉ có một thì những cái tăng trưởng đột biến ở

00:32:58

một vài doanh nghiệp là sẽ trở thành có thể giúp những cổ phiếu đấy trở thành những ngôi sao Thì đấy là cái cách mà tiếp cận để để tìm ra những cổ phiếu mà có thể tăng trưởng đột biến Ví dụ như ngành nhựa vừa rồi ống nhựa vừa rồi chẳng hạn thì bình minh so với nhựa Tiền Phong thì rõ ràng là nhựa Bình Minh có một cái nền tảng tăng trưởng tốt hơn rất nhiều với những yếu tố nội tại của doanh nghiệp Thì đấy là đấy là cách nhìn thế thì liên

00:33:24

quan đến câu hỏi câu hỏi của Nam là có ngành nào bắt đầu có có tia sáng thì chắc là chúng ta phải đợi đến quý 3 quý 4 Ví dụ như ngành chăn nuôi chẳng hạn thì chăn nuôi chúng ta nhìn thấy một ví dụ rất đơn giản thôi là đàn gà đàn lợn thì 3 đến 6 tháng là chỉ có vòng đời thế thôi mà mình nông dân người ta không nuôi thì sẽ không có thịt mà nhu cầu luộc thịt là nhu cầu thiết yếu của người dân Thế thì khi mà cung giảm một cách đột biến cầu thì vẫn cứ tăng trưởng bền

00:33:52

vững Tất nhiên là cầu cho thầy sản phẩm thịt thì không thể tăng nhanh được các bạn vững thì nó sẽ kích hoạt với giá tăng đặc biệt là với những doanh nghiệp mà đã trích lập dự phòng khá là đầy đủ thì sau đó nó sẽ tạo tiềm năng cho cái chuyện lợi nhuận tăng trưởng mạnh trở lại thì đấy chính là những yếu tố giúp cho giá cổ phiếu tăng thì anh Hưng có kinh nghiệm nhiều chắc là không thì tôi vĩ mô đi từ trên xuống anh còn chưa

chúng ta tách ra thị trường làm hai nhóm nhóm thứ nhất là nhóm tài chính, cụ thể là nhóm ngân hàng thì cái định giá của ngân hàng bây giờ đang tầm khoảng 1,4-1,5 lần P/B đánh giá thực ra nhìn thì không phải là cao, đấy nhưng mà cái bức tranh phía trước của ngành ngân hàng thì chúng ta thấy một màu nó hơi xám

00:22:12

tăng trưởng tín dụng thì năm nay tăng trưởng rất là khó và trong cái môi trường tăng trưởng ví dụ khó thì các ngân hàng sẽ phải gianh giat nhau khách hàng tốt và nó làm cho biết lợi nhuận của cái NIM của ngân hàng nó sẽ sẽ giảm xuống các cái trụ chính liên quan đến thu phí của ngân hàng năm nay cũng sẽ giảm Ví dụ như bánh kem qua một loạt các sự kiện gần đây nó cũng sẽ sẽ giảm và đặc biệt hơn nữa là Nếu chúng ta xem báo cáo quý 1 các ngân hàng chúng ta thấy

00:22:37

rằng là định giá tài sản chung với ngoài thị trường thì đi xuống nhưng mà tỷ lệ bao phủ nợ xấu của ngân hàng lại giảm chứng tỏ rằng là các ngân hàng cũng đang co kéo để có được tốc độ tăng trưởng lợi nhuận tốt trong quý 1 vừa rồi thì nếu mà quý 2 và quý 3 tình hình không có tiến triển thì cái tăng trưởng lợi nhuận của ngành ngân hàng sẽ nằm ở trong vùng rủi ro.

đấy là với tài chỉ tài chính khối Phi Tài chính thì bây giờ đang định giá ở cái tầm PE khoảng tầm 15-16 lần trong khi đó thì cái trụ chính của khối Phi Tài chính thì nó liên quan đến bất động sản, Liên quan đến các mặt hàng liên quan đến đến các hàng hóa cơ bản, đó và những cái doanh nghiệp như thế thì cái quý 1 và quý 2 kể cả quý 3 năm ngoái thì cái nền lợi nhuận nó rất là cao, nên chúng ta nhìn xa một chút đến quý 2 quý 3 năm nay thì nó sẽ dành lên đến tầm 18 đến 20 lần PE Nếu mà chúng ta nhìn dự báo thế thì một cái ngành mà chưa nhìn

00:23:34

thấy đầu ra một cái nhóm ngành mà chưa nhìn thấy đầu ra mà định giá 18 20 lần pi nó nằm ở mid cycle thì có gì đó nó nó không hợp lý

Neu nghĩ chung về ngành thì lấy một góc hẹp đây thì đúng là ra đặt hai van de cái này không cần nhấn có thèm cái kết quả lợi nhuận quý 1 của công ty chứng khoán được rồi Đúng là chẳng có một sự và là đột phá so với ki vong của thị

00:49:25

trường hoàn toàn ổn định đến thôi là rất không Ngay cả cuộc đời chứng khoán à công bố Thông tin cấp một thị trường nó ra Rất là tranh tối tranh sáng rất có tên là giả cổ phiếu một công cụ không phải là tốt nhưng mà nhìn rộng hơn chút đấy ra lại thấy rằng là anh cũng tương tự như chả có cái này 21 đã được tăng lên quá cao đến Tân lạc của các ngân hàng để làm tham chiếu thấy rằng nửa đầu năm của mà không Mai mốt giá cổ phiếu ngân hàng nên

00:49:51

tốt mà sau đó thì chững di ngang nha vào trong đó phải tập dần là vì lúc đó chưa gái của tiếng nó thăng hơn rất nhiều so với lại tăng trưởng của ngân hàng là lại cho cho 2021 vào cho nên là nó cũng nó cũng còn luc de day nua những cái này nữa thì mới nói cuộc cái quy khóa gia CTCK cũng tương tự lá như thế là đánh đẩy lên quá cao và thể hiện giá đẩy quá cao đó thực sự nó có những điều thú vị thị trường nhỏ đặc biệt là kế hoạch phát hành không chứng khoán chúng ta sẽ nghe đâu đó nói chuyện OK công ty nó

00:50:18

cũng đi phát hành đã bỏ pin nó đã cổ phiếu đã chặn nó thì đó là cái kính cơ gọi là tính động cơ hội trường tăng lên Thế còn đang nghĩ tới mức mà định giá của công ty chứng khoán của các bức ảnh giá cả bị cả nước hàng thì cá nhân tôi cho rằng nó sẽ bật định là sẽ bắt đầu Mỹ tên kìa cao thế rồi mà tôi nói buồn anh em ấy là giá cao nên tránh đạo cũng chứng khoán Nếu nhìn thấy giá thị trường ý thức Cổ phiếu của mình Tại sao tay phát

00:50:44

hành trên giá thấp đã thị trường Long tục thì nó dành cho xã 12 có thể nó có yếu tố gì đó rất hợp lý ở trong phần trong câu chuyện đó thực tế nếu về cái ngôn ngữ có định giá thì giá cổ phiếu này định giá của công minh chứng khoán khó mà cao hơn để tránh ngân hàng được những gì mình sẽ không còn tất nhiên sẽ có những công ty trên bề mặt vụ ngang hàng nói cái là người đã chỉ quản lý làm sau đấy để còn cả cả Magiê tốt thì phải quay tốt thì giá sẽ cao hơn nữa cả vì

00:51:09

cái quản lý rủi ro khi chứng khoán nó dễ hơn rất nhiều có phản ứng gì đâu này thì chắc chắn là nó sẽ cao hơn nhưng mà kết luận từ các yếu tố rất là được Có những khoản vay đến từ hai phần những công ty dịch vụ thì đa phần lợi nhuận đến từ tuần hoàn Dịch vụ thế mạnh đó nó khá là ổn định có ích rất nhiều khi chứng khoán cái phần luận tự do Kiếm 6 75 được thế thì khi vào thị trường khó đi làm sao kiếm tính là sao cả Định sang hàng công ty có thể kiếm thường của nó bình luận

00:51:33

cho nên đã lên rồi thì phải trả lại tôi giá trị của nó thì nhiên đến tương lai Tôi không nghĩ rằng 520 ở nhà sẽ làm 5 tốt cho cổ phiếu linh hoạt mà nó sẽ như hàng nói tôi đồng ý cho sẽ có sự phân hóa nhưng mà thấy giống hóa đấy nó sẽ đến sau khi cả cái bóng này để nó sẽ bị gọi là gì vậy gọi là thức mặc xác tôi cảm ơn ông tưởng nếu mà chúng ta cứ tiếp tục câu chuyện như thế này nó sẽ rất là giải và chương trình của chúng ta thì cố gắng phấn đấu và mỗi xấu chúng ta

Regularizare

Definiție

OBLIGAȚIA de a pune la dispoziția PMB planurile de rețele

Recepția la terminarea lucrărilor

drum deschis circulației publice

Definiție drum public

Închidere balcoane

Acord asociație pentru modficiări constructive

DOI: 10.1007/s11356-024-34464-7

Resource: ImageJ (RRID:SCR_003070)

Curator: @scibot

SciCrunch record: RRID:SCR_003070

What is this?

Ce poate face primarul față de persoane fizice cu clădiri degradatae

another anecdote

Author response:

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

Reviewing editor’s list of items remaining to be addressed followed by our responses/actions:

(1) The order and organization of supplemental figures and tables is almost impossible to navigate. Please put them in order. 

All the sections from the previous Supplementary files have been divided into individual Supplementary files so that each can be referenced without confusion from the text. All of the references in the body of the text and the author responses have been updated to reflect this change.

(2) The question of sample sizes was partially addressed, with authors stating that cell culture work in iPSCs and PGCLCs was done in replicates of 3. Sertoli and granulosa cells were generated from pooled preps - how many individuals, were they littermates? 

Sertoli and granulosa primary cultures were generated from littermates and each prep used 5 animals (males for Sertoli cells and females for granulosa cells). These changes have been added to the body of the text on pages 39 and 40.

(3) Authors need to discuss the limitations of doing work in triplicates. Their PCA (Supplement Figure 9) reveals that in several cases samples from the same treatment were not discriminated by PC1 and/or PC2. This is especially true in e and f, the variance of which was explained by PC1 for cell type, but for which treatments showed poor discrimination by PC2. Some discussion of the limitations of sample size should be provided.

Additional text has been added to what is now Supplementary file 15 to acknowledge this limitation imposed by the limited number of replicates (three) and the ability to resolve the differences in treatments by PCA in subplots e and f. However, we also note that the differences were sufficient to identify significant DMCs/DMRs/DEGs.

Reviwer 2 also noted a potential weakness that “exposures are more complicated in a whole organism than in an isolated cell line.”

We note that in our revised manuscript we included wording noting that despite the advantages of using an in vitro approach to deduce underlying molecular mechanisms, results of such in vitro studies “ultimately warrant validation of results discerned from studies of in vitro models to ensure they also reflect functions ongoing in the more complex and heterogeneous environment of the intact animal in vivo.” Thus we have endeavored to acknowledge the reviewer’s point.

Reviewer #1 (Public Review): 

Critiques/Comments: 

(1) A problem with in vitro work is that homogeneous cell lines/cultures are, by nature, absent from the rest of the microenvironment. The authors need to discuss this. 

[Addressed on pages: 24-25] – We have added two sentences to the second paragraph of the Discussion section in which we now acknowledge this concern, but also point out that in vitro models of this sort also provide an experimental advantage in that they facilitate a deconvolution of the extensive complexity resident within the intact animal. Nevertheless, we acknowledge that this deconvolution requires ultimate validation of findings obtained within an in vitro model system to ensure they accurately recapitulate functions that occur in the intact animal in vivo.

In response to Reviewer 2’s stated weakness of our study that “The weakness includes the fact that exposures are more complicated in a whole organism than in an isolated cell line,” please note that this added text includes the statement that despite the advantages of using an in vitro approach to deduce underlying molecular mechanisms, results of such in vitro studies “ultimately warrant validation of results discerned from studies of in vitro models to ensure they also reflect functions ongoing in the more complex and heterogeneous environment of the intact animal in vivo.” Thus we have endeavored to acknowledge the reviewer’s point.

(2) What are n's/replicates for each study? Were the same or different samples used to generate the data for RNA sequencing, methylation beadchip analysis, and EM-seq? This clarification is important because if the same cultures were used, this would allow comparisons and correlations within samples.  

Addressed on pages: 39-45 and in new Supplementary file 15 – Additional text has been added in the Methods section to indicate that all samples involving cell culture models which include iPSCs and PGCLCs came from a single XY iPS cell line aliquoted into replicates and all primary cultures which included Sertoli and granulosa cells were generated from pooled tissue preps from mice and then aliquoted into replicates. Finally, all experiments in the study were performed on three replicates. Because this experimental design did indeed allow for comparisons among samples, we have added a new Supplementary file 15

which displays PCA plots showing clustering among control and treatment datasets, respectively, as well as distinctions between each cluster representing each experimental condition.

