Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rare and progressive nervous system disease that destroys nerve cells in the brain and spinal cord, leading to a loss of muscle contro

Rather than the focus on the natural world and conditions of urban living like Burial of the Dead, A Game of Chess highlights wealth, gender imbalances, and love–as represented by cupid. The reference to a game of chess can be connected to the content of the section in two ways: the calculated existence of men and women as separate entities, or the cold and calculated nature of romantic relationships. Eliot begins by describing a scene of immense wealth and ascribing the ostentatious display to a female figure by using the pronoun her in, “The glitter of her jewels rose to meet it”. The woman who is seemingly overcome by wealth is then contrasted with the presence of the nightingale, an animal representation of the violence inflicted upon women. The nightingale, small, animal, and unable to speak, represents the subjugated state of the female race. Oppressed and incapable of responding to the tyranny of men, the woman is silenced and left to an empty, lavish lifestyle.

The weak, tenuous scaffold of this glass structure—fruited vines"—represents tension and fragility in male-female relations (those present within the assigned texts). Within Paradise Lost, most explicitly is this dynamic evident; describing the fruit upon the "Tree of Life:" "...loaden with fairest Fruit,/ Blossoms and Fruits at once of golden hue/ ...of pure now purer aire/ Meets his approach, and to the heart inspire/ Vernal delight and joy, able to drive/ All sadness but despair: now gentle gales/ Fanning their odoriferous wings dispense/ Native perfumes, and whisper whence they stole/ Those balmie spoils." Not only does use the "Tree of Life" act as the ultimate point of pressure and conflict between Adam (man) and Eve (woman), but a bodily dynamic. From the vegetation, inspired in the "heart...[is]/ Vernal delight and joy, able to drive / All sadness but despair... ." The words "Vernal delight" connote Spring and revival, perhaps fertility. With spring, "sadness" is dissolved yet "despair" remains. Now, in conversation with lines 215-225, (line 220-222), "despair" seems to mean or define the opposite of fertility, or of that which defines the loss of "sadness." Given lines 220-222 explain the growth of a "Tree of Knowledge," bringing about ill and Death, "despair" must be the continuation of sexual desire and lust in celibacy.

Witnessing sexual lust and violence in Mandelbaum's The Metamorpjoses of Ovid, and the ultimate destruction of one's humanity following 'rape,' this notion of male-female tension holds relevant. In the play's final scene, Philomela and her sister Procne dismember Itys feeding his body to Tereus. The sister's actions are unique; not only do the two remove Tereus's humanity via filicidal cannibalism, but ever sever his relationship with God. In lines 642-643, this is specifically referenced: Thrace describes himself as “the miserable tomb/ of his own son;” he is a desolate sepulcher, ever removed and dejected by God.

Overall, the poems act as explorations and attempts to disrupt male-centric power & domination; the female body, though fragile like the "fruited vines," defies male divinity in some sacrilegious act.

I am particularly interested in Eliot’s “From satin cases poured in rich profusion; In vials of ivory and coloured glass Unstoppered, lurked her strange synthetic perfumes,” In Baudelaire, perfume is not elegant or pleasant, but suffocating the “hot-house” air feels almost dangerous. The girl died from fatal beauty. She was described as artifial, posed like a doll, and used. Eliot does something similar to this artificalness of perfume: “synthetic” and "drowned the sense of odors" emphasizes that the perfume is artificial. Both poets connect wealth and luxury with suffocation. Almost being lost by temptation, human desire, like in Paradise Lost, the “native perfumes” of Eden seem natural and good, although their sweetness could also suggest temptation! The line in Eliots goes right into "vials of ivory and coloured glass" connects to buadelairs image of bouquets dying in glass coffins, like the girl in the poem.

There are many allusions within this small section, first to Ovid, in the form of the mournful nightingale, and the continual references to fire throughout the section with many connections to Eliot and the sources we read for tonight. First of all, industrail England was fueled by fires and the smoke produced by them, the damages of which were clear to see for all, Eliot included, which connects back to the Wasteland originally referred to. Additionally the anguish of Dido as she burns herself on a funeral pyre because of her deserting lover. In Eliots work, these allusions function as an emphasis on the tragedy the character within the poem is feeling. Because she is sad remembering her past, or perhaps a former lover as well, those who pick up on the references understand the intensity of feeling without Eliot having to outright describe how the chracter herself is "feeling"

Here, Elliot twists John Miltons words in Paradise Lost, transforming a description of beauty and serenity in the garden of eden into one of barbarity. The word sylvan refers to characteristic woods or the countryside, and was used by Milton int he quote "Insuperable highth of loftiest shade, Cedar, and Pine, and Firr, and branching Palm A Silvan Scene, and as the ranks ascend Shade above shade, a woodie Theatre Of stateliest view."Milton describes the garden of eden in all its glory using sylvan as one of its adjectives, yet ultimately this description is used by Elliot in a much different way. Elliot is describing a painting above the narrators mantel, which depicts the rape of Philomel and her transformation. This corrupts the original intent of the reference, and as a result speaks to the corruption of the past and of nature present throughout the book. By using this word which describes a scenic landscape such as the garden of eden to describe this brutality, it reinforces the idea Elliot has already presented of the perversion of nature and humanities corruption of it. Thus, through this deliberate usage of the descriptor sylvan, Elliot reinforces his themes of the corruption of humanity and the destruction of nature using paradise lost and the myth of Philomel.

annotation!

this is a test

these are prtty cool, but technically it would be all three? xD might need to rethink what we say in the regualr banner.

REVIEW: Banner picks: company page and holiday (RME-9 · RME-10) Reviewer: YT · 9/22/2026, 4:40:14 PM Decided 4 of 6

▸ Company page banner [pick one] · C · Both are true [RME-9] · D · One list, three answers [RME-9] · E · Which one [RME-9] · H · One list, three answers (with addresses) [RME-9] (no choice yet)

▸ Halloween 2026 [pick one] · A · The line [RME-10] · C · Trick or treat [RME-10] · D · Haunted inbox [RME-10] (no choice yet)

▸ Q4 holiday banners [keep or cut] KEEP Black Friday · Nov 27 [RME-10] KEEP Cyber Monday · Nov 30 [RME-10] KEEP Christmas · Dec 25 [RME-10] note: but fix design, on my end the list is breoken the x and chekcbox too?

KEEP New Year's Eve · Dec 31 [RME-10]

OVERALL: (none)

don't put liikne on the left, add as rule in branding guidelies, this is the third one so far. bvubt the text is gerat

this is the one i choose. lets make sure (maybe just in quiz it is broken?) but the label icons next to the three email is missing. also we don'T use thinck line bars on boxes, shows as ai too much. so remove the yello line, just put the labelicon in the cicrlce next yo the email

I like this but I would but the ghost a different color so we can see it a bit better?

the valid icon is wrong also it isn'T valid it is to keep. also we have to decide the label icons n also to monitor is missing, even though i understand that the banner is talking abotu an email and what we would say, but it might make people think it is the only status and labels we give. maybe e need to rethink it.

also we need to decide label icon vs text next to it, how much taller/bigger it can be vs text. the icon should be bigger in heght than the text nto same, so we can see it. we also have dark mode of the icon i believe with a fine white line around? we shoudl use that when background isn't white or beige or an o the other light colors

They all look a bit blurry but it may just be because its jsut hte quiz?

« tient-elle à » ou « ne tient-elle qu’à »

ce qui a été

Même remarque que pour Bridel

« c’est pour la raison que » ou « c’est en raison de »

Supprimer toutes les références à Bridel et réattribuer ces citations à Bernanos

à un

répétition

discutable

très allusif

remplacer ces guillemets ASCI par des guillemets anglais (courbes)

emploi impropre : voir CNRTL

Manque dans la bibliographie

Même remarque que plus haut sur l’emploi du prénom

« l’ » devant « on » ne se justifie que par l’euphonie et n’est donc pas nécessaire ici

Mentionner les prénoms des grands savants, comme des grands écrivains, fait un peu scolaire

Excellente mise au point conceptuelle, qui va à l’essentiel

oui

alléger la syntaxe

accoler « prestige » et « pathologie » semble paradoxal

Supprimer le trait d'union

Éviter « un » ... « d'une » ...

I would really like to find the past one in my download folders. It's gonna be a folder with a video on it. That video should be able to give you the date to help you find how we generate them the scripts how we then went to create the voice and the captions and then how it works for the terminal, etc., and one of the things were really gonna need help with for V or maybe she needs to find somebody is how we're gonna do the background music and things like that and I don't mind paying when we've done all the videos paying for a month or two a little bit higher to clone my voice and my face maybe and then my face in the corner, which we then decide my face always there is it just my voice or is it a man's voice or is it a couple of different voices based what we're talking about maybe that's the ship shaped characters you know I don't know. Maybe it's a question for V to decide there's a lot of these questions and thoughts that we could present to her so that she can see well and make a good decision for us once we've kind of confirmed all of this obviously.

