Its so true!

nice wording!

Will do

Brauchen wir hier alle Absprünge im Header oder kann man das reduzieren? Hat man sein Cobranding dann je nach Nutzer:in?

Progressbar ist sehr nah an Scrollbar. könnte man das nach links stellen oder macht das probleme?

Kommt hieri m allgemeinen noch ein Anreiz für das Gewinsspiel in form von Grafik Bild etc? was passiert hier dann?

Hier ist gemischt gut. aber das Pulsieren mit em Schlagschatten finde ich etwas heftig

Auch hier ist der Efffekt wieder ein andrer als auf den ersten slides. einheitlich?

Typofarbe ändert sich wegen HON Lounges? Sieht ein bisschen nach Fehler aus. auch wenns nur detail ist. Würde wenn dann etwas deutlicher einfärben. hintergrund?

HON Lounges und surfer passen finde ich nicht. kommt aber besimmt noch ein bild

Ich finde im ersten bild nach dem Begriff Community braucht es etwas was mehr community sagt. Das ist jetzt das selbe inhatllich wie im Abschnitt "reisen wir zurück"

Ich finds ansich nicht verkehrt das so zu machen in diesem Fall. Können wir es noch deutlicher zu einem einzelfall machen und es sich etwas abheben lassen?

Versal zu gemsicht

Könnten wir ghenerell die Bilder tweaken, dass sie dezente animationen in sich tragen? oder zumindest ganz leichtes aufskalieren? Mit der Scrollbewegung?

CTA Versal sollte eher gemsicht denke ich. Warum erst weiß und dann blau BG Farbe?

Der Typewritereffekt ist anders als der auf den ersten screens. da fadet es eher von unten rein. können wir einheitlicher werden?

Versal eher nur bei den Worten Points und Meilen. Vielleicht aber auch da mal gemischt versuchen

Das läuft noch wie ein nicht gewolltes Element mit. Ist das Teil des Designs?

This equation is not correct since if dy/dx is inserted on the differential equation dy/dx+3y=6x+11 it should be: ((-3e^-2x)+2)+3((e^-3x)+2x+3)=6x+11 Correct?

pricing

Reviewer #3 (Public Review):

Summary:

Day et al. introduced high-throughput expansion microscopy (HiExM), a method facilitating the simultaneous adaptation of expansion microscopy for cells cultured in a 96-well plate format. The distinctive features of this method include 1) the use of a specialized device for delivering a minimal amount (~230 nL) of gel solution to each well of a conventional 96-well plate, and 2) the application of the photochemical initiator, Irgacure 2959, to successfully form and expand the toroidal gel within each well.

Strengths:

This configuration eliminates the need for transferring gels to other dishes or wells, thereby enhancing the throughput and reproducibility of parallel expansion microscopy. This methodological uniqueness indicates the applicability of HiExM in detecting subtle cellular changes on a large scale.

Weaknesses:

To demonstrate the potential utility of HiExM in cell phenotyping, drug studies, and toxicology investigations, the authors treated hiPS-derived cardiomyocytes with a low dose of doxycycline (dox) and quantitatively assessed changes in nuclear morphology. However, this reviewer is not fully convinced of the validity of this specific application. Furthermore, some data about the effect of expansion require reconsideration.

eLife assessment

This important study develops a high throughput version of expansion microscopy that can be performed in 96-well plates. The engineered technology is convincing and compatible with standard microplates and automated microscopes and thus will be of broad interest. However, the application is incomplete and would benefit from additional experiments.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Day et al. present a high-throughput version of expansion microscopy to increase the throughput of this well-established super-resolution imaging technique. Through technical innovations in liquid handling with custom-fabricated tools and modifications to how the expandable hydrogels are polymerized, the authors show robust ~4-fold expansion of cultured cells in 96-well plates. They go on to show that HiExM can be used for applications such as drug screens by testing the effect of doxorubicin on human cardiomyocytes. Interestingly, the effects of this drug on changing DNA organization were only detectable by ExM, demonstrating the utility of HiExM for such studies.

Overall, this is a very well-written manuscript presenting an important technical advance that overcomes a major limitation of ExM - throughput. As a method, HiExM appears extremely useful, and the data generally support the conclusions.

Strengths:

Hi-ExM overcomes a major limitation of ExM by increasing the throughput and reducing the need for manual handling of gels. The authors do an excellent job of explaining each variation introduced to HiExM to make this work and thoroughly characterize the impressive expansion isotropy. The dox experiments are generally well-controlled and the comparison to an alternative stressor (H2O2) significantly strengthens the conclusions.