(3) In Figure 1, it is interesting that the 50 uM BPS dose mainly resulted in hypermethylation whereas 100 uM appears to be mainly hypomethylation. (This is based on the subjective appearance of graphs). The authors should discuss and/or present these data more quantitatively. For example, what percentage of changes were hypo/hypermethylation for each treatment? How many DMRs did each dose induce? For the RNA-seq results, again, what were the number of up/down-regulated genes for each dose?  

Addressed on pages: 6-7 and in new Supplementary files 1-3  – The experiment shown in Figure 1 was designed to 1) serve as proof of principle that cells maintained in culture could be susceptible to EDC-induced epimutagenesis at all, 2) determine if any response observed would be dose-dependent, and 3) identify a minimally effective dose of BPS to be used for the remaining experiments in this study (which we identified as 1 μM). We agree that it is interesting that the 50 µM dose of BPS induced predominantly hypermethylation changes whereas the 1 µM and 100 µM doses induced predominantly hypomethylation changes, but are not in a position to offer a mechanistic explanation for this outcome at this time. As the results shown satisfied our primary objectives of demonstrating that exposure of cells in culture to BPS could indeed induce DNA methylation epimutations, that this occurs in a dose-dependent manner, and that a dose of as low as 1 µM of BPS was sufficient to induce epimutagenesis, the data obtained satisfied all of the initial objectives of this experiment. That said, in response to the reviewer’s request we have now added text on pages 6-7 alluding to new Supplementary files 1-3 indicating the total number of DMCs and DMRs, as well as the number of DEGs, detected in response to exposure to each dose of BPS shown in Figure 1, as well as stratifying those results to indicate the numbers of hyper- and hypomethylation epimutations and up- and down-regulated DEGs induced in response to each dose of BPS. While, as noted above, investigating the mechanistic basis for the difference in responses induced by the 50 µM versus 1 and 100 µM doses of BPS was beyond the scope of the study presented in this manuscript, we do find this result reminiscent of the “U-shaped” response curves often observed in toxicology studies. Importantly, this result does demonstrate the elevated resolution and specificity of analysis facilitated by our in vitro cell culture model system.

(4) Also in Figure 1, were there DMRs or genes in common across the doses? How did DMRs relate to gene expression results? This would be informative in verifying or refuting expectations that greater methylation is often associated with decreased gene expression.  

Addressed on pages: 6-7 and new Supplementary files 1-6 – In general, we observed a coincidence between changes in DNA methylation and changes in gene expression (Supplementary files 1-3). Pertaining directly to the reviewer’s question about the extent to which we observed common DMRs and DEGs across all doses, while we only found 3 overlapping DMRs conserved across all doses tested, we did find an average of 51.25% overlap in DMCs and an average of 80.45% overlap in DEGs across iPSCs exposed to the different doses of BPS shown in Figure 1. In addition, within each dose of BPS tested in iPSCs, we also found that there was an overlap between DMCs and the promoters or gene bodies of many DEGs (Supplementary file 5). Specifically within gene promoters, we observed a correlation between hypermethylated DMCs and decreased gene expression and hypomethylated DMCs and increased gene expression, respectively (Supplementary file 6).

(5) In Figure 2, was there an overlap in the hypo- and/or hyper-methylated DMCs? Please also add more description of the data in 2b to the legend including what the dot sizes/colors mean, etc. Some readers (including me) may not be familiar with this type of data presentation. Some of this comes up in Figure 4, so perhaps allude to this earlier on, or show these data earlier.  

Addressed on pages: 8-9 and new Supplementary file 4 – We observed an average of 11.05% overlapping DMCs between different pairs of cell types, we did not observe any DMCs that were shared among all four cell types. Indeed, this limited overlap of DMCs among different cell types exposed to BPS was the primary motivation for the analysis described in Figure 2. Thus, instead of focusing solely on direct overlap between specific DMCs, we instead examined similarities among the different cell types tested in the occurrence of epimutations within different annotated genomic regions. To better describe this, we have now added additional text to page 9. We have also added more detail to the legend for Figure 2 on page 8 to more clearly explain the significance of the dot sizes and colors, explaining that the dot sizes are indicative of the relative number of differentially methylated probes that were detected within each specific annotated genomic region, and that the dot colors are indicative of the calculated enrichment score reflecting the relative abundance of epimutations occurring within a specific annotated genomic region. The relative score is calculated by iterating down the list of DMCs and increasing a running-sum statistic when encountering a DMC within the specific annotated genomic region of interest and decreasing the sum when the epimutation is not in that annotated region. The magnitude of the increment depends upon the relative occurrence of DMCs within a specific annotated genomic region.

(6) iPSCs were derived from male mice MEFs, and subsequently used to differentiate into PGCLCs. The only cell type from an XX female is the granulosa cells. This might be important, and should be mentioned and its potential significance discussed (briefly).  

Addressed on page: 29 – We have added a new paragraph just before the final paragraph of the Discussion section in which we acknowledge that most of the cell types analyzed during our study were XY-bearing “male” cells and that the manner in which XX-bearing “female” cells might respond to similar exposures could differ from the responses we observed in XY cells. However, we also noted that our assessment of XX-bearing granulosa cells yielded results very similar to those seen in XY Sertoli cells suggesting that, at least for differentiated somatic cell types, there does not appear to be a significant sex-specific difference in response to exposure to a similar dose of the same EDC. That said, we also acknowledged that in cell types in which dosage compensation based on X-chromosome inactivation is not in place, differences between XY- and XX-bearing cells could accrue.

(7) EREs are only one type of hormone response element. The authors make the point that other mechanisms of BPS action are independent of canonical endocrine signaling. Would authors please briefly speculate on the possibility that other endocrine pathways including those utilizing AREs or other HREs may play a role? In other words, it may not be endocrine signaling independent. The statement that the differences between PGCLCs and other cells are largely due to the absence of ERs is overly simplistic.  

Addressed on page: 11 and in a new Supplementary file 8  – Previous reports have indicated that BPS does not have the capacity to bind with the androgen receptor (Pelch et al., 2019; Yang et al., 2024). However there have been reports indicating that BPS can interact with other endocrine receptors including PPARγ and RXRα, which play a role in lipid accumulation and the potential to be linked to obesity phenotypes (Gao et al., 2020; Sharma et al., 2018). To address the reviewer’s comment we assessed the expression of a panel of hormone receptors including PPARγ, RXRα, and AR  in each of the cell types examined in our study and these results are now shown in a new Supplementary file 8. We show that in addition to not expressing either estrogen receptor (ERa or ERb), germ cells also do not express any of the other endocrine receptors we tested including AR, PPARγ, and RXRα. Thus we now note that these results support our suggestion that the induction of epimutations we observed in germ cells in response to exposure to BPS appears to reflect disruption of non-canonical endocrine signaling. We also note that non-canonical endocrine signaling is well established (Brenker et al., 2018; Ozgyin et al., 2015; Song et al., 2011; Thomas and Dong, 2006). Thus we feel the suggestion that the effects of BPS exposure could conceivably reflect either disruption of canonical or non-canonical signaling in any cell type is well justified and that our data suggests that both of these effects appear to have accrued in the cells examined in our study as suggested in the text of our manuscript.

(8) Interpretation of data from the GO analysis is similarly overly simplistic. The pathways identified and discussed (e.g. PI3K/AKT and ubiquitin-like protease pathways) are involved in numerous functions, both endocrine and non-endocrine. Also, are the data shown in Figure 6a from all 4 cell types? I am confused by the heatmap in 6c, which genes were significantly affected by treatment in which cell types?  

Addressed on pages: 19-21 – Per the reviewer’s request, we have added text to indicate that Figure 6a is indeed data from all four cell types examined. We have also modified the text to further clarify that Figure 6c displays the expression of other G-coupled protein receptors which are expressed at similar, if not higher, levels than either ER in all cell types examined, and that these have been shown to have the potential to bind to either 17β-estradiol or BPA in rat models. As alluded to by the reviewer, this is indicative of a wide variety of distinct pathways and/or functions that can potentially be impacted by exposure to an EDC such as BPS. Thus, we have attempted to acknowledge the reviewer’s primary point that BPS may interact with a variety of receptors or other factors involved with a wide variety of different pathways and functions. Importantly, this illustrates the strength of our model system in that it can be used to identify potential impacted target pathways that can then be subsequently pursued further as deemed appropriate.

(9) In Figure 7, what were the 138 genes? Any commonalities among them? 

Addressed on page: 22 and in a new Supplementary files 13 and 14 – We have now added a new supplemental Excel file (Supplementary file 13) that lists the 138 overlapping conserved DEGs that did not become reprogrammed/corrected during the transition from iPSCs to PGCLCs. In addition, we have added new text on page 22 and a new Supplementary file 14 which displays KEGG analysis of pathways associated with these 138 retained DEGs. We find that these genes are primarily involved with cell cycle and apoptosis pathways which, interestingly, have the potential to be linked to cancer development which is often linked to disruptions in chromatin architecture.

(10) The Introduction is very long. The last paragraph, beginning line 105, is a long summary of results and interpretations that better fit in a Discussion section.

Addressed on page: 6 – We have now significantly reduced the length and scope of the final paragraph of the Introduction per the reviewer’s recommendation.

(11) Provide some details on husbandry: e.g. were they bred on-site? What food was given, and how was water treated? These questions are to get at efforts to minimize exposure to other chemicals.  

Addressed on page: 37 – We have added additional text detailing that all mice used in the project were bred onsite, water was non-autoclaved conventional RO water, and our selection of 5V5R extruded feed for mice used in this study which was highly controlled for the presence of isoflavones and has been certified to be used for estrogen-sensitive animal protocols.

Reviewer #2 (Public Review): 

Summary: 

This manuscript uses cell lines representative of germ line cells, somatic cells, and pluripotent cells to address the question of how the endocrine-disrupting compound BPS affects these various cells with respect to gene expression and DNA methylation. They find a relationship between the presence of estrogen receptor gene expression and the number of DNA methylation and gene expression changes. Notably, PGCLCs do not express estrogen receptors and although they do have fewer changes, changes are nevertheless detected, suggesting a nonconical pathway for BPS-induced perturbations. Additionally, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. Epimutations in the somatic and pluripotent cell types were predominantly in enhancer regions whereas that in the germ cell type was predominantly in gene promoters. 

Strengths: 

The strengths of the paper include the use of various cell types to address the sensitivity of the lineages to BPS as well as the observed relationship between the presence of estrogen receptors and changes in gene expression and DNA methylation. 

Weaknesses: 

The weaknesses include the lack of reporting of replicates, superficial bioinformatic analysis, and the fact that exposures are more complicated in a whole organism than in an isolated cell line. 

Recommendations for the authors: please note that you control which revisions to undertake from the public reviews and recommendations for the authors. 

Reviewer #2 (Recommendations For The Authors): 

Overall, this is an intriguing paper but more transparency in the replicates and methods and a more rigorous bioinformatic treatment of the data are required. 

Specific comments: 

(1) End of abstract "These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally following a single exposure to the causative EDC." This is overly speculative for an abstract. There is only epigenetic inheritance following mitosis or differentiation presented in this study. There is no meiosis and therefore no ability to assess multi- or transgenerational inheritance. 

Addressed on page: 2 – We have modified the text at the end of the abstract to more precisely reflect our intended conclusions based on our data. In our view, the ability of induced epimutations to transcend meiosis per se is not as relevant to the mechanism of transgenerational inheritance as their ability to transcend major waves of epigenetic reprogramming that normally occur during development of the germ line. In this regard the transition from pluripotent iPSCs to germline PGCLCs has been shown to recapitulate at least the first portion of normal germline reprogramming, and now our data provide novel insight into the fate of induced epimutations during this process. Specifically, we show that a prevelance of epimutations was conserved during the iPSC à germ cell transition but that very few (< 5%) of the specific epimutations present in the the BPS-exposed iPSCs were retained when those cells were induced to form PGCLCs. Rather, we observed apparent correction of a large majority of the initially induced epimutations during this transition, but this was accompanied by the apparent de novo generation of novel epimutations in the PGCLCs. We suggest, based on other recent reports in the literature, that this is a result of the BPS exposure inducing changes in the chromatin architecture in the exposed iPSCs such that when the normal germline reprogramming mechanism is imposed on this disrupted chromatin template there is both correction of many existing epimutations and the genesis of many novel epimutations. This observation has the potential to explain the long-standing question of why the prevalence of epimutations persists across multiple generations despite the occurrence of epigenetic reprogramming during each generation. Nevertheless, as noted above, we have modified the text at the end of the abstract to temper this interpretation given that it is still somewhat speculative at this point.