Did you find the process we use in order to create the first one? What is email because the script and the video came out really well we did do other video things and I'd like to add and edit maybe even though what his email not the content but like the video maybe just because who might come up with better smarter animations that are grounded and we also need to process right to keep these somewhere because some things like if you're talking about SPF and how it works, where we designed an animated header we might reuse it or we might reuse the base of it in order to make it different and change it, but at least we don't have to re-create things always from scratch, which then uses less tokens and also ensure that everything is correctly branded. This is also a great time to double check that her branding isn't getting confused or you know this organized because we have different projects and then are adding random things we shouldn't be adding.

Even if we might not be publishing, something somewhere the descriptions and whatever should be done on a social media level just because they have different character limit so you can put your L some of them we have to be reminded to add it as like a sticker like on it Instagram stories we have to do it manually so we need all that information organized somewhere in a database because this is a lot of stuff to manage and a lot of steps and we're not gonna be doing a continuously we're gonna start and we're gonna stop everything tracked correctly and we need to learn things as we go so that there's less corrections and back-and-forth

Just making sure we're on the same page it doesn't have to be an actual exact card like we take a long video. We just cut it into like trim it. It can be a rewrite in order for it to be Pierre in a shorter sentence so that it works for social media in places where videos can't be a minute a minute and a half two minutes plus

How about we do it in order and then also we have a page connect here or like whatever drop down I don't know somewhere where we can see which questions we have actually done what part like the square versus the video versus posting it versus the M4 versus the cards versus you know every single step EV all the reviews like everything on the table and if at some point, some questions or combined in the same video for whatever reason that becomes a combined role of videos instead of each line being its own QA/video role in this table page to track process

We will create the first version of the storyboard, but we can add her own ideas when she comes up with better ideas to have animations that are gonna keep people listening you know more of the page or things are gonna be used, but the points to create a storyboard where we talk about normal email things, but find a way to visualize that or explain things by creating mind animation just putting text making a list of examples means whatever makes sense using obviously the information from the email Monarch

vy is going to be the one who confirms the resized didn't break or things have moved and will also be the one scheduling and preparing the post to put online whether it's for the LinkedIn page my personal page, her personal page, Diego, Bruno, or cat's personal page on LinkedIn or TikTok or YouTube shorts or YouTube long format which we're gonna be recruiting the email as a pla then the audio version of that with the video or the exact same but for Spotify and then confirming the descriptions, the PDFs or generating tickets saying hey, in order to post this, we have a description, but I would like to have the description with a lead magnet. The lead magnet she can choose from could be the ones that are still in drafts that I haven't finished which then organizing and pushes me to create them, which is kind of good because it fixes and opens new funnel options on the email monarch pages. It's a task I need to do so might as well create the lead magnets from the ones we already have drafted and kind of almost done, but I actually have to build them using our template system or maybe she has another idea or you help her come up with another idea that would be great for that particular video which would then also I guess be a good thing to add as another option for lead magnet on the actual email almanac page or use it for a social media in general on its own specific learning page right

this is correct title font style with one font dark green and then italics and bolder in teal. can we update on preview on all pages, and then push on prodafter testing othign broke? cause i eealized our headers lost this design style accross the board, and alos different font sizes, etc on pages or even between prod and and preview. we can create as new ticket for TT chat, and then TT can manage and push the pill when time to do it

Изначально мы думали, что преподаватели будут сами создавать стенды студентам по конфигу.

идея была такова: админ создаёт конфиг по которому развёртываются стенды для студентов, а преподы уже конфиг.

по итогу от этого отказались, но всё равно с конфигом удобно развёртывать стенды самоу админу - можно сразу батчами - кучами развёртывать стенды

ДЕТАЛЬНО ПОКАЗАТЬ КАК ЭТО СВЯЗАНО С PROXMOX

Он выбирает node - потом это будет происходить автоматически.

пользователей и название стенда.

ну и всё по итогу собраны стенды

понятно, что для того, чтоб пользователь мог пользоватсья proxmox у него там должен быть акканунт

Через proxor есть возможность получить данные о любом пулле.

скажем так: стенд и золотой стенд - это пулы, но в более прикладном домене. чтоб просто различать пулы с разной целью между собой.

ОТВЕТ

Показать стенд - что по итогу создаётся админом.

eLife Assessment

This work makes an important contribution to understanding the role of calreticulin and the Del52 variant in calcium homeostasis in cells based on convincing evidence. There was some question as to whether the work in vitro fully recapitulated the physiological setting in which the homozygous Del52 variant has been reported to have defects in calcium homeostasis. Nonetheless, the studies are well performed using appropriate methods to address these questions and make a significant contribution to the understanding of calreticulin, and the Del52 variant, function in health and disease.

Reviewer #1 (Public review):

The authors attempted to compare calcium binding properties of wildtype calreticulin with calreticulin deletion mutant (CRTDel52) associated with myeloproliferative neoplasms.

The researchers conducted their study using advanced techniques They found almost no difference in calcium binding between the two proteins and observed no impact on calcium signaling, specifically store-operated calcium entry (SOCE). The study also noted an increase in ER luminal calcium-binding chaperone proteins. Surprisingly, the authors selected flow cytometry as a technique for measurements of ER luminal calcium. Considering limitations of this approach it would be better to use alternative approaches. This is particularly important as previous reports, using cells from MPN patients, indicate reduced ER luminal calcium and effects on SOCE (Blood, 2020). This issue matters because earlier research with MPN patient cells reported reduced ER luminal calcium levels and altered SOCE (Blood, 2020). How do the authors explain the difference between their results and previous findings about lower ER luminal calcium and changed SOCE in MPN patient cells expressing CRTDel52? Other studies have found that unfolded protein responses are activated in MPN cells with CRTDel52 calreticulin (see Blood, 2021), and increased UPR could account for higher levels of some ER resident calcium-binding proteins observed here. Overall, it remains unclear how this work improves our understanding of MPN or clarifies calreticulin's role in MPN pathophysiology.

Comments on revised version.

The authors have addressed the points raised in the original review. However, given the absence of significant differences between the wild-type and mutant proteins, the relevance of this work to MPN pathology remains unclear. The novelty of the study is limited, as calcium has generally not been considered a significant factor in MPN pathology associated with mutant calreticulin.

Reviewer #2 (Public review):

Summary:

Tagoe and colleagues present a thorough analysis of the calcium (Ca2+) binding capacity of calreticulin (CRT), an endoplasmic reticulum (ER) Ca2+-buffer protein, using a mutant version (CRT del52) found in myeloproliferative neoplasms (MPNs). The authors use purified human CRT protein variants, CRT-KO cell lines, and an MPN cell line to elucidate the differing Ca2+ dynamics, both on the level of the protein and on cell-wide Ca2+-governed processes. In sum, the authors provide new insights into CRT that can be applied to both normal and malignant cell biology.

First the authors purify CRT protein and perform isothermal titration calorimetry to quantify the Ca2+ binding capacity of CRT. They use full-length human CRT, CRT del52, and two truncations of CRT (1-339 and 1-351, the former of which should lead to the entire loss of low affinity Ca2+ binding). While CRT del52 has previously been shown to lead to a decrease in Ca2+ binding affinity in other models, the ITC data shows that this is retained in CRT del52.

Next, the authors utilize a CRT-KO cell line with subsequent addition of CRT protein variants to validate these findings with flow cytometric analysis. Cells were transfected with a ratiometric ER Ca2+ probe, and fluorescence indicates that CRT del52 is unable to restore basal ER Ca2+ levels to the same extent as CRT wild-type. To translate these findings to MPNs, the authors perform CRT-KO in a megakaryocytic cell line, where reconstitution with either CRT variant did not cause a difference in cytosolic calcium levels. The authors further test store-operated calcium entry (SOCE), an important process to maintaining ER Ca2+ levels, in these cells, and find that CRT-KO cells have lower SOCE activity, and that this can be slightly recovered with CRT addition.

Finally, the authors ask whether other effects of CRT-KO/reconstitution can affect cellular Ca2+ signaling pathway and levels. RNASeq analysis revealed showed that CRT-KO lead to an increase in various chaperone protein expressions, and that reconstitution with CRT del52 is unable to reduce expression to the same extent as reconstitution with CRT wildtype.

Comments on revised version.

The authors have sufficiently addressed my concerns from the first review.

Author response:

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

eLife Assessment

This study investigates low-affinity Ca2+ binding by WT calreticulin and mutant calreticulin associated with type I myeloproliferative neoplasms, as well as the impact on Ca2+ fluxes in suspension cultures of megakaryocyte-like cells in vitro in response to ER Ca2+ ATPase inhibitors that deplete endoplasmic reticulum (ER) Ca2+ store and open plasma membrane Ca2+ channels through STIM1-Orai interactions. The results are important in that they show that Ca2+ binding by calreticulin and store-operated Ca2+ entry are not fundamentally impacted by the type I deletion mutation in calreticulin, which rules out a direct effect of the calreticulin mutation on its own low-affinity Ca2+ binding and any broad impact on ER Ca2+ regulation. The strength of the data and methods used ranges from solid to convincing, although the use of suspension-based flow cytometric assays to investigate ER Ca2+ levels and Ca2+ entry can be challenged. High-affinity Ca2+ binding sites could be further considered, and possible confounding effects of Abl kinase activity in the megakaryocyte-like cell lines could be offset.