Weaknesses:

(1) Based on the exceedingly small volume of solution used to form the hydrogel in the well, there may be many unexpanded cells in the well and possibly underneath the expanded hydrogel at the end of this. How would this affect the image acquisition, analysis, and interpretation of HiExM data?

(2) It is unclear why the expansion factor is so variable between plates (e.g., Figure 2H). This should be discussed in more detail.

(3) The authors claim that CF dyes are more resistant to bleaching than other dyes. However, in Figure. S3, it appears that half of the CF dyes tested still show bleaching, and no data is shown supporting the claim that Alexa dyes bleach. It would be helpful to include data supporting the claim that Alexa dyes bleach more than CF dyes and the claim that CF dyes in general are resistant to bleaching should be modified to more accurately reflect the data shown.

(4) Related to the above point, it appears that Figure S11 may be missing the figure legend. This makes it hard to understand how HiExM can use other photo-inducible polymerization methods and dyes other than CF dyes.

(5) The use of automated high-content imaging is impressive. However, it is unclear to me how the increased search space across the extended planar area and focal depths in expanded samples is overcome. It would be helpful to explain this automated imaging strategy in more detail.

(6) The general method of imaging pre- and post-expansion is not entirely clear to me. For example, on page 5 the authors state that pre-expansion imaging was done at the center of each gel. Is pre-expansion imaging done after the initial gel polymerization? If so, this would assume that the gelation itself has no effect on cell size and shape if these gelled but not yet expanded cells are used as the reference for calculating expansion factor and isotropy.

(7) In the dox experiments, are only 4 expanded nuclei analyzed? It is unclear in the Figure 3 legend what the replicates are because for the unexpanded cells, it says the number of nuclei but for expanded it only says n=4. If only 4 nuclei are analyzed, this does not play to the strengths of HiExM by having high throughput.

(8) I am not sure if the analysis of dox-treated cells is accurate for the overall phenotype because only a single slice at the midplane is analyzed. It would be helpful to show, at least in one or two example cases, that this trend of changing edge intensity occurs across the whole 3D nucleus.

(9) It would be helpful to provide an actual benchmark of imaging speed or throughput to support the claims on page 8 that HiExM can be combined with autonomous imaging to capture thousands of cells a day. What is the highest throughput you have achieved so far?

Reviewer #2 (Public Review):

Summary:

In the present work, the authors present an engineering solution to sample preparation in 96-well plates for high-throughput super-resolution microscopy via Expansion Microscopy. This is not a trivial problem, as the well cannot be filled with the gel, which would prohibit the expansion of the gel. A device was engineered that can spot a small droplet of hydrogel solution and keep it in place as it polymerizes. It occupies only a small portion of space at the center of each well, the gel can expand into all directions, and imaging and staining can proceed by liquid handling robots and an automated microscope.

Strengths:

In contrast to Reference 8, the authors' system is compatible with standard 96 well imaging plates for high-throughput automated microscopy and automated liquid handling for most parts of the protocol. They thus provide a clear path towards high-throughput exM and high-throughout super-resolution microscopy, which is a timely and important goal.

Weaknesses:

The assay they chose to demonstrate what high-throughput ExM could be useful for, is not very convincing. But for this reviewer that is not important.

Reviewer #1 (Public Review):

Summary:

Zanetti et al. use biophysical and cellular assays to investigate the interaction of the birnavirus VP3 protein with the early endosome lipid PI3P. The major novel finding is that the association of the VP3 protein with an anionic lipid (PI3P) appears to be important for viral replication, as evidenced through a cellular assay on FFUs.

Strengths:

Supports previously published claims that VP3 may associate with early endosomes and bind to PI3P-containing membranes. The claim that mutating a single residue (R200) critically affects early endosome binding and that the same mutation also inhibits viral replication suggests a very important role for this binding in the viral life cycle.

Weaknesses:

The manuscript is relatively narrowly focused: one bimolecular interaction between a host cell lipid and one protein of an unusual avian virus (VP3-PI3P). Aspects of this interaction have been described previously. Additional data would strengthen claims about the specificity and some technical issues should be addressed. Many of the core claims would benefit from additional experimental support to improve consistency.

eLife assessment

This study presents valuable information on the mechanism of how birnavirus VP3 protein interacts with PI3P in early endosomes. Evidence supporting the proposed two-stage mechanism is incomplete and would benefit from additional supporting experiments, and additional experimentation would also address concerns about data consistency.