(2) Doses used in the experiments. One needs to be careful when stating that the dose used is "below FDA's suggested safe environmental level established for BPA" because a different bisphenol is being used here (BPA vs BPS) and the safe level is that which the entire organism experiences. It is likely that cell lines experience a higher effective dose.  

Addressed on pages: 3, 5, and 26 – We have now made a point of noting that our reference to an EPA-recommended “safe dose” of BPA was for humans and/or intact animals. Changes to this effect have been made in the second and sixth paragraphs of the Introduction section. In addition, we have added text at the end of the fourth paragraph of the Discussion section acknowledging that, as the reviewer suggests, the same dose of an EDC could exert greater effects on cells in a homogeneous culture than on the same cell type within an intact animal given the potential for mitigating metabolic effects in the latter. However, we also note that the ability we demonstrated to quantify the effects of such exposures on the basis of numbers of epimutations (DMCs or DMRs) induced could potentially be used in future studies to study this question by assessing the effects of a specific dose of a specific EDC on a specific cell type when exposed either within a homogeneous culture or within an intact animal.

(3) Figure 1: In the dose response, what was the overlap in DMCs and DEGs among the 3 doses? Are the responses additive, synergistic, or completely non-overlapping? This is an important point that should be addressed. 

Addressed on page: 6-7 and in Supplementary files 1-5 – Please see our response to Reviewer 1 critique #4 above where we address similar concerns. While we do find overlap among different cell types with respect to the DMCs, DMRs, and DEGs displayed in Figure 1, we found the effect to be only partially additive as opposed to synergistic in any apparent manner. The fold increase in DMCs, DMRs, and DEGs resulting from exposure to doses of 1 μM or 50 μM ranged from 2.5x to 4.4x, which was well below the 50x increase that would have been expected from a strictly additive effect, and the effect increased even less, if at all, in response to exposure to doses of 50 μM versus 100 μM BPS. Finally, as now noted in the Discussion section on page 25, our conclusion is that these results display a limited dose-dependent effect that was partially additive but also plateaued at the highest doses tested.

(4) Methods: How many times was each exposure performed on a given cell type? This information should be in the figure legends and methods. In the case of multiple exposures for a given line, do the biological replicates agree? 

Addressed on pages: 39-45 and in new Supplementary file 15 –  Please see our response to Reviewer 1 critique #2 where we address similar concerns with newly added text and analysis. We now note repeatedly on pages 39-45 that each analysis was conducted on three replicate samples, and we display the similarity among those replicates graphically in a new Supplementary file 15.

(5) DNA methylation analyses. Very little analysis is presented on the BeadChip array other than hypermethylated/hypomethylated and genomic regions of DMCs. What is the range of methylation changes? Does it vary between hypo vs. hyper DMCs? How many array experiments were performed (biological replicates) and what stats were used to determine the DMCs? Are there DMCs in common among the various cell types? As an example, if more meaningful analysis, one can plot the %5mC over a given array for comparisons between control and treated cell types. For more granularity, the %5mC can be presented according to the element type (enhancers vs promoters). 

Addressed on pages: 10 and 39-45 and in new Supplementary files 1-5, 15 –  Please see our response to Reviewer 1 critique #2 above where we address similar concerns regarding the number of biological replicates used in this study. DMCs on the Infinium array are identified using mixed linear models. This general supervised learning framework identifies CpG loci at which differential methylation is associated with known control vs. treated co-variates. CpG probes on the array were defined as having differential changes that met both p-value and FDR (≤ 0.05) significant thresholds between treatment and control samples for each cell type analyzed. The range of medians across all samples was 0.0278 to 0.0059 for hypermethylated beta values and -0.0179 to -0.0033 for hypomethylated beta values. As noted above, we did observe an overlap in DMCs between cell types. Thus, we observed an average of 11.05% overlapping DMCs between two or more cell types but we did not observe any DMCs shared between all four cell types. We have added additional text on page 9 and new Supplementary files 1-5 to now more clearly describe that this limited similarity in direct overlap of DMCs was the underlying motivation for the analysis described in Figure 2. Finally, the enrichment dot plots shown in Figure 2 provide the information the reviewer requested regarding the %5mC observed at different annotated genomic element types.

(6) The investigators correlate the number of DMCs in a given cell type with the presence of estrogen receptors. Does the correlation extend to the methylation difference (delta beta) at the statistically different probes?

Addressed in a new Supplementary file 7 – We have added a new Supplementary file 7 in which we provide data addressing this question. In brief, we find that the delta betas of probes enriched at enhancer regions and associated with relative proximity to ERE elements in Sertoli cells, granulosa cells, and iPSCs appear very similar to those associated with DMCs not located within these enriched regions. However, when we compared the similarity of the two data sets with goodness of fit tests, we found these relatively small differences were, in fact, statistically significant based on a two-sample Kolmogorov-Smirnov test. These observed significant differences appear to indicate that there is higher variability among the delta betas associated with hypomethylated, but not hypermethylation changes occurring at DMCs associated with enhancers, potentially suggesting a greater tendency for exposure to BPS to induce hypomethylation rather than hypermethylation changes, at least in these specific regions.

(7) Methylation changes relative to EREs are presented in multiple figures. Are other sequences enriched in the DMCs? 

Addressed in a new Supplementary file 11. We profiled the genomic sequence within 500 bp of cell type-specific enriched DMCs that were either associated with enhancer regions in Sertoli, granulosa, or iPS cells or transcription factor binding sites in PGCLCs for the identification of higher abundance motif sequences. We then compared any motifs identified with the JASPAR database to potentially find transcription factors that could be binding to these regions. Interestingly we found that the two most common motifs across all cell types were associated with either the chromatin remodeling transcription factor HMG1A or the pluripotency factor KLF4.

(8) Please present a correlation plot between the methylation differences and the adjacent DEGs. Again, the absence of consideration of the absolute changes in methylation and gene expression minimizes the impact of the data. 

Addressed on pages 6, 7, and 17 and in a new Supplementary file 6 – We analyzed the relationship between DMCs at DEGs promoter regions and the corresponding change in expression of that DEG. Our data support a relationship between up-regulated genes showing decreased methylation in promoter regions and down-regulated genes showing increased methylation at promoter regions, although there were some exceptions to this relationship.

(9) EM-Seq is mentioned in Figure 7 and in the material and methods. Where is it used in this study? 

Addressed on page 22 – We now note in the text on page 22 that EM-seq was used during experiments assessing the propagation of BPS-induced epimutations during the iPSC à EpiLC à PGCLC cell state transitions to gather higher resolution data of changes to DNA methylation differences at the whole-epigenome level.

References

Brenker C, Rehfeld A, Schiffer C, Kierzek M, Kaupp UB, Skakkebæk NE, Strünker T. 2018. Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals. Hum Reprod 33:1915–1923. doi:10.1093/humrep/dey275

Gao P, Wang L, Yang N, Wen J, Zhao M, Su G, Zhang J, Weng D. 2020. Peroxisome proliferator-activated receptor gamma (PPARγ) activation and metabolism disturbance induced by bisphenol A and its replacement analog bisphenol S using in vitro macrophages and in vivo mouse models. Environ Int 134. doi:10.1016/J.ENVINT.2019.105328

Ozgyin L, Erdos E, Bojcsuk D, Balint BL. 2015. Nuclear receptors in transgenerational epigenetic inheritance. Prog Biophys Mol Biol. doi:10.1016/j.pbiomolbio.2015.02.012

Pelch KE, Li Y, Perera L, Thayer KA, Korach KS. 2019. Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles. Toxicol Sci 172:23–37. doi:10.1093/TOXSCI/KFZ173

Sharma S, Ahmad S, Khan MF, Parvez S, Raisuddin S. 2018. In silico molecular interaction of bisphenol analogues with human nuclear receptors reveals their stronger affinity vs. classical bisphenol A. Toxicol Mech Methods 28:660–669. doi:10.1080/15376516.2018.1491663

Song K-H, Lee K, Choi H-S. 2011. Endocrine Disrupter Bisphenol A Induces Orphan Nuclear Receptor Nur77 Gene Expression and Steroidogenesis in Mouse Testicular Leydig Cells. Endocrinology 143:2208–2215. doi:10.1210/endo.143.6.8847

Thomas P, Dong J. 2006. Binding and activation of the seven-transmembrane estrogen receptor GPR30 by environmental estrogens: A potential novel mechanism of endocrine disruption. J Steroid Biochem Mol Biol 102:175–179. doi:10.1016/j.jsbmb.2006.09.017

Yang Z, Wang L, Yang Y, Pang X, Sun Y, Liang Y, Cao H. 2024. Screening of the Antagonistic Activity of Potential Bisphenol A Alternatives toward the Androgen Receptor Using Machine Learning and Molecular Dynamics Simulation. Environ Sci Technol 58:2817–2829. doi:10.1021/ACS.EST.3C09779/ASSET/IMAGES/LARGE/ES3C09779_0004.JPEG

DOI: 10.1016/j.scr.2024.103493

Resource: (R and D Systems Cat# MAB1368, RRID:AB_357658)

Curator: @scibot

SciCrunch record: RRID:AB_357658

What is this?

DOI: 10.1242/bio.059525

Resource: (BDSC Cat# 8164,RRID:BDSC_8164)

Curator: @mpairish

SciCrunch record: RRID:BDSC_8164

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DOI: 10.1016/j.celrep.2024.114406

Resource: eulerr (RRID:SCR_022753)

Curator: @scibot

SciCrunch record: RRID:SCR_022753

What is this?

https://www.reddit.com/r/typewriters/comments/1dz7kdy/is_this_really_the_smallest_typeface_ever_made/

I've heard of Olympia SG1s with 17 CPI typefaces and some seem to think that 20 CPI machines may exist.

Unul din puținele cursuri gratuite cu puncte EMC

DOI: 10.1186/s12864-024-10423-x

Resource: (DSHB Cat# ro-62C2A8 anti-rough, RRID:AB_528456)

Curator: @evieth

SciCrunch record: RRID:AB_528456

What is this?

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

(1) A problem with in vitro work is that homogeneous cell lines/cultures are, by nature, absent from the rest of the microenvironment. The authors need to discuss this. 

We have added two sentences to the second paragraph of the Discussion section in which we now acknowledge this concern, but also point out that in vitro models of this sort also provide an experimental advantage in that they facilitate a deconvolution of the extensive complexity resident within the intact animal. Nevertheless, we acknowledge that this deconvolution requires ultimate validation of findings obtained within an in vitro model system to ensure they accurately recapitulate functions that occur in the intact animal in vivo.

(2) What are n's/replicates for each study? Were the same or different samples used to generate the data for RNA sequencing, methylation beadchip analysis, and EM-seq? This clarification is important because if the same cultures were used, this would allow comparisons and correlations within samples.  

Additional text has been added in the Methods section to indicate that all samples involving cell culture models which include iPSCs and PGCLCs came from a single XY iPS cell line aliquoted into replicates and all primary cultures which included Sertoli and granulosa cells were generated from pooled tissue preps from mice and then aliquoted into replicates. Finally, all experiments in the study were performed on three replicates. Because this experimental design did indeed allow for comparisons among samples, we have added a new Supplement figure 9 which displays PCA plots showing clustering among control and treatment datasets, respectively, as well as distinctions between each cluster representing each experimental condition.

(3) In Figure 1, it is interesting that the 50 uM BPS dose mainly resulted in hypermethylation whereas 100 uM appears to be mainly hypomethylation. (This is based on the subjective appearance of graphs). The authors should discuss and/or present these data more quantitatively. For example, what percentage of changes were hypo/hypermethylation for each treatment? How many DMRs did each dose induce? For the RNA-seq results, again, what were the number of up/down-regulated genes for each dose?  

The experiment shown in Figure 1 was designed to 1) serve as proof of principle that cells maintained in culture could be susceptible to EDC-induced epimutagenesis at all, 2) determine if any response observed would be dose-dependent, and 3) identify a minimally effective dose of BPS to be used for the remaining experiments in this study (which we identified as 1 μM). We agree that it is interesting that the 50 µM dose of BPS induced predominantly hypermethylation changes whereas the 1 µM and 100 µM doses induced predominantly hypomethylation changes, but are not in a position to offer a mechanistic explanation for this outcome at this time. As the results shown satisfied our primary objectives of demonstrating that exposure of cells in culture to BPS could indeed induce DNA methylation epimutations, that this occurs in a dose-dependent manner, and that a dose of as low as 1 µM of BPS was sufficient to induce epimutagenesis, the data obtained satisfied all of the initial objectives of this experiment. That said, in response to the reviewer’s request we have now added text on pages 6-7 alluding to new Supplemental tables 1-3 indicating the total number of DMCs and DMRs, as well as the number of DEGs, detected in response to exposure to each dose of BPS shown in Figure 1, as well as stratifying those results to indicate the numbers of hyper- and hypomethylation epimutations and up- and down-regulated DEGs induced in response to each dose of BPS. While, as noted above, investigating the mechanistic basis for the difference in responses induced by the 50 µM versus 1 and 100 µM doses of BPS was beyond the scope of the study presented in this manuscript, we do find this result reminiscent of the “U-shaped” response curves often observed in toxicology studies. Importantly, this result does demonstrate the elevated resolution and specificity of analysis facilitated by our in vitro cell culture model system.