The authors thank the editors and the reviewers for the summary, comments and many helpful suggestions. In the revised manuscript, we have used fluorimetry for more precise ER calcium measurements (new Figure 5), clarified the high-affinity calcium binding site concern based on our previous work, and addressed possible effects of BCR-ABL translocation kinase activity upon cellular calcium signaling using the drug Imatinib (new supplements to Figure 6 and 7).

Public Reviews:

Reviewer #1 (Public review):

The researchers conducted their study using advanced techniques. They found almost no difference in calcium binding between the two proteins and observed no impact on calcium signaling, specifically store-operated calcium entry (SOCE). The study also noted an increase in ER luminal calcium-binding chaperone proteins. Surprisingly, the authors selected flow cytometry as a technique for measurements of ER luminal calcium. Considering the limitations of this approach, it would be better to use alternative approaches.

Thank you for this suggestion. We have undertaken fluorimetry-based ER calcium measurements, which are shown in a new Figure 5. These also indicate similar ER calcium levels in CRT-KO HEK293T cells, compared to those reconstituted with wild-type CRT and CRT_Del52.

This is particularly important as previous reports, using cells from MPN patients, indicate reduced ER luminal calcium and effects on SOCE (Blood, 2020). This issue matters because earlier research with MPN patient cells reported reduced ER luminal calcium levels and altered SOCE (Blood, 2020). How do the authors explain the difference between their results and previous findings about lower ER luminal calcium and changed SOCE in MPN patient cells expressing CRTDel52?

We thank the reviewer for asking for these clarifications. We have revised the discussion to address some of these points and also clarify the findings of the referenced study (Di Buduo et al., 2020) which did not directly measure ER calcium levels. We also discuss findings from a related study with cultured megakaryocytes from patients that indicated different effects of type I vs type II mutations (Pietra et al., 2016). In the absence of engineered controls, sample-to-sample heterogeneities in primary cells make it difficult to attribute any measured differences as direct effects of CRT mutations. Different from these experiments, by using purified proteins and ITC, our studies show that the Del52 mutant has calcium-binding characteristics resembling that of the wild-type protein. Additionally, through genetic manipulations in cell lines, our studies directly address the effects of calreticulin KO and its Del52 mutation upon ER luminal and cytosolic calcium levels, and cellular SOCE signals. We did not measure significant differences in any of these parameters between the KO cells and those reconstituted with wild-type calreticulin or the Del52 mutant. As noted by the editors, these results show that Ca2+ binding by calreticulin and SOCE in a cell are not fundamentally impacted by the type I deletion mutation.

Other studies have found that unfolded protein responses are activated in MPN cells with CRTDel52 calreticulin (see Blood, 2021), and increased UPR could account for higher levels of some ER-resident calcium-binding proteins observed here.

These points are addressed in the discussion. Either protein misfolding in cells with wild-type calreticulin deficiency or the sensing of cellular calcium perturbations could induce the expression of ER calcium-binding proteins in calreticulin-deficient cells, although we favor the latter model for the reason specified in the discussion. Regardless of the precise mechanisms underlying the expression changes in calcium-binding proteins, the upregulated factors are predicted to compensate for calreticulin deficiency and contribute to the maintenance of the overall cellular calcium homeostasis.

Overall, it remains unclear how this work improves our understanding of MPN or clarifies calreticulin's role in MPN pathophysiology.

Multiple studies referenced in the manuscript have suggested links between altered calcium signaling/binding by CRT mutants and MPN pathogenesis. Our studies indicate that ER and cytosolic calcium levels and SOCE are not directly impacted by the MPN type I CALR mutation, points noted in the abstract and discussion. Thus, calcium signaling may not play a specific role in MPN CALR mutant pathology via suggested mechanisms. We are confident that readers will find these results important for better understanding the role of calreticulin type I mutations in MPN.

Reviewer #1 (Recommendations for the authors):

This study aimed to express, purify, and evaluate low-affinity calcium binding by a calreticulin deletion mutant (CRTdel52) that is linked to myeloproliferative neoplasms (MPN). The researchers performed cell imaging, flow cytometry, and isothermal titration calorimetry to compare calcium binding between wild-type calreticulin and CRTDel52. They assessed cytosolic calcium levels and store-operated calcium entry (SOCE) in HET293T cells (CRT knocked-out background) and in megakaryoblastic MEG-1 cells. Additionally, they examined changes in the abundance of endoplasmic reticulum (ER) resident calcium-binding proteins in cells expressing either wild-type or mutant CRT.

This study is well executed but lacks clear relevance to MPN, and it is not clear how this work advances our knowledge of calreticulin biology. No differences were found in calcium binding between wild-type calreticulin and CRTDel52, nor was SOCE impacted. They noticed, however, a compensatory increase in the abundance of some ER resident calcium-binding proteins. The lack of any significant changes in calcium behavior between wild type and CRTDel52 is not surprising based on the known amino acid sequence of calreticulin and calreticulin mutant and based on our knowledge about CRT calcium binding in general. Consequently, it is not clear how this work advances our understanding of the pathophysiology of MPN. Calcium may not play a critical role in the MPN pathology; instead, CRTDel52 secretion and receptor signaling appear more central. Further research should address how these findings relate specifically to MPN and cell biology, in general.

Our current studies demonstrate increased expression of other calcium-binding proteins in the context of heterozygous MPN type I CALR mutations (Figure 8C) or conditions resembling homozygous MPN type I CALR mutations (Figures 8D-8G). These results, together with findings of maintained ER and cytosolic calcium levels and SOCE signals (Figures 4-7 and Figure 6, supplemental Figure 2 and Figure 7, supplemental Figure 1), indicate that altered calcium binding/signaling by Del52 does not directly contribute to MPN pathology.

What is the biological or pathophysiological relevance of the CRTDel52-KDEL construct?

The KDEL sequence is important for the ER retention of CRT (Sonnichsen et al., 1994), and its addition was expected to at least partially remedy the ER retention defect of CRT_Del52. This point is clarified in the revised results section.

The rise in ER calcium-binding proteins is noteworthy but anticipated, given likely genetic changes from UPR pathway activation in these cells. Is this relevant to MPN?

We suggest that increased expression of other calcium-binding proteins in the context of heterozygous MPN type I CALR mutations (Figure 8C) or conditions resembling homozygous MPN type I CALR mutations (Figures 8D-8G) would contribute to the maintenance of the cell’s calcium signaling capacity.

How do the authors explain the difference between their results and previous findings about lower ER luminal calcium and changed SOCE in MPN patient cells expressing CRTDel52?

The findings related to SOCE are addressed in the points discussed above and in the revised discussion. Related to ER luminal calcium, the study by Ibarra et al. (Ibarra et al., 2022) reported that CRT_Del52 overexpressed in U2OS cells (expressing endogenous CRT) had reduced ER calcium levels compared to the same cells expressing WT CRT or CRT_Ins5. Those measurements did not use a ratiometric ER calcium probe, and additionally it is possible that the expression of compensatory calcium-binding proteins is more muted in cells expressing endogenous wild-type CRT.

Other studies have found that unfolded protein responses are activated in MPN cells with CRTDel52 calreticulin (see Blood, 2021), and increased UPR could account for higher levels of some ER-resident calcium-binding proteins observed here.

We agree that increased UPR could account for higher levels of some ER-resident calcium-binding proteins. As noted in the revised discussion, regardless of the precise mechanisms underlying the expression changes in calcium-binding proteins, the upregulated factors are predicted to compensate for calreticulin deficiency and contribute to the maintenance of the overall cellular calcium homeostasis.

It is not clear why flow cytometry was a choice of technique for measurements of ER calcium. Pacific Blue was detected at 405 nm excitation and 452-455 nm emission, while unbound probe signals appeared in the AmCyan channel (405 nm excitation/498 nm emission). It is unnecessary to mention fluorochrome labels (like Pacific Blue or AmCyan) for channels that are not in use. Only the channels actually utilized need to be specified: The calcium-bound GEM-CEPIA1er probe's signal was collected using the Pacific Blue channel (excitation at 405 nm, emission at 452-455 nm), while the unbound probe's signal was detected using the AmCyan channel (excitation at 405 nm, emission at 498 nm). Since it's not possible to monitor emission only at a specific wavelength with a Fortessa, could this be a different channel that is being recorded?

Additionally, due to the similar spectra and potential for bleed-through between Pacific blue and Amcyan, compensation is likely necessary. Therefore, you should include single-stained control cells containing only one probe for proper reporting. Additionally, only the "GEM-CEPIA1er probe" is displayed, while the second probe is referred to solely as "unbound".