Reviewer #2 (Public Review):

Summary:

Birnavirus replication factories form alongside early endosomes (EEs) in the host cell cytoplasm. Previous work from the Delgui lab has shown that the VP3 protein of the birnavirus strain infectious bursal disease virus (IBDV) interacts with phosphatidylinositol-3-phosphate (PI3P) within the EE membrane (Gimenez et al., 2018, 2020). Here, Zanetti et al. extend this previous work by biochemically mapping the specific determinants within IBDV VP3 that are required for PI3P binding in vitro, and they employ in silico simulations to propose a biophysical model for VP3-PI3P interactions.

Strengths:

The manuscript is generally well-written, and much of the data is rigorous and solid. The results provide deep knowledge into how birnaviruses might nucleate factories in association with EEs. The combination of approaches (biochemical, imaging, and computational) employed to investigate VP3-PI3P interactions is deemed a strength.

Weaknesses:

(1) Concerns about the sources, sizes, and amounts of recombinant proteins used for co-flotation: Figures 1A, 1B, 1G, and 4A show the results of co-flotation experiments in which recombinant proteins (control His-FYVE v. either full length or mutant His VP3) were either found to be associated with membranes (top) or non-associated (bottom). However, in some experiments, the total amounts of protein in the top + bottom fractions do not appear to be consistent in control v. experimental conditions. For instance, the Figure 4A western blot of His-2xFYVE following co-flotation with PI3P+ membranes shows almost no detectable protein in either top or bottom fractions. Reading the paper, it was difficult to understand which source of protein was used for each experiment (i.e., E. coli or baculovirus-expressed), and this information is contradicted in several places (see lines 358-359 v. 383-384). Also, both the control protein and the His-VP3-FL proteins show up as several bands in the western blots, but they don't appear to be consistent with the sizes of the proteins stated on lines 383-384. For example, line 383 states that His-VP3-FL is ~43 kDa, but the blots show triplet bands that are all below the 35 kDa marker (Figures 1B and 1G). Mass spectrometry information is shown in the supplemental data (describing the different bands for His-VP3-FL) but this is not mentioned in the actual manuscript, causing confusion. Finally, the results appear to differ throughout the paper (see Figures 1B v. 1G and 1A v. 4A).

(2) Possible "other" effects of the R200D mutation on the VP3 protein. The authors performed mutagenesis to identify which residues within patch 2 on VP3 are important for association with PI3P. They found that a VP3 mutant with an engineered R200D change (i) did not associate with PI3P membranes in co-floatation assays, and (ii) did not co-localize with EE markers in transfected cells. Moreover, this mutation resulted in the loss of IBDV viability in reverse genetics studies. The authors interpret these results to indicate that this residue is important for "mediating VP3-PI3P interaction" (line 211) and that this interaction is essential for viral replication. However, it seems possible that this mutation abrogated other aspects of VP3 function (e.g., dimerization or other protein/RNA interactions) aside from or in addition to PI3P binding. Such possibilities are not mentioned by the authors.

(3) Interpretations from computational simulations. The authors performed computational simulations on the VP3 structure to infer how the protein might interact with membranes. Such computational approaches are powerful hypothesis-generating tools. However, additional biochemical evidence beyond what is presented would be required to support the authors' claims that they "unveiled a two-stage modular mechanism" for VP3-PI3P interactions (see lines 55-59). Moreover, given the biochemical data presented for R200D VP3, it was surprising that the authors did not perform computational simulations on this mutant. The inclusion of such an experiment would help tie together the in vitro and in silico data and strengthen the manuscript.

Reviewer #3 (Public Review):

Summary:

infectious bursal disease virus (IBDV) is a birnavirus and an important avian pathogen. Interestingly, IBDV appears to be a unique dsRNA virus that uses early endosomes for RNA replication that is more common for +ssRNA viruses such as for example SARS-CoV-2.

This work builds on previous studies showing that IBDV VP3 interacts with PIP3 during virus replication. The authors provide further biophysical evidence for the interaction and map the interacting domain on VP3.

Strengths:

Detailed characterization of the interaction between VP3 and PIP3 identified R200D mutation as critical for the interaction. Cryo-EM data show that VP3 leads to membrane deformation.

Weaknesses:

The work does not directly show that the identified R200 residues are directly involved in VP3-early endosome recruitment during infection. The majority of work is done with transfected VP3 protein (or in vitro) and not in virus-infected cells.

Additional controls such as the use of PIP3 antagonizing drugs in infected cells together with a colocalization study of VP3 with early endosomes would strengthen the study.