(4) Also in Figure 1, were there DMRs or genes in common across the doses? How did DMRs relate to gene expression results? This would be informative in verifying or refuting expectations that greater methylation is often associated with decreased gene expression.  

In general, we observed a coincidence between changes in DNA methylation and changes in gene expression (Supplement Tables 1-3). Pertaining directly to the reviewer’s question about the extent to which we observed common DMRs and DEGs across all doses, while we only found 3 overlapping DMRs conserved across all doses tested, we did find an average of 51.25% overlap in DMCs and an average of 80.45% overlap in DEGs across iPSCs exposed to the different doses of BPS shown in Figure 1. In addition, within each dose of BPS tested in iPSCs, we also found that there was an overlap between DMCs and the promoters or gene bodies of many DEGs (Supplement Table 4). Specifically within gene promoters, we observed a correlation between hypermethylated DMCs and decreased gene expression and hypomethylated DMCs and increased gene expression, respectively (Supplement Figure 2).

(5) In Figure 2, was there an overlap in the hypo- and/or hyper-methylated DMCs? Please also add more description of the data in 2b to the legend including what the dot sizes/colors mean, etc. Some readers (including me) may not be familiar with this type of data presentation. Some of this comes up in Figure 4, so perhaps allude to this earlier on, or show these data earlier.  

We observed an average of 11.05% overlapping DMCs between different pairs of cell types, we did not observe any DMCs that were shared among all four cell types. Indeed, this limited overlap of DMCs among different cell types exposed to BPS was the primary motivation for the analysis described in Figure 2. Thus, instead of focusing solely on direct overlap between specific DMCs, we instead examined similarities among the different cell types tested in the occurrence of epimutations within different annotated genomic regions. To better describe this, we have now added additional text to page 9. We have also added more detail to the legend for Figure 2 on page 8 to more clearly explain the significance of the dot sizes and colors, explaining that the dot sizes are indicative of the relative number of differentially methylated probes that were detected within each specific annotated genomic region, and that the dot colors are indicative of the calculated enrichment score reflecting the relative abundance of epimutations occurring within a specific annotated genomic region. The relative score is calculated by iterating down the list of DMCs and increasing a running-sum statistic when encountering a DMC within the specific annotated genomic region of interest and decreasing the sum when the epimutation is not in that annotated region. The magnitude of the increment depends upon the relative occurrence of DMCs within a specific annotated genomic region.

(6) iPSCs were derived from male mice MEFs, and subsequently used to differentiate into PGCLCs. The only cell type from an XX female is the granulosa cells. This might be important, and should be mentioned and its potential significance discussed (briefly).  

We have added a new paragraph just before the final paragraph of the Discussion section in which we acknowledge that most of the cell types analyzed during our study were XY-bearing “male” cells and that the manner in which XX-bearing “female” cells might respond to similar exposures could differ from the responses we observed in XY cells. However, we also noted that our assessment of XX-bearing granulosa cells yielded results very similar to those seen in XY Sertoli cells suggesting that, at least for differentiated somatic cell types, there does not appear to be a significant sex-specific difference in response to exposure to a similar dose of the same EDC. That said, we also acknowledged that in cell types in which dosage compensation based on X-chromosome inactivation is not in place, differences between XY- and XX-bearing cells could accrue.

(7) EREs are only one type of hormone response element. The authors make the point that other mechanisms of BPS action are independent of canonical endocrine signaling. Would authors please briefly speculate on the possibility that other endocrine pathways including those utilizing AREs or other HREs may play a role? In other words, it may not be endocrine signaling independent. The statement that the differences between PGCLCs and other cells are largely due to the absence of ERs is overly simplistic.  

Previous reports have indicated that BPS does not have the capacity to bind with the androgen receptor (Pelch et al., 2019; Yang et al., 2024). However there have been reports indicating that BPS can interact with other endocrine receptors including PPARγ and RXRα, which play a role in lipid accumulation and the potential to be linked to obesity phenotypes (Gao et al., 2020; Sharma et al., 2018). To address the reviewer’s comment we assessed the expression of a panel of hormone receptors including PPARγ, RXRα, and AR  in each of the cell types examined in our study and these results are now shown in a new Supplent Figure 4. We show that in addition to not expressing either estrogen receptor (ERa or ERb), germ cells also do not express any of the other endocrine receptors we tested including AR, PPARγ, and RXRα. Thus we now note that these results support our suggestion that the induction of epimutations we observed in germ cells in response to exposure to BPS appears to reflect disruption of non-canonical endocrine signaling. We also note that non-canonical endocrine signaling is well established (Brenker et al., 2018; Ozgyin et al., 2015; Song et al., 2011; Thomas and Dong, 2006). Thus we feel the suggestion that the effects of BPS exposure could conceivably reflect either disruption of canonical or non-canonical signaling in any cell type is well justified and that our data suggests that both of these effects appear to have accrued in the cells examined in our study as suggested in the text of our manuscript.

(8) Interpretation of data from the GO analysis is similarly overly simplistic. The pathways identified and discussed (e.g. PI3K/AKT and ubiquitin-like protease pathways) are involved in numerous functions, both endocrine and non-endocrine. Also, are the data shown in Figure 6a from all 4 cell types? I am confused by the heatmap in 6c, which genes were significantly affected by treatment in which cell types?  

Per the reviewer’s request, we have added text to indicate that Figure 6a is indeed data from all four cell types examined. We have also modified the text to further clarify that Figure 6c displays the expression of other G-coupled protein receptors which are expressed at similar, if not higher, levels than either ER in all cell types examined, and that these have been shown to have the potential to bind to either 17β-estradiol or BPA in rat models. As alluded to by the reviewer, this is indicative of a wide variety of distinct pathways and/or functions that can potentially be impacted by exposure to an EDC such as BPS. Thus, we have attempted to acknowledge the reviewer’s primary point that BPS may interact with a variety of receptors or other factors involved with a wide variety of different pathways and functions. Importantly, this illustrates the strength of our model system in that it can be used to identify potential impacted target pathways that can then be subsequently pursued further as deemed appropriate.

(9) In Figure 7, what were the 138 genes? Any commonalities among them? 

We have now added a new supplemental Excel file that lists the 138 overlapping conserved DEGs that did not become reprogrammed/corrected during the transition from iPSCs to PGCLCs. In addition, we have added new text on page 22 and a new Supplemental Figure 8 which displays KEGG analysis of pathways associated with these 138 retained DEGs. We find that these genes are primarily involved with cell cycle and apoptosis pathways which, interestingly, have the potential to be linked to cancer development which is often linked to disruptions in chromatin architecture.

(10) The Introduction is very long. The last paragraph, beginning line 105, is a long summary of results and interpretations that better fit in a Discussion section.

We have now significantly reduced the length and scope of the final paragraph of the Introduction per the reviewer’s recommendation.

(11) Provide some details on husbandry: e.g. were they bred on-site? What food was given, and how was water treated? These questions are to get at efforts to minimize exposure to other chemicals.  

We have added additional text detailing that all mice used in the project were bred onsite, water was non-autoclaved conventional RO water, and our selection of 5V5R extruded feed for mice used in this study which was highly controlled for the presence of isoflavones and has been certified to be used for estrogen-sensitive animal protocols.

Reviewer #2 (Public Review):

Summary:

This manuscript uses cell lines representative of germ line cells, somatic cells, and pluripotent cells to address the question of how the endocrine-disrupting compound BPS affects these various cells with respect to gene expression and DNA methylation. They find a relationship between the presence of estrogen receptor gene expression and the number of DNA methylation and gene expression changes. Notably, PGCLCs do not express estrogen receptors and although they do have fewer changes, changes are nevertheless detected, suggesting a nonconical pathway for BPS-induced perturbations. Additionally, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. Epimutations in the somatic and pluripotent cell types were predominantly in enhancer regions whereas that in the germ cell type was predominantly in gene promoters.

Strengths:

The strengths of the paper include the use of various cell types to address the sensitivity of the lineages to BPS as well as the observed relationship between the presence of estrogen receptors and changes in gene expression and DNA methylation.

Weaknesses:

The weaknesses include the lack of reporting of replicates, superficial bioinformatic analysis, and the fact that exposures are more complicated in a whole organism than in an isolated cell line.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

Overall, this is an intriguing paper but more transparency in the replicates and methods and a more rigorous bioinformatic treatment of the data are required.

Specific comments:

(1) End of abstract "These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally following a single exposure to the causative EDC." This is overly speculative for an abstract. There is only epigenetic inheritance following mitosis or differentiation presented in this study. There is no meiosis and therefore no ability to assess multi- or transgenerational inheritance. 

We have modified the text at the end of the abstract to more precisely reflect our intended conclusions based on our data. In our view, the ability of induced epimutations to transcend meiosis per se is not as relevant to the mechanism of transgenerational inheritance as their ability to transcend major waves of epigenetic reprogramming that normally occur during development of the germ line. In this regard the transition from pluripotent iPSCs to germline PGCLCs has been shown to recapitulate at least the first portion of normal germline reprogramming, and now our data provide novel insight into the fate of induced epimutations during this process. Specifically, we show that a prevelance of epimutations was conserved during the iPSC à germ cell transition but that very few (< 5%) of the specific epimutations present in the the BPS-exposed iPSCs were retained when those cells were induced to form PGCLCs. Rather, we observed apparent correction of a large majority of the initially induced epimutations during this transition, but this was accompanied by the apparent de novo generation of novel epimutations in the PGCLCs. We suggest, based on other recent reports in the literature, that this is a result of the BPS exposure inducing changes in the chromatin architecture in the exposed iPSCs such that when the normal germline reprogramming mechanism is imposed on this disrupted chromatin template there is both correction of many existing epimutations and the genesis of many novel epimutations. This observation has the potential to explain the long-standing question of why the prevalence of epimutations persists across multiple generations despite the occurrence of epigenetic reprogramming during each generation. Nevertheless, as noted above, we have modified the text at the end of the abstract to temper this interpretation given that it is still somewhat speculative at this point.

(2) Doses used in the experiments. One needs to be careful when stating that the dose used is "below FDA's suggested safe environmental level established for BPA" because a different bisphenol is being used here (BPA vs BPS) and the safe level is that which the entire organism experiences. It is likely that cell lines experience a higher effective dose.  

We have now made a point of noting that our reference to an EPA-recommended “safe dose” of BPA was for humans and/or intact animals. Changes to this effect have been made in the second and sixth paragraphs of the Introduction section. In addition, we have added text at the end of the fourth paragraph of the Discussion section acknowledging that, as the reviewer suggests, the same dose of an EDC could exert greater effects on cells in a homogeneous culture than on the same cell type within an intact animal given the potential for mitigating metabolic effects in the latter. However, we also note that the ability we demonstrated to quantify the effects of such exposures on the basis of numbers of epimutations (DMCs or DMRs) induced could potentially be used in future studies to study this question by assessing the effects of a specific dose of a specific EDC on a specific cell type when exposed either within a homogeneous culture or within an intact animal.

(3) Figure 1: In the dose response, what was the overlap in DMCs and DEGs among the 3 doses? Are the responses additive, synergistic, or completely non-overlapping? This is an important point that should be addressed. 

Please see our response to Reviewer 1 critique #4 above where we address similar concerns. While we do find overlap among different cell types with respect to the DMCs, DMRs, and DEGs displayed in Figure 1, we found the effect to be only partially additive as opposed to synergistic in any apparent manner. The fold increase in DMCs, DMRs, and DEGs resulting from exposure to doses of 1 μM or 50 μM ranged from 2.5x to 4.4x, which was well below the 50x increase that would have been expected from a strictly additive effect, and the effect increased even less, if at all, in response to exposure to doses of 50 μM versus 100 μM BPS. Finally, as now noted in the Discussion section on page 25, our conclusion is that these results display a limited dose-dependent effect that was partially additive but also plateaued at the highest doses tested.

(4) Methods: How many times was each exposure performed on a given cell type? This information should be in the figure legends and methods. In the case of multiple exposures for a given line, do the biological replicates agree? 