A single genetically encoded GEM-CEPIA1er probe (Suzuki et al., 2014) was used for measuring both the bound and unbound signals. The GEM-CEPIA1er probe was excited with the 405 nm violet laser. In the methods section of the revised manuscript, the GEM-CEPIA1er probe wording is included for describing both the bound and unbound signal collections. Additionally, we have undertaken new spectrofluorimetric experiments (new Figure 5), which allow for the distinct emission peaks to be recorded corresponding to the Ca²⁺-bound and Ca²⁺-unbound signals. Similar results were obtained as reported for the flow cytometry-based experiments.

Increased expression of wild-type calreticulin compared to parental cells should impact on ER calcium content and dynamics in back-transfected HEK293T-KO or MEG-1 cells. Direct ER calcium measurements in HEK293 cells with various calreticulin constructs would significantly strengthen this presentation.

Our experiments were structured to compare calcium signaling in cells expressing only wild-type CRT or CRT_Del52 (resembling homozygous type I MPN CALR mutations) compared to CRT-KO cells. The over-expression of CRT in the reconstituted cells compared to endogenous expression level is a limitation of our study which we have acknowledged in the revised manuscript discussion. Understanding the effects of over-expression of wild-type CRT vs the CRT_Del52 mutant upon ER and cytosolic calcium signals and SOCE is interesting, but beyond the scope of the present study.

The authors should examine the immunolocalization of CRTDel52 and wild-type protein in HEK293 cells.

Previous published studies from another lab showed that CRT_Del52 is secreted from HEK cells and that the addition of a KDEL sequence to CRT_Del52 reduces secretion and induces its increased intracellular accumulation (Arshad and Cresswell, 2018). This point is noted in the revised results section and the reference is cited. This appears to be the general theme in primary cells and cell lines. Previous studies and our own prior published studies have shown that CRT_Del52 (but not wild-type CRT) is detectable in the media of cell lines and patient serum as well on the cell surface of primary cells and cell lines (Kaur et al., 2024, Venkatesan et al., 2021, Pecquet et al., 2023).

SDS-PAGE of purified proteins is overloaded, and chromatograms show extra peaks or shoulders, making protein quality assessment uncertain.

Representative chromatograms, peaks corresponding to protein monomers used for ITC analyses and the relevant gels are clarified in the revised manuscript. In new analyses since the original submission, intact protein mass spectrometry was undertaken for CRT_Del52. The results indicate a 35-42 amino acid truncation in different preparations. The truncated proteins would still include acidic residues (between 340–351) previously implicated in low-affinity calcium binding by murine CRT that are shared between wild-type and CRT_Del52. This new information is now included in the revised results section.

Analysis of SOCE in calreticulin-deficient cells and cells reconstituted with calreticulin or overexpressing the protein has already been reported (PMID12324449).

The indicated reference and additional related papers examining effects of CRT deficiency and overexpression on cellular calcium signaling (Arnaudeau et al., 2002, Bastianutto et al., 1995, Mery et al., 1996, Nakamura et al., 2001) are cited in the revised manuscript.

Reviewer #2 (Public review):

Tagoe and colleagues present a thorough analysis of the calcium (Ca2+) binding capacity of calreticulin (CRT), an endoplasmic reticulum (ER) Ca2+-buffer protein, using a mutant version (CRT del52) found in myeloproliferative neoplasms (MPNs). The authors use purified human CRT protein variants, CRT-KO cell lines, and an MPN cell line to elucidate the differing Ca2+ dynamics, both on the level of the protein and on cell-wide Ca2+-governed processes. In sum, the authors provide new insights into CRT that can be applied to both normal and malignant cell biology.

First, the authors purify CRT protein and perform isothermal titration calorimetry to quantify the Ca2+ binding capacity of CRT. They use full-length human CRT, CRT del52, and two truncations of CRT (1-339 and 1-351, the former of which should lead to the entire loss of low-affinity Ca2+ binding). While CRT del52 has previously been shown to lead to a decrease in Ca2+ binding affinity in other models, the ITC data show that this is retained in CRT del52.

Next, the authors utilize a CRT-KO cell line with subsequent addition of CRT protein variants to validate these findings with flow cytometric analysis. Cells were transfected with a ratiometric ER Ca2+ probe, and fluorescence indicates that CRT del52 is unable to restore basal ER Ca2+ levels to the same extent as CRT wild-type. To translate these findings to MPNs, the authors perform CRT-KO in a megakaryocytic cell line, where reconstitution with either CRT variant did not cause a difference in cytosolic calcium levels. The authors further test store-operated calcium entry (SOCE), an important process for maintaining ER Ca2+ levels, in these cells, and find that CRT-KO cells have lower SOCE activity, and that this can be slightly recovered with CRT addition.

Finally, the authors ask whether other effects of CRT-KO/reconstitution can affect the cellular Ca2+ signaling pathway and levels. RNASeq analysis revealed that CRT-KO leads to an increase in various chaperone protein expressions, and that reconstitution with CRT del52 is unable to reduce expression to the same extent as reconstitution with CRT wildtype.

Strengths:

The authors provide new insights into CRT that can be applied to both normal and malignant cell biology.

We thank the reviewer for the recognition that this study is important for our understanding of both normal and malignant cell biology.

Weaknesses:

(1) The authors should consider discussing the high-affinity Ca2+ binding site more in the introduction. Can they show a proof-of-concept experiment that validates that incubation of recombinant CRT reduces the function of that high-affinity Ca2+ binding site?

In a previous study (Wijeyesakere et al., 2011), we showed that at a starting calcium concentration of 0 mM and with CaCl₂ injections to a final concentration of 70-80 mM the measured K_D value was 16.6 mM for calcium binding to wild type murine calreticulin, (which has ~95% sequence identity with human calreticulin), corresponding to the high-affinity site. On the other hand, at a starting calcium concentration of 50-100 mM and CaCl₂ injections to a final concentration of 700-850 mM, the measured K_D value for calcium binding to wild-type murine calreticulin was 590 mM (corresponding to the low-affinity sites). We did not observe the high-affinity sites when the starting calcium concentration was 50 mM and calcium injections were at 33 mM each; similar conditions are used in the present study. These points are clarified in the revised manuscript in the results section.

(2) For Figure 2B, do you have an explanation for why the purified proteins run higher than predicted (48-52kDa) - are these proteins still tagged with pGB1?

Yes, the purified proteins shown in Figure 2B retained a GB1 tag. This point is clarified in the revised methods.

(3) The MEG-01 cell line has the BCR:ABL1 translocation, while CRT mutations are strictly found in BCR:ABL1 negative MPNs. Could these experiments be repeated in these cells treated with imatinib to decrease these effects, or see if basal MEG-01 Ca2+ levels/activity are changed with or without imatinib?

Thank you for this important point. We have assessed cytosolic calcium levels in MEG-01 cells that were treated or not treated with imatinib in new Figure 6, supplemental Figures 1 and 2 and Figure 7, supplemental Figure 1) and show that the prior results hold in imatinib-treated cells.

References

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eLife Assessment

In this important study, Abdelnaby and colleagues systematically examine the four native Orai channel isoforms (Orai1α, Orai1β, Orai2, and Orai3) and find that all can couple to NFAT1/4 activation, with NFAT responses largely scaling with the magnitude of Ca2+ influx (Orai1β > Orai1α >> Orai2 > Orai3). The study shows that in primary CD4⁺ T cells, Orai1α and Orai1β are functionally redundant, restoring SOCE, NFAT activation, cytokine production, and highly similar transcriptional programs. Human genetic data further show that selective loss of Orai1α does not cause CRAC channelopathy and instead slightly enhances SOCE and NFAT activation. Overall, the findings are significant, and the strength of the evidence supporting the conclusions is convincing.

Reviewer #1 (Public review):

Summary:

This study quantifies the ability of the four isoforms of the calcium-release calcium-activated (CRAC) channel Orai to mediate calcium entry and transcriptional responses. By genetically invalidating each isoform and by separately re-expressing them in Orai-deficient human embryonic kidney cells, the authors show that the rates of calcium entry across the four native Orai calcium channel isoforms (Orai1α/β, Orai2, Orai3) correlate with the degree of NFAT activation. They further show that the two alternatively translated isoforms Orai1α and Orai1β are interchangeable, as their expression in Orai1-deficient primary mouse T cells induces identical cytokine responses and transcriptional programmes, and that individuals bearing frameshift mutations causing a loss of the Orai1α isoform do not exhibit immune, muscular, or dermatologic features and have preserved T cells' Ca2+ and transcriptional responses.

Strengths:

The data are of high quality, relying on clean cell line and mouse knockout models to link calcium entry rates directly to NFAT activation, and human data from homozygous and heterozygous frameshift mutation carriers confirm that Orai1α and Orai1β are functionally redundant in vivo.

Weaknesses:

An acknowledged limitation is that some conclusions depend on transient overexpression experiments. The authors should explicitly address whether Orai1α could possess non-redundant functions under unique physiological environments not captured by these assays.