In addition, it would be advisable to include a control for cryo-EM using liposomes that do not contain PIP3 but are incubated with HIS-VP3-FL. This would allow ruling out any unspecific binding that might not be detected on WB.

The authors also do not propose how their findings could be translated into drug development that could be applied to protect poultry during an outbreak. The title of the manuscript is broad and would improve with rewording so that it captures what the authors achieved.

zero-cost tooling

from - !search.telegram - iroh

replace 'current' to 'actual'

remove 'a'

!search.telegram - iroh

Reviewer #1 (Public Review):

Summary:

This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibits increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:

This report describes a mouse model where LRRK1 and LRRK2 genes are conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons, dopaminergic (DAergic) neurons in the substantia nigra (SN) and noradrenergic neurons in the Locus Coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in LRRK double Knock Out (DKO) was cell autonomous. To rigorously test this question, the authors generated a double conditional KO allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. This was beyond what is usually required as most investigators might just have combined one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in most DAergic neurons in SN and that LRRK levels are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DA neurons is average at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death and activated microglia. The degeneration is limited to DA neurons as LC neurons are not lost, and this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed rigorously, and the results were statistically sound and compelling.

Weakness:

I only have a few minor comments. First, in PD and other degenerative conditions, axon and terminal loss occur prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also the case with the Allen Brain Atlas profile. Thus, the authors should discuss the discrepancy, as they imply significant LRRK1 expression in DA neurons.

Revision:

I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

eLife assessment

This current revision builds on observations in validated conditional double KO (cDKO) mice for LRRK1 and LRRK2 that will be useful for the field, given that LRRK2 is widely expressed in the brain and periphery, and many divergent phenotypes have been attributed previously to LRRK2 expression. The manuscript presents solid data demonstrating that it is the loss of LRRK1 and LRRK2 expression within the SNpc DA cells that is not well tolerated, as it was previously unclear from past work whether neurodegeneration in the LRRK double Knock Out (DKO) was cell autonomous or the result of loss of LRRK1/LRRK2 expression in other types of cells. Future studies may pursue the biochemical mechanisms underlying the reason for the apoptotic cells noted in this study, as here, the LRRK1/LRRK2 KO mice did not replicate the dramatic increase in autophagic vacuole numbers previously noted in the germline global LRRK1/LRRK2 KO mice.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shen and collaborators described the generation of conditional double knockout (cDKO) mice lacking LRRK1 and LRRK2 selectively in DAT-positive dopaminergic neurons. The Authors asked whether selective deletion of both LRRK isoforms could lead to a Parkinsonian phenotype, as previously reported by the same group in germline double LRRK1 and LRRK2 knockout mice (PMID: 29056298). Indeed, cDKO mice developed a late reduction of TH+ neurons in SNpc that partially correlated with the reduction of NeuN+ cells. This was associated with increased apoptotic cell and microglial cell numbers in SNpc. Unlike the constitutive DKO mice described earlier, cDKO mice did not replicate the dramatic increase in autophagic vacuole numbers. The study supports the authors' hypothesis that loss of function rather than gain of function of LRRK2 leads to Parkinson's Disease.

Strengths:

For the first time, the study described a model in which both the Parkinson's disease-associated gene LRRK2 and its homolog LRRK1 are deleted selectively in dopaminergic neurons. This offers a new tool to understand the physiopathological role of LRRK2 and the compensating role of LRRK1 in modulating dopaminergic cell function.

Weaknesses:

The model has no construct validity since loss of function mutations of LRRK2 are well tolerated in humans and do not lead to Parkinson's disease. The evidence of a Parkinsonian phenotype in these conditional knockout mice is limited and should be considered preliminary.

Reviewer #3 (Public Review):

Kang, Huang, and colleagues have provided new data to address concerns regarding confirmation of LRRK1 and LRRK2 deletion in their mouse model and the functional impact of the modest loss of TH+ neurons observed in the substantia nigra of their double KO mice. In the revised manuscript, the new data around the characterization of the germline-deleted LRRK1 and LRRK2 mice add confidence that LRRK1 and LRRK2 can be deleted using the genetic approach. They have also added new text to the discussion to try and address some of the comments and questions raised regarding how LRRK1/2 loss may impact cell survival and the implications of this work for PD-linked variants in LRRK2 and therapeutic approaches targeting LRRK2. The new data provides additional support for the author's claims.