Please see our response to Reviewer 1 critique #2 where we address similar concerns with newly added text and analysis. We now note repeatedly on pages 39-45 that each analysis was conducted on three replicate samples, and we display the similarity among those replicates graphically in a new Supplement Figure 9.

(5) DNA methylation analyses. Very little analysis is presented on the BeadChip array other than hypermethylated/hypomethylated and genomic regions of DMCs. What is the range of methylation changes? Does it vary between hypo vs. hyper DMCs? How many array experiments were performed (biological replicates) and what stats were used to determine the DMCs? Are there DMCs in common among the various cell types? As an example, if more meaningful analysis, one can plot the %5mC over a given array for comparisons between control and treated cell types. For more granularity, the %5mC can be presented according to the element type (enhancers vs promoters). 

Please see our response to Reviewer 1 critique #2 above where we address similar concerns regarding the number of biological replicates used in this study. DMCs on the Infinium array are identified using mixed linear models. This general supervised learning framework identifies CpG loci at which differential methylation is associated with known control vs. treated co-variates. CpG probes on the array were defined as having differential changes that met both p-value and FDR (≤ 0.05) significant thresholds between treatment and control samples for each cell type analyzed. The range of medians across all samples was 0.0278 to 0.0059 for hypermethylated beta values and -0.0179 to -0.0033 for hypomethylated beta values. As noted above, we did observe an overlap in DMCs between cell types. Thus, we observed an average of 11.05% overlapping DMCs between two or more cell types but we did not observe any DMCs shared between all four cell types. We have added additional text on page 9 and new Supplement Tables 1-4 and Supplement Figure 1 to now more clearly describe that this limited similarity in direct overlap of DMCs was the underlying motivation for the analysis described in Figure 2. Finally, the enrichment dot plots shown in Figure 2 provide the information the reviewer requested regarding the %5mC observed at different annotated genomic element types.

(6) The investigators correlate the number of DMCs in a given cell type with the presence of estrogen receptors. Does the correlation extend to the methylation difference (delta beta) at the statistically different probes?

We have added a new Supplement Figure 3 in which we provide data addressing this question. In brief, we find that the delta betas of probes enriched at enhancer regions and associated with relative proximity to ERE elements in Sertoli cells, granulosa cells, and iPSCs appear very similar to those associated with DMCs not located within these enriched regions. However, when we compared the similarity of the two data sets with goodness of fit tests, we found these relatively small differences were, in fact, statistically significant based on a two-sample Kolmogorov-Smirnov test. These observed significant differences appear to indicate that there is higher variability among the delta betas associated with hypomethylated, but not hypermethylation changes occurring at DMCs associated with enhancers, potentially suggesting a greater tendency for exposure to BPS to induce hypomethylation rather than hypermethylation changes, at least in these specific regions.

(7) Methylation changes relative to EREs are presented in multiple figures. Are other sequences enriched in the DMCs? 

We profiled the genomic sequence within 500 bp of cell type-specific enriched DMCs that were either associated with enhancer regions in Sertoli, granulosa, or iPS cells or transcription factor binding sites in PGCLCs for the identification of higher abundance motif sequences. We then compared any motifs identified with the JASPAR database to potentially find transcription factors that could be binding to these regions. Interestingly we found that the two most common motifs across all cell types were associated with either the chromatin remodeling transcription factor HMG1A or the pluripotency factor KLF4.

(8) Please present a correlation plot between the methylation differences and the adjacent DEGs. Again, the absence of consideration of the absolute changes in methylation and gene expression minimizes the impact of the data. 

We analyzed the relationship between DMCs at DEGs promoter regions and the corresponding change in expression of that DEG. Our data support a relationship between up-regulated genes showing decreased methylation in promoter regions and down-regulated genes showing increased methylation at promoter regions, although there were some exceptions to this relationship.

(9) EM-Seq is mentioned in Figure 7 and in the material and methods. Where is it used in this study? 

We now note in the text on page 22 that EM-seq was used during experiments assessing the propagation of BPS-induced epimutations during the iPSC à EpiLC à PGCLC cell state transitions to gather higher resolution data of changes to DNA methylation differences at the whole-epigenome level.

References

Brenker C, Rehfeld A, Schiffer C, Kierzek M, Kaupp UB, Skakkebæk NE, Strünker T. 2018. Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals. Hum Reprod 33:1915–1923. doi:10.1093/humrep/dey275

Gao P, Wang L, Yang N, Wen J, Zhao M, Su G, Zhang J, Weng D. 2020. Peroxisome proliferator-activated receptor gamma (PPARγ) activation and metabolism disturbance induced by bisphenol A and its replacement analog bisphenol S using in vitro macrophages and in vivo mouse models. Environ Int 134. doi:10.1016/J.ENVINT.2019.105328

Ozgyin L, Erdos E, Bojcsuk D, Balint BL. 2015. Nuclear receptors in transgenerational epigenetic inheritance. Prog Biophys Mol Biol. doi:10.1016/j.pbiomolbio.2015.02.012

Pelch KE, Li Y, Perera L, Thayer KA, Korach KS. 2019. Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles. Toxicol Sci 172:23–37. doi:10.1093/TOXSCI/KFZ173

Sharma S, Ahmad S, Khan MF, Parvez S, Raisuddin S. 2018. In silico molecular interaction of bisphenol analogues with human nuclear receptors reveals their stronger affinity vs. classical bisphenol A. Toxicol Mech Methods 28:660–669. doi:10.1080/15376516.2018.1491663

Song K-H, Lee K, Choi H-S. 2011. Endocrine Disrupter Bisphenol A Induces Orphan Nuclear Receptor Nur77 Gene Expression and Steroidogenesis in Mouse Testicular Leydig Cells. Endocrinology 143:2208–2215. doi:10.1210/endo.143.6.8847

Thomas P, Dong J. 2006. Binding and activation of the seven-transmembrane estrogen receptor GPR30 by environmental estrogens: A potential novel mechanism of endocrine disruption. J Steroid Biochem Mol Biol 102:175–179. doi:10.1016/j.jsbmb.2006.09.017

Yang Z, Wang L, Yang Y, Pang X, Sun Y, Liang Y, Cao H. 2024. Screening of the Antagonistic Activity of Potential Bisphenol A Alternatives toward the Androgen Receptor Using Machine Learning and Molecular Dynamics Simulation. Environ Sci Technol 58:2817–2829. doi:10.1021/ACS.EST.3C09779/ASSET/IMAGES/LARGE/ES3C09779_0004.JPEG

not good - means we need more creatine bound AAS to even get in skeletal muscle b/c o.w. it wont be carried in blood

Đã có một số nghiên cứu cung cấp những lý giải mang tính lý thuyết và các phân tích cho việc LLMs có thể học từ các ngữ cảnh như thế nào. Tuy nhiên, lý do chính xác cho khả năng này của LLMs là chưa được rõ ràng, từ đó khiến cho việc chọn được các ngữ cảnh tối ưu trở nên khó khăn.

DOI: 10.1101/2024.04.28.591518

Resource: (BD Biosciences Cat# 553142, RRID:AB_394657)

Curator: @scibot

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What is this?

Reviewer #1 (Public Review):

Summary:

The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by (1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, (2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), (3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, (4) better defines how the vasculature, nervous, and immune system integrates with the OR.

Strengths:

The data is beautifully presented and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.

Weaknesses:

It is not always clear what the novel findings are that this manuscript is presenting. It appears to be largely similar to the analysis done by McKey et al. (2022) but with more time points and molecular markers. The novelty of the present study's findings needs to be better articulated.

Reviewer #2 (Public Review):

A large number of ovarian experiments have been conducted - especially in morphological and molecular biology studies - specifically removing the ovarian membrane. This experiment is a good supplement to existing knowledge and plays an important role in early ovarian development and the regulation of ovarian homeostasis during the estrous cycle. There are also innovations in research ideas and methods, which will meet the requirements of experimental design and provide inspiration for other researchers.

This reviewer did not identify any major issues with the article. However, the following points could be further clarified:

(1) Is there any comparative data on the proteomics of RO and rete testis in early development? With some molecular markers also derived from rete testis, it would be better to provide the data or references.

(2) Although the size of RO and its components is quite small and difficult to operate, the researchers in this article had already been able to perform intracavitary injection of EOR and extract EOR or CR for mass spectrometry analysis. Therefore, can EOR, CR, or IOR be damaged or removed, providing further strong evidence of ovarian development function?

(3) Although IOR is shown on the schematic diagram, it cannot be observed in the immunohistochemistry pictures in Figure 1 and Figure 3. The authors should provide a detailed explanation.

Reviewer #3 (Public Review):

Summary:

The rete ovarii (RO) has long been disregarded as a non-functional structure within the ovary. In their study, Anbarci and colleagues have delineated the markers and developmental dynamics of three distinct regions of the RO - the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). Notably focusing on the EOR, the authors presented evidence illustrating that the EOR forms a convoluted tubular structure culminating in a dilated tip. Intriguingly, microinjections into this tip revealed luminal flow towards the ovary containing potentially secreted functional proteins. Additionally, the EOR cells exhibit associations with vasculature, macrophages, and neuronal projections, proposing the notion that the RO may play a functional role in ovarian development during critical ovariogenesis stages. By identifying marker genes within the RO, the authors have also suggested that the RO could serve as a potential structure linking the ovary with the neuronal system.

Strengths:

Overall, the reviewer commends the authors for their systematic research on the RO, shedding light on this overlooked structure in developing ovaries. Furthermore, the authors have proposed a series of hypotheses that are both captivating and scientifically significant, with the potential to reshape our understanding of ovarian development through future investigations.

Weaknesses:

There is a lack of conclusive data supporting many conclusions in the manuscript. Therefore, the paper's overall conclusions should be moderated until functional validations are conducted.

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adnotare

comment

te rog să vezi acest paragraf

This sort of reminds me of the dress code issues mentioned in last weeks lectures. When we discussed that female students are always at fault on what they are wearing and not what the male students are thinking. Society has always had scenarios like these come up, causing innocent individuals to be the victim.

Aviz favorabil - unanimitate

Aviz favorabil - 2 abtineri

AMANARE

to-docdrop

Thái độ mới đối với trẻ em và nền giáo dục của chúng bắt đầu phát triển vào cuối thế kỷ 17, khi nhiều nhà giáo dục kêu gọi quan tâm nhiều hơn đến những nhu cầu đặc biệt của trẻ và khi khái niệm về niềm vui học tập ngày càng được chấp nhận rộng rãi hơn.3 Biểu hiện rõ ràng nhất của sự phát triển ý tưởng này là các bài viết của triết gia John Locke (1632–1704) và Jean-Jacques Rousseau (1712–78). Năm 1693, Locke viết trong Một số suy nghĩ liên quan đến giáo dục rằng “trẻ em nên được đối xử như những sinh vật có lý trí…. Chúng không được bị cản trở để trở thành trẻ con, cũng như không được chơi và làm như trẻ con, mà là bị bệnh.” Rousseau coi tuổi thơ như một đứa trẻ. một trạng thái trong sáng và tự nhiên - khác biệt với trạng thái trưởng thành - và tin rằng mục tiêu trọng tâm của giáo dục phải là bảo tồn bản chất nguyên thủy của trẻ. Ông cũng tin rằng điều cần thiết là giáo viên phải nhìn sự việc như trẻ em nhìn.5 Các tác phẩm của Locke và Rousseau đã ảnh hưởng đến các nhà giáo dục Anh, và ý tưởng của họ cuối cùng đã dẫn tới một cách tiếp cận giáo dục nhân đạo hơn, trong đó niềm vui được coi là một phương tiện hỗ trợ cho việc học.

In

galima dar sukinėti 2-ro veidr. rotorių.

Kudos

Este un muzeu creativ și actual.

Clear water means clear mind!

DOI: 10.1101/2024.03.06.583750

Resource: (DSHB Cat# ro-62C2A8 anti-rough, RRID:AB_528456)

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What is this?

Trong trường hợp này, người dùng có thể muốn có câu trả lời rõ ràng hơn bằng cách đặt ra 1 câu trả lời theo sau câu trả lời trước "So that used up my credit, right ?". Nếu topic này bị loại khỏi stack thì nó không thể làm rõ dc thứ mà người dùng muốn là gì.

7.This paper uses logos. As a whole, the analysis is objective, using many quotes and design suggestions. The discussion section also uses logos because it is derived from analysis rather than emotion.

This work has been published in GigaScience Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giad115), and has published the reviews under the same license. These are as follows.