Reviewer #2 (Public review):

Summary:

The authors aim to assess the differential role of Orai isoforms for mediating SOCE and NFAT1 and/or NFAT4 nuclear translocation primarily in the context of T cells. For this purpose, they have used genetically modified cells and appropriate human genetic conditions. All isoforms were expressed individually (with deletions of other isoforms) at roughly equivalent levels so that data across isoforms could be compared unambiguously. Their experiments convincingly identify Orai1 as the main driver of SOCE and NFAT1/4 translocation in the HEK293 cell line and in primary T cells. The two Orai1 isoforms appear functionally equivalent, and loss of the longer isoform (Orai1α) is compensated in humans by the presence of the shorter isoform (Orai1β).

Strengths:

Overall, their judicious use of appropriate knockouts, mutants and expression constructs allows for unambiguous interpretation of data regarding the key role of Orai1α and Orai1β in T cell physiology. They also demonstrate a role for Orai3 in driving SOCE in a breast cancer cell line.

Weaknesses:

The main shortcoming of this manuscript is its inability to place the findings in a context that would be of interest to a broader audience. It would be helpful to provide a metanalysis from existing public databases (human and/or murine) of the known expression in various cell types and tissues of Orai1α, Orai1β, Orai2 and Orai3. This could suggest possible roles for each Orai isoform in tissues other than immune cells. Further, the physiological relevance of the two NFAT isoforms studied, NFAT1 and NFAT4, needs some elaboration, both in the context of T cells and other tissues.

Reviewer #3 (Public review):

Summary:

The work by Abdelnaby et al. investigated the different Orai isoforms, and also especially Orai1 alpha and beta for NFAT 1 and NFAT 4 translocation, but also its impact in genetically modified T-cells. This is a very well-performed work that manages to monitor NFAT nuclear/cytosol ratio even in a time-dependent manner. Overall, this study has been very well performed, is clearly written and discusses the literature very accurately.

Strengths:

For monitoring NFAT translocation, the authors have managed to observe this pattern even in a time-dependent manner. The authors may like to describe how this analysis was done with more details, as I believe some software assistance was needed to distinguish the nuclear to cytosolic region for a huge number of cells. Clearly, this analysis provides a very clear picture of the time course of translocation and allows for a precise statistical analysis.

Remarkably, NFAT1 and NFAT4 translocation was not substantially different for Orai1 alpha- and Orai1 beta-mediated Ca2+ signals. This has also been observed previously by Zhang et al. for NFAT1 from the same laboratory. Clearly, this is in contrast to the work of reference 38 (Kar et al.). All these publications have been performed with a huge amount of data, and I believe that clearly stating this difference in results from previous work is an additional important aspect of this manuscript.

In line with the similar NFAT translocation efficiency of Orai1 alpha- or beta-containing cells, the authors carefully evaluated differentially expressed genes in CD4+ T cells. They did not find a substantial difference in these cells that were transduced with Orai1 alpha or beta - clear orthogonal evidence for a conserved function of Orai1 alpha and beta for transcriptional activation.

Weaknesses:

This reviewer identified no obvious weaknesses in this work.

Reviewer #4 (Public review):

Summary

Orai1, Orai2 and Orai3 are the pore-forming subunits of CRAC channels, and Orai1 itself exists as two N-terminally distinct translational isoforms, Orai1α and Orai1β. Despite well-documented differences in the biophysical properties of these four proteins (Ca²⁺-dependent inactivation, expression pattern, evolutionary conservation), it has been unclear whether these differences translate into distinct transcriptional outputs through the Ca²⁺-calcineurin-NFAT axis, or whether NFAT responses are simply scaled by the amount of Ca²⁺ that each channel variant lets through. The authors address this using HEK293 cell lines engineered by CRISPR/Cas9 to retain only a single native Orai homologue (OraiDKO lines) or none at all (Orai-TKO, reconstituted at near-native levels with a weak TK promoter), and extend their findings to primary murine CD4⁺ T cells lacking endogenous Orai1, as well as to human primary T cells and population genetic datasets from individuals carrying naturally occurring loss-offunction alleles that selectively eliminate Orai1α while sparing Orai1β. Across these systems, the authors report a consistent rank order of NFAT1/NFAT4 activation (Orai1β {greater than or equal to} Orai1α >> Orai2 > Orai3) that mirrors the rank order of SOCE amplitude, and show that a fast Ca²⁺ chelator (BAPTA) but not a slow one (EGTA) blocks NFAT1 activation, consistent with a requirement for local, channel-proximal Ca²⁺ signals rather than global cytosolic Ca²⁺ elevation. Human individuals homozygous for Orai1αselective null alleles are clinically unaffected and show normal or even enhanced SOCE/NFAT responses, contrasting sharply with the severe CRAC channelopathy phenotype produced by mutations affecting residues shared by both isoforms. Together, the data support a model in which the graded strength of SOCE, rather than isoform-specific coupling machinery, is the principal determinant of NFAT activation and downstream gene expression.

Strengths

The central strength of this study is its genetic strategy. Rather than relying on overexpression of individual Orai/STIM constructs in a background where multiple endogenous Orai paralogues are still present (a common confound in this field), the authors generated HEK293 clones that retain only one native Orai isoform, and separately reconstituted Orai-TKO cells with individual isoforms at nearendogenous expression levels using a weak TK promoter, validated by immunofluorescence and Western blotting. This design substantially reduces the risk that observed differences reflect artifacts of Orai/STIM stoichiometry rather than genuine isoform-intrinsic properties, a concern the authors explicitly raise and address (citing prior work showing that Orai/STIM overexpression itself can alter regulator sensitivity).

The multi-tier validation strategy is unusually thorough for this type of mechanistic question. The same qualitative conclusion, that NFAT activation scales with SOCE magnitude rather than isoform identity, is supported independently by (i) engineered HEK293 lines under both maximal (thapsigargin) and physiological (carbachol) stimulation, (ii) an orthogonal cellular system (MCF7 breast cancer cells natively dominated by Orai3) in which a "weak" channel is shown to support robust NFAT1 activation when sufficiently abundant, (iii) reconstitution of Orai1-deficient primary murine CD4⁺ T cells with matched, near endogenous levels of Orai1α or Orai1β, assessed by SOCE, endogenous NFAT1 localization (ImageStream), cytokine production, and genome-wide RNA-seq, and (iv) human population genetics across four independent cohorts (UK Biobank, gnomAD, Qatar Biobank, All of Us) combined with direct cellular phenotyping of human T cells from individuals with defined Orai1α genotypes. The convergence of cell line, mouse, and human genetic data on a single coherent model considerably strengthens confidence in the conclusions beyond what any single approach could provide.

The chelator experiment (Figure 4) is a clean, well-controlled test of local versus global Ca²⁺ signaling, using the classical BAPTA/EGTA differential kinetic-buffering logic, and the result (BAPTA-sensitive, EGTA-insensitive NFAT1 activation for both isoforms) is internally consistent and clearly presented.

The RNA-seq analysis (Figure 6) is a valuable addition, showing near-identical global transcriptional programs driven by Orai1α and Orai1β (log2 fold-change correlation R ≈ 0.92) and demonstrating that the larger number of nominal differentially expressed genes for Orai1α is attributable to statistical thresholding rather than a qualitatively distinct transcriptional signature. This substantially strengthens the claim that isoform identity has little independent influence on the transcriptional output of SOCE beyond its effect on Ca²⁺ influx magnitude.

Finally, the human genetic component is a genuine strength that elevates the physiological relevance of the paper considerably. Directly testing individuals who are natural "knockouts" for one Orai1 isoform but not the other is a powerful complement to the reductionist cell biology, and the finding that Orai1αnull carriers are clinically well and immunologically not compromised (indeed showing modestly enhanced SOCE/NFAT signaling) provides an unusually direct refutation of a specific published mechanistic model (the AKAP79-Orai1α N-terminus coupling hypothesis).

Weaknesses

Some tension remains between the HEK293/human T cell data, where Orai1β is moderately but consistently more efficient than Orai1α at driving SOCE and NFAT activation, and the murine primary CD4⁺ T cell data, where the two isoforms are functionally indistinguishable. The authors offer a plausible explanation, that retroviral expression levels in T cells were high enough to mask subtle isoform differences, and they partially address this by gating on Amtlow (lower-expressing) cells.

The TK-promoter reconstitution system, while a clear improvement over CMV-driven overexpression and validated as achieving comparable expression across isoforms, still involves ectopic expression in an Orai-null background rather than truly endogenous expression from the native locus. Describing this system as achieving "near-native" levels is reasonable given the Western blot validation shown in the supplement, but readers would benefit from the manuscript being explicit that this is a reconstitution model rather than unperturbed endogenous expression, and from a brief discussion of what residual differences (in, for example, membrane trafficking, promoter-driven transcript stability, or subtle stoichiometric mismatch with STIM) could still confound isoform comparisons.

Physiological cells normally co-express Orai1α and Orai1β at varying ratios (as the authors themselves show in Figure S2 across T helper subsets), and it is not established whether heteromeric Orai1α/Orai1β channels (which have been reported by others to form) behave as a simple functional average of the two homomeric channels or display emergent properties. The current study's conclusions rest entirely on isoform-pure systems, and this leaves open whether the "SOCE magnitude" model generalizes cleanly to the mixed populations of channels present in most native cells.