Reviewer 1 Carole Goble - Original Submission

This work reports a multi-omics data analysis workflow packaged as a RO-Crate, an implementation of a FAIR Digital Object.We limit our comments to the technical aspects of the Research Object and workflow packaging. The scientific validity of the omics analysis itself is outside our expertise.The paper is comprehensive and the background grounding in the current state of the art is excellent and thorough. The paper is an excellent exemplar of the future of data analysis reporting for FAIR and reproducible computational methods, and the amount of work impressive. We congratulate the authors.WorkflowHub entry https://workflowhub.eu/workflows/402?version=5# gives a comprehensive report of the Nextflow workflow and its multiple versions, all the files including the R scripts and the synthetic data. The RO-Crate rendering looks correct and version-locking the R containers is following best practice(https://github.com/Xomics/ACTIONdemonstrator_workflow/blob/main/nextflow.config#L44)T he paper also highlights the amount of work needed to make such a pipeline to be both metadata machine processable and metadata human readable.To make this pipeline reproducible requires a mixture of notebooks submitted as supplementary materials, the Nextflow workflow with its R scripts represented as an RO-Crate in WorkflowHub and a README is linked to the container recipes in https://github.com/Xomics/Docker_containers and then another Documentation.md file. There seems to be the potential for duplicated effort in reporting the necessary metadata describing the workflow that could be highlighted in the Discussion as a steer to the digital object community.- Could the ROCrate approach be widened beyond the current Workflow RO-Crate, and would there be value in streamlining the metadata, or is this just an artefact of the need for multiple descriptions and ease of publishing. If the JSON within the RO-Crate was more richly annotated, could some of the Documentation.md be avoided altogether, and is that even desirable?- The README includes the container/software packaging and is not linked from the RO-Crate (and there isn't an obvious property to link to it yet). Could these be RO-Crates too?- The notebooks in the supplementary files could also be registered in WorkflowHub and linked to the Nextflow workflow (see https://workflowhub.eu/workflows?filter%5Bworkflow_type%5D=jupyter).- Is it feasible and desirable to have a single RO-Crate linked to many other RO-Crates to represent the whole reproducible pipeline in full?In the discussion the FAIR principles verification through different practices and approaches would be more helpful if it was more precise. Comments seem to be limited to the Workflow RO-Crate and use of ontologies for machine readability. As highlighted in table 1 there is more to FAIR software & workflows than this.Minor remarksKey points- We here demonstrate the implementation multiomics data -> We here demonstrate an implementation of an multi-omics data.Background- The documentation of dependencies is highlighted as a prerequisite for software interoperability. In the FAIR4RS principles I2 also highlights qualified references to other objects - presumably other software or installation requirements. This highlights the relationship between software interoperability and software portability. It seems that dependencies more relate to portability rather than interoperability.- "Based on the FDO concept, the RO-Crate approach was specified". This is a confusing statement. ROCrates have been recognised as an implementation approach for the FDO concept as proposed by the FDO Forum. For more discussion on FDO and the Linked Data approach promoted by RO-Crates see https://arxiv.org/abs/2306.07436. However, RO-Crates are not based in the FDO - they are based on the Research Object packaging work that emerged from the EU Wf4ever project, (see https://doi.org/10.1016/j.future.2011.08.004 from 2013).- It is better to describe the RO-Crate metadata file as " It contains all contextual and non-contextual related data to re-run the workflow". Instead of "It can additionally contain data on which the workflow can be run."Workflow Implementation- At the beginning of the last paragraph, "Besides the workflow and the synthetic data set" replace with "As well as the workflow and the synthetic data set".- https://workflowhub.eu/workflows/402?version=5# gives a very nice pictorial overview of the workflow that you may consider including in the paper itself.

tihs is personal

Foarte atractiv prețul

sidi is sun because her marriage to Baroka will be a ritual; ro benefit him his fertility but diminish her light

Membrane charge has little to do with rejecting charged solutes in RO

Personally, I had great history classes and great instructors in high school. They taught history from all perspectives. They did not teach the one sided stories which I am very thankful. I believe schools and curriculums are evolving and putting more emphasis on what they are teaching their students.

The statement describes a significant change in the behavior of Boston students, both boys and girls. Initially, these students engaged frequently in fights and verbal confrontations, often resorting to yelling and calling each other derogatory names like "stupid." However, there has been a noticeable shift in their behavior, with a reduction in these aggressive interactions. The mention of them stopping "yelling at each other so much and calling each other stupid" implies a positive development towards more peaceful and respectful interactions among the students. This change could indicate improved social dynamics, better conflict resolution skills, or the success of interventions aimed at reducing hostility in the school environment.

Tekstlikhet med en masteroppgave fra 2019, som meldt av NRK: https://www.nrk.no/norge/flere-tekstlikheter-funnet-i-helseminister-kjerkols-masteroppgave-1.16725113

Acesta este un mai pas înainte, o diferență ....

Often overlooks other factors that contribute to a nation's military capabilities

This is understandable. Private institutions probably prepared them to upper division classes or higher level math.

This just proves that having access to private education immediately sets you above other students academically. I know that private schools succeed because they have a rigorous system, but my question is, why don't public schools try to achieve the same thing? Is it just a way to set lower-income students back?

DOI: 10.1016/j.cmet.2023.12.016

Resource: (Cell Signaling Technology Cat# 4053, RRID:AB_1904096)

Curator: @scibot

SciCrunch record: RRID:AB_1904096

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

In this manuscript, Grossmann et al. present a new potential pathway that regulates PLK4 levels in cells mediated by the CRL4^DCAF1 E3 ubiquitin ligase complex (CUL4A/B-DDB1-DCAF1). PLK4 plays a crucial role in centriole assembly acting as a master regulator of the centriole biogenesis and thus contributes to centriole number control. Centriole numbers need to be tightly regulated as deviations could lead to aneuploidy and potentially cancer. At the onset of centriole assembly in G1/S, PLK4 is focusing into a single point on each parental centriole together with STIL and SAS6 defining the site of procentriole formation. For this process to happen, PLK4 trans-phosphorylates itself creating a binding site for SCF^β-TrCP E3 ubiquitin ligase that targets PLK4 for ubiquitination and degradation by the proteasome. The authors identified by co-IP and mass spectrometry the CRL4DCAF1 E3 ubiquitin ligase complex as a potential regulator of PLK4. They show that CRL4^DCAF1 E3 ubiquitin ligase complex binds to PLK4 and targets it for degradation. Furthermore, the authors present data where knockdown of DCAF1 leads to increased levels of PLK4 and centriole amplification. Using AlphaFold and followed by IPs with PLK4 point-mutants, they propose that DCAF1 binds to the dimer of PLK4 at PB1-PB2 at a similar site where Cep192/Cep152 bind. Then, they move on to show that CRL4^DCAF1 E3 ubiquitin ligase complex ubiquitylates PLK4 predominantly in G2 phase. Lastly, they propose that DCAF1 regulates the interaction of PLK4 with STIL and that it is required to prevent premature centriole disengagement in G2 phase. The manuscript is written in a clear and concise manner while the experimental approaches are sound and well described. The experimental data are well presented with a good number of replicates in most cases. However, some of the conclusions are drawn from marginal differences in the data and without statistical tests (cases indicated in detail below). I believe that this work is of interest to the scientific community, but it would require revisions to address the following major and minor comments.

Major comments:

• The key finding of the paper is that PLK4 co-IPs with DCAF1 and DDB1 that are core components of CUL4A/B E3 ubiquitin ligase. However, the only evidence that this interaction between PLK4 and DCAF1 is direct relies on the ubiquitylation assay performed in E. coli. This experiment was performed only once, and no quantitation is performed (Fig 4A). Given that the components are overexpressed in this heterologous system, it is very plausible to have a non-specific interaction between the DCAF1-Acidic domain and PLK4-PB1-PB2 considering that native binders to this region (Cep192/Cep152) are absent. The PLK4-DCAF1 model that was generated with AlphaFold suggests that this interaction is plausible but stronger verification that the interaction is direct are necessary in this reviewer's point of view. This could be performed for instance by purifying proteins (or fragments of them) to test binding in vitro, or through IPs of full-length proteins from bacterial extracts. If the interaction between PLK4 and DCAF1 is indeed direct, then the authors would need to provide an explanation of the feasibility of this interaction given that the binding site is occupied by Cep192 or Cep152 at the centrioles. Based on the current knowledge, PLK4 is loaded to the centriole through the interaction with Cep192 which is then switched to interaction with Cep152. For the DCAF1 to be able to bind to PLK4 it would need to outcompete Cep152. Thus, in order to prove that DCAF1 can control PLK4 at the centrosome, evidence would need to be provided that this interaction is possible. If that is not the case, then the most likely alternative is that DCAF1 interactions with cytoplasmic pool of PLK4, thus only indirectly controlling the PLK4 levels at centrioles. A plausible alternative interpretation of the data provided would be that DCAF1-Acidic domain could bind weakly and perhaps non-specifically to PLK4 but in human cells the interaction is mediated through another component such as Cep152 or Cep192 (which are also present in the MS data). Based on the AlphaFold model, the authors introduced point mutations that abolish the PLK4-DCAF1 interaction, but this effect could just as easily be an indirect effect due to abolishing of the PLK4-Cep152/Cep192 interaction.
• The authors state that DCAF1 depletion with siRNA or shRNA leads to increased level of PLK4 which triggers centriole overduplication. However, this statement is not entirely supported by the data provided. Firstly, in the western blots shown (Fig2A, 2D) the increase in PLK4 levels is hardly visible. Given that this is a key finding, stronger evidence would need to be provided. Furthermore, the quantification of the PLK4 levels upon siRNA mediated DCAF1 depletion are confusing as siDCAF1#2 leads to higher PLK4 levels than siDCAF1#1 despite being less effective in DCAF1 depletion (Fig 2A). More importantly, the quantification on the HeLa tet-on shDCAF1, that are used in many experiments, is missing an important statistical test (Fig 2D). Similarly, no statistical test is performed on the quantification of centriole numbers (Fig 2F) which puts to question the conclusion that "CRL4DCAF1 might function to keep PLK4 protein levels low, thus preventing centriole overduplication". Moreover, GFP-PLK4 levels shown in Fig 6A seem unaltered (if not lowered) upon DCAF1 depletion. Lastly, DCAF1 overexpression does not seem to decrease PLK4 levels as shown in Fig 6B. In that experiment, though, PLK4 is also overexpressed. In order to support the proposed function that CRL4DCAF1 keeps PLK4 levels low, it would be useful to also investigate whether overexpression of DCAF1 would lead to further decrease of PLK4 levels.
• In page 7, the authors mention: "A premature onset of centriole duplication in the absence of DCAF1 should also result in increased numbers of already disengaged centrioles in G2 phase." This premise is not correct as it is inverted to the current knowledge. It is the premature disengagement that licences for premature centriole duplication (or as often stated as re-duplication) rather than the premature onset of centriole duplication that causes disengagement. This is also what the authors correctly state in the discussion. In the data presented (Fig 6C) the authors observe centriole disengagement upon DCAF1 depletion using expansion microscopy, but no re-duplication is visible in the images provided. This is contrary to the overduplication claim made earlier on (Fig 2F). As such, the data presented do not fully support the drawn conclusion that DCAF1 controls PLK4 levels in G2 to prevent unscheduled centriole duplication. The authors would also need to investigate whether the prolonged use of Cdk1 inhibitor RO-3306 to synchronise the cells in G2 in addition to DCAF1 depletion contributes to the centriole disengagement that is observed, considering that Cdk1-Cyclin B acts also on PLK4-STIL complex.
• The mechanism proposed by the authors is that DCAF1 maintains PLK4 at low levels throughout G2 which prevents premature disengagement. Subsequently, low PLK4 levels prevent binding and activation of STIL impeding premature initiation of centriole duplication. However, this would not happen since centrioles remain engaged at this stage. Overall, some of the aspects of the proposed mechanism are not fully supported by the data presented. In addition, the proposed mechanism does not offer a suitable mechanistic explanation of how lower PLK4 levels by CRL4^DCAF1 mediated ubiquitylation and degradation prevent centriole disengagement.