Several of the human genetic subgroup analyses involve modest sample sizes. The number of homozygous carriers of the two Orai1α-specific truncating variants varies substantially by cohort (for example, only 2 and 0 homozygotes identified in QBB), and the UK Biobank clinical phenotype comparison is based on 24 mutation carriers versus 984 controls. While the overall pattern (absence of enrichment for CRAC channelopathy associated diagnostic codes) is reassuring and consistent across the core infection, ectodermal, and myopathy categories, the confidence intervals in these comparisons are necessarily wide, and the absence of statistically significant differences in a modestly sized cohort should be interpreted as consistent with, rather than definitive proof of, phenotypic equivalence. Likewise, the direct cellular characterization of NFAT translocation and SOCE in human T cells from wild-type, heterozygous, and homozygous Orai1α-null Qatar Genome Project donors (Figure 8) is based on a single individual per genotype, which, although understandable given the rarity of the relevant genotype, limits the ability to distinguish genotype effects from inter-individual variability.

The manuscript proposes that NFAT activation is governed principally by the magnitude of local, channel-proximal Ca²⁺ signals rather than isoform-specific decoding machinery, but does not fully address whether isoform-specific differences in Ca²⁺-dependent inactivation kinetics (which shape the temporal profile, not just the amplitude, of the Ca²⁺ signal) could themselves constitute a form of isoform-specific "coding" that is conceptually distinct from, but difficult to fully disentangle from, simple amplitude scaling. A brief discussion of how the SOCE magnitude and Ca²⁺ signal kinetics/duration are related, and whether they can be cleanly separated in this experimental system, would help readers judge the boundaries of the magnitude-based model.

To better parallel the first half of this sentence, this might be framed as Oak "growing slower" than Maple after it gets 75 units wide. The way you said it is still true, but since you started the sentence by saying when Oak "grows faster", saying specifically when that changes makes it clearer what point you are trying to make.

This paragraph overall is a great interpretation 👍

這邊不太理解，我可以理解一個字的大小至少要可以寫得下 c 乘上 log n也就是說至少這個字可以表示出它的 index我這樣理解沒錯吧，可是這一段話我不理解的地方就是說我知道要設定一個上限，不能允許超能力，但是就算把一百萬個數塞進一個超寬的字裡面 那要如何用一條指令，直接對這個字操作，然後這樣子會等於一部數據，所以我不理解的是就算我把很多個數塞進一個超寬的字裡面那我的想像中也是需要經過很多個 instruction 才可以處理全部的數據為什麼這邊可以說一條指令，就可以對一個字操作，然後處理全部數據 ?

可是這邊我就看不太懂，因為我收到的資訊是矛盾的，比如說你前面說常見的64位機器，一個字是64，64個 bit，但是你下面又說 CLRS 規定，一個字是這樣子，那到底是，到底是怎麼樣子

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@juanesco ¿te llega este mensaje?

加点亲密戏，女主一进来就被男主按在门前抱着

19集和20集有些冗长，最好在不改变情节的情况下缩减一些，然后注意可以增加一些吸血鬼被抽血挣扎，那种恐怖实验室的画面，来减缓走剧情带来的无趣

这个时候男主没有什么吸血鬼的特征，最好换上现代一点的衣服很帅的那种

这段要写一个比较激烈的打斗戏，不然画面很平庸

女主前面应该是比较不愿意的，但是在男主的诱惑下才扑上去

Di sini sudah oke, teruskan kerja kalian

Bagian ini di berikan catatan lebih baik ya

I feel like this entire paragraph can relate to some of the topics we touched in our reflection posts from last Thursday. It also ties in with the perspective of the eldest daughter staying home and eventually becoming a caregiver. The traditionalist policies that pressure women to adopt these roles are also meant to keep them there. By staying home as a caregiver there is less of a chance that a woman will be able to have financial liberty and that it will become more difficult to leave if the she chooses to.

One of my favorite university lectures was on the topic of banned/challenged books. We had to theorize why certain books were banned/challenged and then we'd look up the real reason it was banned. It will never cease to amazing the amount of books that were banned/challenged due to lgbtq+ content. Furthermore, we're seeing more protest and outrage over events like drag story hour. Events like this are means to create inclusive spaces for all ages. But instead they are being twisted into something they're not through the narrative that queer rights are dangerous to children.

Although this is a very disappointing and disheartening statistic, I was not surprised to read it. These past few years, a clear shift backwards in equality for all minorities, not just in regards to gender equality, has been felt everywhere from worldwide politics to representation on our TV screens. What strikes me about this is when people often say there is nothing left for feminists to fight for as women already have everything they could want (for example, the right to work, vote, etc.). However, even before this cultural shift from the last decade or so, this statement was never true, as it completely disregarded women of colour from all around the world and the struggles they continue to face that white women do not have to worry about. This is why intersectionality is important, as some people's perceptions of feminism often only includes white women. The current regression in feminism also proves why this statement of gender inequality being solved is not and should not ever be true; once people start thinking the problem is solved, nothing will be done to continue to fix it. Nobody will care anymore, and slowly, the progress will be undone, as we have seen and will continue to see. Many people believe women are only discriminated against in countries that are called "third-world" countries, which again brings up the conversation of intersectionality and racism as the patriarchy clearly still exists in the so-called "first-world, progressive" countries. The rights to abortion are slowly being taken away from many countries, including America, and domestic abuse and rape cases are often brushed under the rug, even allowing male celebrities who are known abusers and rapists to continue to walk around in the public spotlight and live their careers as if nothing has happened. I really wanted to highlight this line as it emphasises just how serious the setbacks on gender equality have been (and continue to be), and also the importance of intersectionality, as feminism means making sure no woman is free until every woman is free.

The article mentions the importance of intersectionality and I think this point does a great job at representing how different axes of power often come together to put down people of intersecting identities. The (most often) white UK citizens have recently been rallying against immigration because they believe the (notably non-white) migrants have been solely responsible for the rise of crime rates, including rape. They frame it as wanting to protect the women and children of their country, just like the article states. However, these are often the same people (the right wing population) who vote against women's rights to abortion. It raises the following question: if they truly cared about the women and children of their country, why would they willingly put these same women through unwanted experiences, resulting in children that would grow up in households where they are unwanted, especially if the child is a product of rape? It can foster in an unhealthy and deeply mentally damaging environment for all involved. This proves that their issue is not with the lack of protection over the women and children of the country, but actually with the rise of Black and people of colour immigrating there (including women and children), resulting in both the upholding of patriarchal ideals and racist rhetoric as they pin all the blame on people of colour while simultaneously contributing to the control held over women's bodies.

I truly commend the bravery of the many people around the world who aide in the needs of their community through the means of health clinics, hotlines, shelters and awareness campaigns. I know The Satanic Temple has been one NGO that is very vocal about women's right to healthcare with the USA. Under the fundamentalist tenet "III. Bodily Autonomy: One’s body is inviolable, subject to one’s own will alone." the group argues that their pseudo-religion gives everyone choice and that this must be held in respect to the USA's first amendment the right to hold religious belief and to practice that faith without government interference or establishment. I really appreciate the clever work around/ legal loophole this collective found as a non-theistic organization registered as a religion. I question how much longer The Satanic Temple will exist within the USA if this article states that many groups similar in mission are being defunded or criminalized in other places.

This sentence represents what I believe is a major part as to why it feels as though our society has moved backwards in the last few years in regards to the patriarchy and womens roles. Social media makes up a lot of todays culture all around the world, and with the rise of children gaining full access to social media at such young ages, it has acquired a huge hand in how peoples mindsets are developed both in relation to themselves and everyone around them. This sentence of the article delves into that idea and how both men and women online are influencing impressionable audiences into falling for patriarchal roles. In my opinion, this is also partially due to many people online being incapable of thinking deeper for themselves and instead taking everything at face value, telling themselves and everyone around them that adult men convincing younger men and boys into adopting misogynistic behaviours and women promoting tradwife lifestyles is not that serious and "just a joke". This connects to another sentence earlier in the introduction of the article stating how anti-gender actions can quickly go from "meme to ministry". Things that are said to be "just a joke" are never truly just jokes; they have real impacts on our society and affect peoples mindsets, which in turn results in more patriarchal rules taking over on higher political levels. This aversion to thinking deeper about misogyny and the patriarchy and how we see it in our every day lives comes from people not wanting to leave their comfort zone and admitting that it is, in fact, affecting their every day lives; for example, going online and seeing men make comments about how showing emotions is lame, and women being unable to admit many of the things they do to "maintain" their body (shaving, feeling the need to wear makeup every day, etc.) stems from patriarchal ideals. This has led us to where we are at a cultural level today.

I think something interesting to bring to this conversation is the idea of church vs state. While religion does play a very important part in many peoples lives, I don't think that it should have a place in legislature. Especially in North America where our populations are so diverse there is no 'one religion.' But, regardless, our governing bodies have intergrated the Catholic/Christian value system within our governance., leaving no option for autonomy.