Minor comments

• In Fig S2A authors need to indicate the expected size of the expressed protein. In its current form blot is difficult to be assessed. More specifically, it is unclear what is the result on the IP with the PLK4 fragment (1-879) since the more intense band in the input in not the same as in in the IP with Flag.
• In Fig 1C, S2B, S3B, it would be helpful to have a summary of the interactions observed next to each construct. This is commonly represented with (-, +, ++, +++) depending on the amounts present in the IP.
• In Fig 1D, even though not statistically significant, there seems to be a reduction in the IP of AA and PEST. Do the authors have some suggestion why that might be?
• Authors used two different cell lines in the experiments presented in Fig2A and Fig 2B. Given that depletion of siDCAF5 is provided as a control of having no effect in the PLK4 levels I would expect to have the experiment performed on same cell line.
• No statistical test is provided in the comparison on PLK4 levels upon siRNA treatment coupled with CHX (Fig 2C).
• In the quantification of the PLK4 levels at the centrosomes (Fig 2E), it is not specified whether a background subtraction step was performed prior to the normalisation to the untreated control.
• In the blot shown in Fig S3, no input is visible in the lane with expression of the Acidic domain.
• Authors claim that both WD40 and acidic domain contribute to binding of PLK4 because WD40-Acidic is more efficient in binding PLK4 that Acidic domain alone. However, in the blot provided, WD40 alone does not interact with PLK4. Thus, the most likely explanation would be that Acidic domain is the major interactor and WD40 has only minor contribution or it offers a stabilisation role to the acidic domain.
• Regarding the AlphaFold model provided, and in addition to the comments above, some further clarifications and controls would need to be provided. AlphaFold is a powerful tool but not without its caveats and needs to be used with caution. The authors need to provide a description on how they used AlphaFold to generate the model presented. Typically, AlphaFold produces 5 output models. At which site was DCAF1-Acidic domain positioned in the other output models? Based on what criteria the model shown was selected? Also, a confidence score for the model should be provided.
• The authors compare their PLK4-DCAF1 AlphaFold model with the structure of PLK4-CEP192 complex but not with the PLK4-Cep152. What is the explanation for this? Given that Cep152 is reported to have higher affinity than Cep192 (Park SY et al., 2014) it would be important to be included in the comparisons performed.
• The phrase "An overlay between the two structures revealed that ..." is not accurate as one is a merely a model. There are also other instances in the text that the model is referred to as 'structure' which is not correct.
• Please provide a citation for "Poisson-Boltzmann solver (APBS)".
• In Fig 3A ribbon representations are too small to see DCAF1 in the printout.
• The mutations designed might affect the folding of the PBs and thus no interaction is observed. Authors could test how the mutations would affect PB1-PB2 and also design one or two mutants that are in the vicinity but not in the interaction interface to serve as true negative controls in addition to the PLK4-WT. Do these mutants localise to centrioles or also the interaction with Cep192/Cep152 is affected?
• There is no statistical test in the quantification in Fig 3D, but it is not critical as the difference is very clear and certainly statistically significant.
• Authors state that DCAF1 strongly interacts with PLK4 during interphase but only weakly in mitosis with quantification in Fig 5A but there is no statistical test.
• Based on the data shown in Fig 5B, authors state that PLK4 is predominantly ubiquitylated by CRL4DCAF1 in G2 phase. However, in the blot shown, PLK4 seems to be in more abundance in G2 that might explain the apparent higher ubiquitylation. Furthermore, the experiment was performed once and no quantification of the ubiquitylation is performed. Lastly, there are no evidence that this apparent higher ubiquitylation in G2 is mediated by CRL4DCAF1.
• In Fig 6A, STIL levels upon DCAF1 depletion seem to be lower, is there any potential explanation for that? No statistical test is performed for the STIL/GFP-PLK4 levels difference in siGL2 versus siDCAF1. The authors should provide a justification for over-expressing PLK4 in this experiment. Similarly, in Fig 6B, the authors use overexpression of both PLK4 and DCAF1 and no statistical test is performed.
• Authors report in Fig 6C disengaged centrioles. How are disengaged centriole defined, is it based on a distance cut-off or loss of orthogonality? In the images provided, this reviewer's impression is that in the (+) Dox condition, there are two parental centrioles that have separated rather disengaged procentriole. Do the images come from the same cell?
• Based on the data presented, would overexpression of PLK4 in G2 would result in centriole disengagement? This is something that the authors would optionally check.
• The quantification of rootletin as an additional confirmation of centriole disengagement is puzzling to me as I would expect an increase rather than decrease of its levels. As centrioles disengage, a new link would need to form and thus the expected increase in its levels. However, new rootlet might form only later in mitosis. Also, given that the cells are synchronised in G2 the quantification is more complex. In late G2, centrioles separate in order to move to opposite poles to form the mitotic spindle. This would result in removal of the rootlet that might reflect the reduction the authors report. Ideally the quantification should be limited to cells in late G2 (that centrioles have separated) stained with Centrin 2 to allow for a quantification per centriole pair.
• In the discussion, authors state "It is conceivable that increasing amounts of PLK4 during mitosis, when the interaction between CRL4DCAF1 and PLK4 is weak, might capture STIL from binding to CDK1 initiating the interaction between PLK4 and STIL". In mitosis CDK1-Cyclin B binds to STIL and prevents formation of the PLK4-STIL complex, thus inhibiting untimely onset of centriole biogenesis (Zitouni et al., 2016). In addition, the authors show that total PLK4 levels are low in mitosis (Fig 5A). The conclusion drawn are not in line with the current literature.
• The addition of a graphical representation of the proposed mechanism would be beneficial to the readers.
• A reference for the Ac.Tubulin antibody used is missing.
• Please provide a citation for FiJi.

Referees cross-commenting

I find the comments by the other two reviewers to be valid, clear, insightful, and complementary to those made by this reviewer. There is a good convergence between the reviewers on the critical aspects in this manuscript that require attention. Following revisions this study will contribute to the understanding of regulatory mechanisms acting at the centrioles.

Significance

Centriole number control is an important aspect that is relevant not only to the centrosome research field but is also related to cilia, cells signaling, and cancer research. This work presents a novel pathway involved in the regulation of PLK4 levels in cells mediated by the CRL4^DCAF1 E3 ubiquitin ligase complex (CUL4A/B-DDB1-DCAF1). The authors present extensive data to characterise when and how DCAF1 interactions with PLK4 to lowers its levels through ubiquitination and subsequent degradation by the proteasome. However, the effects from various treatments are often minor. The study from Grossmann et al. comes to complement already known pathways of controlling centriole numbers, at G1/S through SCFβ-TrCP E3 ubiquitin ligase mediated PLK4 degradation, and in mitosis by CDK1-Cyclin B through STIL 'capturing' to block centriole reduplication. Given that certain aspects of the manuscript are revised, and an updated and more thorough mechanism is proposed and supported, it will contribute to the conceptual advancement or our understanding of centriole number control across the cell cycle. It could potentially also contribute to the ubiquitin research field of research, but it is hard for me to assess this as it is not my field of expertise.

Schwa 撲朔迷離的音，很煩！

When phonetically realised, schwa (/ə/), also called e caduc ('dropped e') and e muet ('mute e'), is a mid-central vowel with some rounding.[22] Many authors consider its value to be [œ],[37][38] while Geoff Lindsey suggests [ɵ].[39][40] Fagyal, Kibbee & Jenkins (2006) state, more specifically, that it merges with /ø/ before high vowels and glides:

netteté /nɛtəte/ → [nɛ.tø.te] ('clarity'), atelier /atəlje/ → [a.tø.lje] ('workshop'), in phrase-final stressed position:

dis-le ! /di lə/ → [di.ˈlø] ('say it'), and that it merges with /œ/ elsewhere.[41] However, some speakers make a clear distinction, and it exhibits special phonological behavior that warrants considering it a distinct phoneme. Furthermore, the merger occurs mainly in the French of France; in Quebec, /ø/ and /ə/ are still distinguished.[42]

The main characteristic of French schwa is its "instability": the fact that under certain conditions it has no phonetic realization.

That is usually the case when it follows a single consonant in a medial syllable: appeler /apəle/ → [ap.le] ('to call'), It is occasionally mute in word-final position: porte /pɔʁtə/ → [pɔʁt] ('door'). Word-final schwas are optionally pronounced if preceded by two or more consonants and followed by a consonant-initial word: une porte fermée /yn(ə) pɔʁt(ə) fɛʁme/ → [yn.pɔʁ.t(ə).fɛʁ.me] ('a closed door'). In the future and conditional forms of -er verbs, however, the schwa is sometimes deleted even after two consonants[citation needed]: tu garderais /ty ɡaʁdəʁɛ/ → [ty.ɡaʁ.d(ə.)ʁɛ] ('you would guard'), nous brusquerons [les choses] /nu bʁyskəʁɔ̃/ → [nu.bʁys.k(ə.)ʁɔ̃] ('we will precipitate [things]'). On the other hand, it is pronounced word-internally when it follows more pronounced consonants that cannot be combined into a complex onset with the initial consonants of the next syllable: gredin /ɡʁədɛ̃/ → [ɡʁə.dɛ̃] ('scoundrel'), sept petits /sɛt pəti/ → [sɛt.pə.ti] ('seven little ones').[43] In French versification, word-final schwa is always elided before another vowel and at the ends of verses. It is pronounced before a following consonant-initial word.[44] For example, une grande femme fut ici, [yn ɡʁɑ̃d fam fy.t‿i.si] in ordinary speech, would in verse be pronounced [y.nə ɡʁɑ̃.də fa.mə fy.t‿i.si], with the /ə/ enunciated at the end of each word.

Schwa cannot normally be realised as a front vowel ([œ]) in closed syllables. In such contexts in inflectional and derivational morphology, schwa usually alternates with the front vowel /ɛ/:

harceler /aʁsəle/ → [aʁ.sœ.le] ('to harass'), with il harcèle /il aʁsɛl/ → [i.laʁ.sɛl] ('[he] harasses').[45] A three-way alternation can be observed, in a few cases, for a number of speakers:

appeler /apəle/ → [ap.le] ('to call'), j'appelle /ʒ‿apɛl/ → [ʒa.pɛl] ('I call'), appellation /apelasjɔ̃/ → [a.pe.la.sjɔ̃] ('brand'), which can also be pronounced [a.pɛ.la.sjɔ̃].[46] Instances of orthographic ⟨e⟩ that do not exhibit the behaviour described above may be better analysed as corresponding to the stable, full vowel /œ/. The enclitic pronoun le, for example, always keeps its vowel in contexts like donnez-le-moi /dɔne lə mwa/ → [dɔ.ne.lœ.mwa] ('give it to me') for which schwa deletion would normally apply (giving *[dɔ.nɛl.mwa]), and it counts as a full syllable for the determination of stress.

Cases of word-internal stable ⟨e⟩ are more subject to variation among speakers, but, for example, un rebelle /œ̃ ʁəbɛl/ ('a rebel') must be pronounced with a full vowel in contrast to un rebond /œ̃ ʁəbɔ̃/ → or [œ̃ʁ.bɔ̃] ('a bounce').

google.search.image "virtual personal digital assistant"

DOI: 10.1016/j.molcel.2023.11.015

Resource: (Cell Signaling Technology Cat# 67824, RRID:AB_2799736)

Curator: @scibot

SciCrunch record: RRID:AB_2799736

What is this?

INCIDENTE DE DESLOCAMENTO DE COMPETÊNCIA Nº 1 - PA

CONSTITUCIONAL. PENAL E PROCESSUAL PENAL. HOMICÍDIO DOLOSO QUALIFICADO. (VÍTIMA IRMÃ DOROTHY STANG). CRIME PRATICADO COM GRAVE VIOLAÇÃO AOS DIREITOS HUMANOS. INCIDENTE DE DESLOCAMENTO DE COMPETÊNCIA – IDC. INÉPCIA DA PEÇA INAUGURAL. NORMA CONSTITUCIONAL DE EFICÁCIA CONTIDA. PRELIMINARES REJEITADAS. VIOLAÇÃO AO PRINCÍPIO DO JUIZ NATURAL E À AUTONOMIA DA UNIDADE DA FEDERAÇÃO. APLICAÇÃO DO PRINCÍPIO DA PROPORCIONALIDADE. RISCO DE DESCUMPRIMENTO DE TRATADO INTERNACIONAL FIRMADO PELO BRASIL SOBRE A MATÉRIA NÃO CONFIGURADO NA HIPÓTESE. INDEFERIMENTO DO PEDIDO. 1. Todo homicídio doloso, independentemente da condição pessoal da vítima e/ou da repercussão do fato no cenário nacional ou internacional, representa grave violação ao maior e mais importante de todos os direitos do ser humano, que é o direito à vida, previsto no art. 4º, nº 1, da Convenção Americana sobre Direitos Humanos, da qual o Brasil é signatário por força do Decreto nº 678, de 6/11/1992, razão por que não há falar em inépcia da peça inaugural. 2. Dada a amplitude e a magnitude da expressão “direitos humanos”, é verossímil que o constituinte derivado tenha optado por não definir o rol dos crimes que passariam para a competência da Justiça Federal, sob pena de restringir os casos de incidência do dispositivo (CF, art. 109, § 5º), afastando-o de sua finalidade precípua, que é assegurar o cumprimento de obrigações decorrentes de tratados internacionais firmados pelo Brasil sobre a matéria, examinando-se cada situação de fato, suas circunstâncias e peculiaridades detidamente, motivo pelo qual não há falar em norma de eficácia limitada. Ademais, não é próprio de texto constitucional tais definições. 3. Aparente incompatibilidade do IDC, criado pela Emenda Constitucional nº 45/2004, com qualquer outro princípio constitucional ou com a sistemática processual em vigor deve ser resolvida aplicando-se os princípios da proporcionalidade e da razoabilidade. 4. Na espécie, as autoridades estaduais encontram-se empenhadas na apuração dos fatos que resultaram na morte da missionária norte-americana Dorothy Stang, com o objetivo de punir os responsáveis, refletindo a intenção de o Estado do Pará dar resposta eficiente à violação do maior e mais importante dos direitos humanos, o que afasta a necessidade de deslocamento da competência originária para a Justiça Federal, de forma subsidiária, sob pena, inclusive, de dificultar o andamento do processo criminal e atrasar o seu desfecho, utilizando-se o instrumento criado pela aludida norma em desfavor de seu fim, que é combater a impunidade dos crimes praticados com grave violação de direitos humanos. 5. O deslocamento de competência – em que a existência de crime praticado com grave violação aos direitos humanos é pressuposto de admissibilidade do pedido – deve atender ao princípio da proporcionalidade (adequação, necessidade e proporcionalidade em sentido estrito), compreendido na demonstração concreta de risco de descumprimento de obrigações decorrentes de tratados internacionais firmados pelo Brasil, resultante da inércia, negligência, falta de vontade política ou de condições reais do Estado-membro, por suas instituições, em proceder à devida persecução penal. No caso, não há a cumulatividade de tais requisitos, a justificar que se acolha o incidente. 6. Pedido indeferido, sem prejuízo do disposto no art. 1º, inc. III, da Lei nº 10.446, de 8/5/2002.

• Informativo nº 790
• 10 de outubro de 2023.
• TERCEIRA SEÇÃO
• Processo: IDC 22-RO, Rel. Ministro Messod Azulay Neto, Terceira Seção, por unanimidade, julgado em 23/8/2023, DJe 25/8/2023.

Ramo do Direito DIREITO CONSTITUCIONAL, DIREITO PROCESSUAL PENAL

Paz, Justiça e Instituições EficazesTema <br /> Incidente de deslocamento de competência (IDC). Deferimento parcial. Art. 109, §5°, da CF/1988. Medida constitucional excepcional. Requisitos cumulativos. Presença. Conflito agrário em Rondônia. Grave violação a direitos humanos. Ineficácia das instâncias locais e risco de responsabilização internacional.

DESTAQUE - A Terceira Seção deferiu, parcialmente, o incidente de deslocamento de competência para que a investigação, o processamento e o julgamento dos mandantes, intermediários e executores dos assassinatos de vítimas, em sua maioria, lideranças de movimentos em prol dos trabalhadores rurais, e responsáveis por denúncias de grilagem de terras e de extração ilegal de madeira, ocorridos em contexto de conflito agrário instalado no Estado de Rondônia, sejam deslocados para o âmbito da Justiça Federal daquele Estado.

INFORMAÇÕES DO INTEIRO TEOR - O art. 109, § 5º, da Constituição Federal, estabelece que, nas "hipóteses de grave violação de direitos humanos, o Procurador-Geral da República, com a finalidade de assegurar o cumprimento de obrigações decorrentes de tratados internacionais de direitos humanos dos quais o Brasil seja parte, poderá suscitar, perante o Superior Tribunal de Justiça, em qualquer fase do inquérito ou processo, incidente de deslocamento de competência para a Justiça Federal".

• Conforme se extrai do IDC n. 1, os requisitos do incidente de deslocamento de competência são: a) grave violação de direitos humanos; b) necessidade de assegurar o cumprimento, pelo Brasil, de obrigações decorrentes de tratados internacionais; c) incapacidade - oriunda de inércia, omissão, ineficácia, negligência, falta de vontade política, de condições pessoais e/ou materiais, etc. - de o Estado Membro, por suas instituições e autoridades, levar a cabo, em toda a sua extensão, a persecução penal (IDC n. 1/PA, Terceira Seção do STJ).

Colhe-se da doutrina relativa ao tema, bem como da análise dos casos de deslocamento de competência decididos neste Superior Tribunal de Justiça, que os requisitos são cumulativos, não bastando a constatação de ineficiência dos mecanismos existentes para apuração e punição por parte dos órgãos persecutórios estaduais. É imprescindível que se demonstrem a gravidade das violações aos direitos humanos, a incapacidade de o Estado-Membro atuar, bem como, o risco de responsabilização do país perante órgãos internacionais. - Tudo isso emoldurado pela proporcionalidade, sob pena de se banalizar a medida constitucional e de se incorrer em risco de violar o princípio do juiz e do promotor natural, criando-se verdadeiros tribunais de exceção. Além de ferir o art. 34 da Constituição Federal, por se proceder à intervenção da União nos Estados Membros fora das situações previstas no mencionado dispositivo constitucional.

• Estão preenchidos todos os requisitos de ordem constitucional, legal e aqueles irradiados da jurisprudência deste STJ, que autorizam o deslocamento de competência da esfera estadual para a federal (relativamente a seis inquéritos não solucionados), eis que evidenciada a grave violação de direitos humanos, a possibilidade de responsabilização do Brasil em razão de descumprimento a obrigações contraídas em tratados internacionais e a incapacidade de órgão locais darem respostas efetivas às demandas.

Create flashcard - set 2 - words not built from word parts -

Anaphylaxis (ăn-ă-fĭ-LĂK-sĭs) - a severe, potentially life-threatening allergic reaction that can cause bronchoconstriction, swelling of the throat, airway obstruction, decreased cardiac output and shock.

Benign - cells that grow and divide in a more controlled manner, remaining localized to a certain area.

Cancer - abnormal cells that overgrow and have the ability to invade and harm tissues.

Graft - transplanted, donated tissue or organ.

Hypersensitivity (hī-pĕr-sĕn-sĭ-TĬV-ĭ-tē) - an exaggerated immune response triggered by exposure to a specific antigen.

Immunocompromised (ĭm-yū-nō-KŎM-prō-mīzd) - an individual with an impaired or weakened immune system.

Malignant - cancer cells that exhibit uncontrolled growth and rapidly divide, subsequently invading and causing damage to normal functioning cells.

Metastasize - spreading of cancer cells through the blood or lymphatic system.

Opportunistic infection (ŏp-ŏr-tū-NĬS-tĭk ĭn-FĔK-shŏn) -infections that occur more often or are more severe in people with weakened immune systems.

Pathogens (PĂTH-ō-jĕn) - bacteria, viruses, or other microorganisms that can cause infection and disease.

Seroconversion (sē-rō-kŏn-VĔR-zhŏn) - the presence of detectable anti-HIV antibodies in a person’s blood indicating a positive HIV test.

Flashcard 4 - allergist - change to a physician who specializes in the study and treatment of allergies.

Flashcard 9 - autoimmune disease - listen to the pronunciation - appears to be missing the "im" in immune

Flashcard 21 - innate immune response - change definiton. Current definition - rapid but relatively non-specific immune response (add that we are born with). Need to include the meaning of innate - born with in the definition.

Flashcard 22 intersititial - spelled wrong - should be: interstitial

Flashcard 23 - intersititial space - spelled wrong - should be interstitial space

Flashcard 29 thymus - change to thymus gland to be consistent with the chapter.

Create flashcard (set 2 - words not built from word parts)

seroconversion (sē-rō-kŏn-VĔR-zhŏn) - the presence of detectable anti-HIV antibodies in a person’s blood indicating a positive HIV test.

DOI: 10.7554/eLife.88819

Resource: ImageJ (RRID:SCR_003070)

Curator: @scibot

SciCrunch record: RRID:SCR_003070

What is this?

linguistic origins of a double-ness to communication: what is said and what is meant

Advertencia: no se debe intentar la posición <posición y rana>, con una posible patología de la cadera al menos que lo indique el médico, después de revisar la radiografía ap. de la pelvis * El tamaño depende el tamaño el paciente * Posición: En decúbito supino. Pelvis centrada en el RC y el RO; utilizar protecciones gonadales en hombres y mujeres. AP: extender las piernas y realizar rotación interna 15°. Lateral en posición de rana: flexionar rodillas y caderas, colocar juntos y abducir ambas piernas, fijar con cinta adhesiva y bolsas da arena. * Distancia foco película 100-120 cm (40 x48”)

Words from the anatomy section:

erythr/o/cytes (ĕr-Ĭ-thrō-sītz) - red blood cell (RBCs).

hem/o/rrhage (HEM-ŏ-rāj) - excessive flow of blood

hepat/o/megaly (hep-a-tō-MĒG-ă-lē) - enlargement of the liver.

leuk/o/cytes (LOO-kō-sītz) - white blood cell (WBCs).

Words from the physiology section:

fibrin/o/lysis - breakdown of fibrin (within blood clots).

Words from the disease section:

embol/us (ĒM-bŏ-lŭs) - a portion of a thrombus breaks free from the vessel wall and travels through the blood stream

hemat/oma (hĕm-ă-TŌ-mă) - tumor of blood (collection of blood outside the blood vessel caused by an injury).

leuk/o/cyt/osis (lū-kō-sī-TŌ-sĭs) - abnormal condition of excessive white blood cell.

thromb/o/cyt/osis (thrŏm-bō-sī-TŌ-sĭs) abnormal condition (abnormally high number of platelets in the blood) which can cause a bloot clot.

flashcard 12 has the wrong phoentics atherosclerosis (ar-tēr-ē-ō-sklĕ-RŌ-sĭs)

It should be atherosclerosis (ath-ĕ-rō-sklĕ-RŌ-sĭs).

Flascard 33 is pronounced wrong - redo for 2nd edition plasmapheresis (plăz-mă-fĕr-Ē-sĭs)

Flashcard 36 - splenomegaly (splē-nō-mē-GĀ-lē) should be (sple-nō-MEG-a-lē)

Move flashcards lymphadenitis (24), lymphadenopathy (25), thymectomy(43) and thymoma(44) to chapter 11 - lymphatic system

Îmi place în mode dosebit această emisiune.

Flashcard 15 - gynecology - include abbreviation - should be gynecology (GYN)

flashcard 17 - gynopathic - incorrect definition - states "pertaining to the women"; should be pertaining to diseases specific to women

Flashcard 36 - menarche - should be in set 2 - words not made from word part according to the references I have

Flashcard 37 - redo - menometrorrhagia - missing the ro sound - reference: https://www.howtopronounce.com/menometrorrhagia

definition

Children learn morals through socialization

Why are heroes important?

După cum vezi, la noi NU există pe piață!

• RE 588322
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 16/06/2010
• Publicação: 03/09/2010

Recurso Extraordinário 1. Repercussão geral reconhecida. 2. Alegação de inconstitucionalidade da taxa de renovação de localização e de funcionamento do Município de Porto Velho. 3. Suposta violação ao artigo 145, inciso II, da Constituição, ao fundamento de não existir comprovação do efetivo exercício do poder de polícia. 4. O texto constitucional diferencia as taxas decorrentes do exercício do poder de polícia daquelas de utilização de serviços específicos e divisíveis, facultando apenas a estas a prestação potencial do serviço público. 5. A regularidade do exercício do poder de polícia é imprescindível para a cobrança da taxa de localização e fiscalização. 6. À luz da jurisprudência deste Supremo Tribunal Federal, a existência do órgão administrativo não é condição para o reconhecimento da constitucionalidade da cobrança da taxa de localização e fiscalização, mas constitui um dos elementos admitidos para se inferir o efetivo exercício do poder de polícia, exigido constitucionalmente. Precedentes. 7. O Tribunal de Justiça de Rondônia assentou que o Município de Porto Velho, que criou a taxa objeto do litígio, é dotado de aparato fiscal necessário ao exercício do poder de polícia. 8. Configurada a existência de instrumentos necessários e do efetivo exercício do poder de polícia. 9. É constitucional taxa de renovação de funcionamento e localização municipal, desde que efetivo o exercício do poder de polícia, demonstrado pela existência de órgão e estrutura competentes para o respectivo exercício, tal como verificado na espécie quanto ao Município de Porto Velho/RO 10. Recurso extraordinário ao qual se nega provimento.

Tema - 217 - Comprovação do poder de polícia para cobrança de taxa de localização e funcionamento.

Tese - É constitucional taxa de renovação de funcionamento e localização municipal, desde que efetivo o exercício do poder de polícia, demonstrado pela existência de órgão e estrutura competentes para o respectivo exercício.

Aici testez adnotarea.

Only engaging with the text through a removed lens leads the reader to not think of how they feel about the content of an author's argument

His mind couldn't rest, it couldn't settle, he never felt safe.