The far right often disguises their need for control and power over women and their bodies as a concern for a fetus. In reality, these people only care until the child is born, then, they are someone else's problem, as they can no longer justify exercising their power by giving a "voice to the voiceless." After the child is born they speak about the supposed resources that are available yet, often can't proceed to name a single one.

remove costs

Case#: patient, 12, Somali, onset 5yo

DiseaseAssertion: STGD

FamilyInfo: parents and 5 siblings did not report visual issues

CasePresentingHPOs: HP:0000007, HP:0011504, HP:0000608

CaseHPOFreeText: BCVA 20/135 OD; 20/100 OS. Red-green color deficit. Bull's eye maculopathy, but no pallor of optic disc. Normal peripheral retina. Loss of macular photoreceptor layer; severely reduced cone function.

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method: whole exome sequencing

PreviouslyPublished: n/a

Variant: NM_000350.3:c.5882G>A p.(Gly1961Glu) ; NM_000350.3:c.634C>T p.(Arg212Cys)

ClinVar: 7888; 7898

CAID: n/a

SupplementalData: n/a

Per ClinVar entry, this variant was associated with this paper. however, after reading through the genotypes, this variant was not found. Likely this paper was mentioned to support the statement that "Loss-of-function variants in ABCA4 are known to be pathogenic"

PMID: 29765157

Gene: ABCA4

HGNC ID: 34

Case#: Braun Family 8 Proband (from left to right, top to bottom of available pedigrees), female

DiseaseAssertion: Stargardt

FamilyInfo: Unaffected carrier parents. c.5196+1137G>A maternally inherited; c.4577C>T (p.T1526M) paternally inherited

CasePresentingHPOs:

CaseHPOFreeText: "five or more of the following features of ABCA4-associated retinal disease: decreased visual acuity before age 20, decreased visual acuity as the first visual symptom, symmetrical fundus findings, pisciform flecks, beaten metal macular atrophy, bulls-eye maculopathy, peripapillary sparing, vermillion fundus, masked choroid on fluorescein angiography, nummular pigment overlying extensive macular atrophy, central outer retinal atrophy on optical coherence tomography and central scotomas on Goldmann perimetry."

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: one plausible disease-causing mutation detected in ABCA4 after assessing the entire coding sequence and canonical retinal splice junctions with automated bidirectional Sanger sequencing using an ABI 3730 sequencer

PreviouslyPublished: n/a

Variant: c.5196+1137G>A; c.4577C>T (p.T1526M)

ClinVar: 438100

CAID: CA26843511

SupplementalData: pedigree in fig s2

PMID: 29706639

Gene: ABCA4

HGNCID: HGNC:34

MonDO: MONDO:0019353

Bioinformatic analyses of an SQL-based database containing 12272 variants that appear in 178 IRD genes in 5706 individuals of Ashkenazi Jewish origin based on the gnomAD database (version 2) and variants that were published in the scientific literature that was extracted from HGMD. Authors extracted information regarding IRD variants from various sources (including data of 5706 Ashkenazi Jewish (AJ) samples and a large cohort of Israeli patients with IRDs) to estimate carrier frequency of IRD mutations in different subpopulations in Israel. Two major databases aiming to estimate carrier frequency of IRD mutations in the Israeli population (Fig. 1): “gnomAD-AJ-IRD DB” containing data of 5706 AJ controls extracted from gnomAD and “HW-IRD DB” containing data extracted from our cohort of Israeli patients with IRDs.

See Fig 2 for breakdown of variants analyzed.

The final DB (IRDB) (Fig. 1 and Table S7) includes all 399 variants from “gnomAD-AJ-IRD DB” and “HW-IRD DB” that were considered here as pathogenic mutations in 111 known IRD genes.

To establish the “HW-IRD DB” (Fig. 1), we collected data on Israeli IRD patients with a known cause of disease (a cohort of >2000 IRD families). The HW-IRD DB includes 289 pathogenic mutations (Fig. 1) that were identified in IRD patients who have biallelic variants.

SupplementalData: S7, carrier frequency data for each mutation in all nine studied subpopulations. Carrier frequency was calculated as 2pq where p = 1 − q and q was calculated as the root square of the number of homozygous patients plus half the number of compound heterozygous patients divided by the population size

Variant: NM_000350.2:c.4895dup,p.Asn1632fs

CAID: CA915941330

Case: Ashkenazi Jewish patient with inherited retinal disease, STGD, CRD

CasePresentingHPOs: HP:0000548 (Cone/cone-rod dystrophy, CRD)

CaseHPOFreeText: Stargardt disease (STGD)

Case#: Family MD-0242 Proband, 18yo at onset, Spanish

DiseaseAssertion: AR Stargardt

FamilyInfo: n/a

CasePresentingHPOs:

CaseHPOFreeText: STGD diagnosed based on "bilateral central vision loss; fundus presenting with a beaten-bronze appearance and/or the presence of orange-yellow flecks in the retina from the posterior pole to the mid-periphery; fluorescein angiography showing typical dark choroid; and normal to subnormal electroretinogram (ERGs)."

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: Haplotype analysis, ABCR400 microarray, direct sequencing for confirmation

PreviouslyPublished: n/a

Variant: c.1715G>C p.Arg572Pro

ClinVar: 99073

CAID: CA226919

SupplementalData:

This paper is not publicly available. Requested from library since it is said to contain this variant.

This variant is in table 3 as a "potentially neutral polymorphism or change of unknown significance" but it is unclear which patient this is associated with, what their phenotype is, what their genotype is

Case#: Patient #15, Moorfields Eye Hospital, London, UK, female, 39yo at onset, 51yo at report,

DiseaseAssertion: ABCA4-Associated Retinopathy, clinical diagnosis of STG

FamilyInfo: no additional family-member WGS data available

CasePresentingHPOs:

CaseHPOFreeText: BCVA= OD: 6/9, OS: 6/9, Fishmann Classification=2 (fleck-like lesions anterior to the vascular arcades and/or nasal to the optic disc), early changes to foveal photoreceptors, Extent of FAF abnormalities with Regard to Vascular Arcades: beyond. ffERG Group: 1(normal). PERG: Abnormal. FAF in Fig. 1B. characteristic yellow-white pisciform flecks in the RPE of the posterior pole that were hyperautofluorescent on FAF imaging or progressive atrophy of the macular RPE

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: Haplotype analysis, ABCA4 mutation screening was performed by next-generation sequencing, sequenced as part of the retinal panel at the Molecular Vision Lab

PreviouslyPublished:

Variant: c.5196+1137G>A p.[=,M1733Efs∗78] c.[1715G>A;2588G>C] p.[(R572Q;G863A,G863del]

ClinVar: 7900

CAID: CA226918

SupplementalData:

This paper is listed under the ClinVar citations for this variant, but only as a paper that references other variants in the initiator codon that have been observed in individuals with ABCA4-related conditions

Case#: Case 3, male, onset at 24yo, Italy

DiseaseAssertion: STGD1

FamilyInfo: third-degree consanguineous parents; both parents healthy heterozygous carriers of the ABCA4 variant

CasePresentingHPOs: HP:0000529, HP:0007754, HP:0000518

CaseHPOFreeText: progressive deterioration of vision at age 24. Bilateral macular dystrophy and diffuse lens opacities on ophthalmologic examination. OCT, ERG, and VEP consistent with Stargardt maculopathy.

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method: NGS (custom enrichment panel), Illumina NextSeq550; variant confirmed by Sanger sequencing.

PreviouslyPublished: n/a

Variant: NM_000350.3(ABCA4):c.2828G>A (p.Arg943Gln), homozygous; rs1801581

ClinVar: Variation ID: 7913

CAID: n/a

SupplementalData: n/a

PMID: 34008801

Gene: ABCA4

HGNC ID: 34

Case#: DNAID 072884/Pat255, female

DiseaseAssertion: STGD1

FamilyInfo: n/a

CasePresentingHPOs:

CaseHPOFreeText: "In classic STGD1, loss of central vision starts around the second decade of life, but both early- and late-onset subtypes have been extensively described." No patient-specific details provided

CaseNotHPOs:

CaseNotHPOFreeText:

GenotypingMethod: smMIPs sequencing of the complete ABCA4 locus

PreviouslyPublished: n/a

Variant: c.1519G>T (p.Asp507Tyr); c.5312+3A>T (p.Asn1734Glyfs*14) phase not confirmed

CAID: CA958508

SupplementalData: tables s9 and s11

PMID: 22334370

Gene: ABCA4

HGNC ID: 34

Disease: Retinitis Pigmentosa

Case#: a 29-year-old man

DiseaseAssertion: Stargardt Disease

FamilyInfo: NR

CasePresentingHPOs: HP:0007663

CaseHPOFreeText: 20/70 visual acuity in both eyes

CaseNotHPOs: NR

CaseNotHPOFreeText: NR

Genotyping Method: ABCA4 microarray (ABCR5000 chip)

PreviouslyPublished: NR

Variant: NM_000350.3:c.5882G>A, p.G1961E and c.5018+2C>T (rare splice variant)

ClinVar: 7888, NR

CAID: CA119132, NR

SupplementalData: No CAID or ClinVar ID were found for the rare splice variant c.5018+2C>T

This variant is listed in supplementary tables S2 and S3. Proband TI-50 is a "solved" patient, meaning the phenotype matches the genotype. Homozygous female with MD/CORD- all that is provided.

ABCA4 expression in HEK293 cells showed equivalent expression of wild type and this variant, indicating that this variant does not impact protein function (BS3_Supporting; PMIDs).

Case#: Patient#010382, Chinese, male, 29yo at onset

DiseaseAssertion: stargardt

FamilyInfo: n/a

CasePresentingHPOs: STGD1 diagnosis based on the following criteria: "a bilateral central vision defect; fundus displaying a beaten-bronze appearance and/or orange-yellow flecks in the retina from the macula to the midperiphery; fluorescein angiography presenting with a typical dark choroid; and normal to subnormal ERG results." BCVA=0.4/ 0.6

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

PreviouslyPublished: n/a

Variant: p.P2097S; c.6050G>A p.(Cys2017Tyr) phase unknown

ClinVar: 2202780;

CAID: CA341277622;

SupplementalData: supplementary table S4 has phenotype information

PMID: 28118664

Gene: ABCA4

HGNC ID: 34

Text is a PDF, but this paper is the previously published paper referenced in PMID: 9973280. Pedigree is found here

these options should be different based ont he esp and how things are paid /scale f paid, or plans etc. we havethis info

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Ecology.<br /> See the peer reviews and the recommendation.

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Ecology.<br /> See the peer reviews and the recommendation.

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wild to me that a father being a father is considered "child care" separate from a mother caring for her child. This is not an "arrangement", it is parenthood.

I am glad she finally stood up for her self.

why would she not tell him?

I like how he talks about how his mom was loyal

I like his use of a very common analogy to show how even through the apartheid was there.

I wonder the gun rules like is it different In every country.

It is cool to se how we have similar cultural events happening on different sides of the world.

I feel like every country should see the importance of other history of other countries.

I feel like he should have just gave up and excepted his lose and told her he was going to be late

its cool to see how there style was back then and in a different country.

Wish we got some more characterization of Tom other than his ability to persuade people and he hustle.

I like his use of a very common analogy to show How even though apartheid was abolished. There was still lingering oppression and still systemic issues that played their community.

I wonder what their gun regulations are like And how they compare to the US

It’s cool to see you similar cultural events happening on different side of the planet and how they interact/affect each other. Is it just by chance this cultural shift about where you’re from happen to be influential when an apartheid it was shutting down or was there an External event that influenced Both?

10.1016/j.omtn.2026.102989

Hi all, please note that this article has been revised and published in Neurobiology of Learning and Memory. Published version DOI: 10.1016/j.nlm.2026/108208 Published version link: https://www.sciencedirect.com/science/article/pii/S1074742726000791

Hi all, please note that this article has been revised and published in Molecular Therapy Nucleic Acids. Published version DOI: 10.1016/j.omtn.2026.102989 Published version link: https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(26)00160-5

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Here it is again. Seek help as needed. What a fantastic resource. This has been my big take away. I did not realize we had access to so many resources until today.

I suspect Getting Help is what I need to grasp. There weren't many resources back when I was in college. Technology help will likely be a weekly occurrence. But how fantastic that students have so many resources to help them succeed. I took the time to find those numbers to call for help and wrote them down.

This stood out because it is so easy to get caught up in deadlines and assignments. I am overwhelmed by the technology requirements. I lost an hour just trying to figure out how to download Microsoft and textbooks. My daughter said, Mom, just go outside for a minute and walk through your gardens. It will help you calm down. It did help. This is important and easy to overlook, especially at my age and having spent most of my life putting myself last. Life is so precious and I don't want to spend it overwhelmed. But I really want to enjoy the education process too. So, this really stood out to me. It was an excellent reminder I wish I'd seen earlier.

understanding history can help people make better decisions in the present and future.

1

Iago's private speech reveals the major strategy he plans to set into motion against Othello, involving confusing Othello until he second guesses what his own eyes are telling him. He plans to avoid accusing Des. since that would be obvious. This ability to make characters interpret the things that happen in front of them incorrectly is way to powerful and in the wrong hands, Iago is incredibly dangerous. - ChatGPT

When Othello arrives and openly expresses his happiness at seeing Desdemona, the relationship appears stronger than ever. This makes Iago’s later manipulation more significant. The audience can see that Othello has genuine trust in Desdemona, meaning Iago will have to gradually replace that trust with doubt rather than simply create suspicion from nothing. - ChatGPT

Iaog's jokes about Cassio's behavior begin turning ordinary actions into possible evidence of romantic interest. This is one of the first clear examples of how he plans to create jealousy, he doesn't even need to start any drama. Instead, he can make innocent events suspicious by managing how Othello interprets them. He is a force to be reconed with.

Iago's conversation here with Desdemona shows just how easily he can hide his personality. He speaks cleverly to entertain her while still making insulting observations about women. A director could have Iago become more animated around Des. to show how much he enjoys manipulating people without making his intentions super obvious to the characters.

Iago jokes about women in a way that kind of seems harmless on the surface but also reveals his cynical view on relationships. His comments are important because the audience has already heard his secret plans. What sounds like him cracking jokes to the other characters is really much more serious to the audience, which is really cool to think about.

Desdemona's arrival gives the scene a much lighter feeling than the opening. She speaks nonchalantly and appears genuinely excited to see Othello again. In an adaptation, her energy could be played up as a sharp contrast to Iago's darker attitude, making it super easy for the audience to recognize the difference between Desdemona's openness and Iago's secret intentions.

When the characters learn that the Turkish fleet has been destroyed by the storm, the immediate military threat disappears, but Shakespear does an amazing job at keeping the scene active. He simply shifts the characters attention from their enemy to the personal conflict between Othello, Desdemona, and Iago. - Copilot

Montano's uncertainty about the Turkish fleet shows just how little control the characters have over their situation. Even thought the character believe they are preparing for war, the storm disrupted their plans, which creates an interesting contrast with Othello, who has been extremely confident and in control throughout act 1.

Shakespeare opens the scene with a storm at sea, immediatly creating a sense of danger and uncertainty. A production could emphasize the chaos through loud wind and waves before the characters appear. This also connecters to the larger danger that was established in act 1, where Othello's marriage is already threatened by outside forces.

I have never thought to do this but I think it would be helpful to be able to focus in on the specific information you are trying to edit. Otherwise, it can get scrambled together.

This is super helpful advice because the longer time you give your writing before you revisit it helps give you a fresh set of eyes to reread and see things you may have missed. When I don't give myself enough time, I am still in the same head space, but time helps to point out issues and places for improvement.

I think this is specifically important for a peer review because when someone you trust to give you criticism, they can be honest and give necessary feedback without any hurt feelings as you know it is only to better your work. It is extremely helpful getting second opinions.

Sometimes especially for me when writing, I can forget about the coherence of it. As I write so many research papers, there is a lack of coherence needed as it is strictly informational, but it is important to make sure it reads smoothly and the information flows well.

The author describes waiting for Dr. H. while preparing to leave. She expects the doctor to arrive too late, but instead he arrives just when she needs him. This creates a sense of irony, because the she expects disappointment but receives exactly the help she was hoping for. I find it interesting that she points out that she is usually disappointed and that doctors tend to not show up when they are supposed to, where as the nurse is doing everything. She also mentions that the doctor looked at her credentials, which i think points to a larger message of how people feel and treated people under them or if they were "worthy" of doing something.

outside evidence/research

personal experience

personal experience

personal experience

personal experience

personal experience

interview/converstation with another person

personal experience

Interview/conversation with another person

Interview/conversation with another person

Interview/conversation with other person

other persons experience

outside research

personal experience

personal experience

personal experience

outside research/evidence

personal experience

personal experience

Personal experience

Personal experience

I appreciate how the writer explains her choices to challenge the norms because this is how you write something new, with new ideas and stories that occur but aren't commonly discussed. It seems refreshing.

I think this is super interesting as most movies I can think of, the happy ending a woman is given is to become a wife and mother and the other ending is usually death. I think it says a lot as a society that across cultures and over the years, this is still the only choice.

Suppose there is no such partition. Then, (1) \(\nexists X_i \)such that \(X_i \cup C_i \subset H_j\). However, (1) contradicts property 2 of the separator. Note that vertices can belong to both H_1 and H_2, but that doesn't mean that they have to belong to A_j.

more interest should be taken in who owns the land, how is it maintained

one way is all graduating urban planners must have a certain amoutn of knowledge of EJ, fieldwork of EJ, and prove X amoutn of time working with EJ post graduation

I think there is no "overarching narrative" of progress because the human mind is so complex and so is mental illness. We have made discoveries of reliable therapies and improved our knowledge of how the mind works. Obviously, this guy is trying to sell his book and his criticisms are valid, but I feel like he's completely ignoring a lot of the positive strides we've made.