Reviewer #1 (Public Review):

Summary:

The authors present a detailed study of a nearly complete Entomophthora muscae genome assembly and annotation, along with comparative analyses among related and non-related entomopathogenic fungi. The genome is one of the largest fungal genomes sequenced, and the authors document the proliferation and evolution of transposons and presence/absence of related genetic machinery to explore how this may have occurred. There has also been an expansion in gene number, which appears to contain many "novel" genes unique to E. muscae. Functionally, the authors were interested in CAZymes, proteases, circadian clock related genes (due to entomopathogenicity/ host manipulation), other insect pathogen specific genes, and secondary metabolites. There are many interesting findings including expansions in trahalases, unique insulinase and another peptidase, and some evidence for RIP in Entomophthoralean fungi. The authors performed a separate study examining E. muscae species complex and related strains. Specifically, morphological traits were measured for strains and then compared to the 28S+ITS-based phylogeny, showing little informativeness of these morpho characters with high levels of overlap.

This work represents a big leap forward in genomics of non-Dikarya fungi and large fungal genomes. Most of the gene homologs have been studied in species that diverged hundreds of millions of years ago, and therefore using standard comparative genomic approaches are not trivial and still relatively little is known. This paper provides many new hypotheses and potential avenues of research about fungal genome size expansion, entomopathogenesis in zygomycetes, and cellular functions like RIP and circadian mechanisms.

Strengths:

There are many strengths to this study. It represents a massive amount of work and a very thorough functional analysis of the gene content in these fungi (which are largely unsequenced and definitely understudied). Too often comparative genomic work will focus on one aspect and leave the reader wondering about all the other ways genome(s) are unique or different from others. This study really dove in and explored the relevant aspects of the E. muscae genome.

The authors used both a priori and emergent properties to shape their analyses (by searching for specific genes of interest and by analyzing genes underrepresented, expanded, or unique to their chosen taxa), enabling a detailed review of the genomic architecture and content. Specifically, I'm impressed by the analysis of missing genes (pFAMs) in E. muscae, none of which are enriched in relatives, suggesting this fungus is really different not by gene loss, but by its gene expansions.

Analyzing species-level boundaries and the data underlying those (genetic or morphological) is not something frequently presented in comparative genomic studies, however, here it is a welcome addition as the target species of the study is part of a species complex where morphology can be misleading and genetic data is infrequently collected in conjunction with the morphological data.

Weaknesses:

The conclusions of this paper are well supported, and I think the clarifications and improvements made to the manuscript in the revision process have greatly improved the paper.

Reviewer #2 (Public Review):

Summary:

This study reveals that short-term social isolation increases social behavior at a reunion, and a population of hypothalamic preoptic area neurons become active after social interaction following short-term isolation (POAiso neurons). Effectively utilizing a TRAP activity-dependent labeling method, the authors inhibit or activate the POAiso neurons and find that these neurons are involved in controlling various social behaviors, including ultrasonic vocalization, investigation, and mounting in both male and female mice. This work suggests a complex role for the POA in regulating multiple aspects of social behavior, beyond solely controlling male sexual behaviors.

Strengths:

A few studies have shown that optogenetic activation of the POA in females promotes vocalization and mounting behavior, similar to the effects observed in males. However, those were the results of artificially stimulating POA neurons, and it was unknown whether POA neurons play a role in naturally occurring female social behaviors. This paper clearly demonstrates that there exists a population of POA neurons that are necessary for naturally evoked female social vocalizations and mounting behaviors.

Weaknesses:

The authors conclude that "In the current study, we identify and characterize a population of preoptic hypothalamic neurons that contribute to the effects of short-term social isolation on the social behaviors of mice." This is an interesting hypothesis, but in my opinion, critical control experiments are missing to support this claim.

All the activity-dependent labeling experiments with TRAP mice, including the subsequent neural activity manipulation experiments (Figures 2, 3, 4, 5E-F), were conducted by labeling neurons only in socially isolated animals, not group-housed animals. The authors labeled neurons after 30-minute social interactions, raising the possibility that the labeled neurons simply represent a "social interaction/behavior population" (mediating mounting and USVs in females and males) rather than a set of neurons specific to social isolation.

I strongly recommend including experimental groups that involve labeling neurons after 30-minute social interactions in group-housed female or male mice and inhibit TRAPed neurons after social isolation or activate TRAPed neurons after group housing. If manipulating the group-housed TRAP neurons has similar effects to manipulating the isolated TRAP neurons, it would suggest the current labeling paradigm is not isolating neurons specific to the effect of social isolation per se. Rather, the neurons may mediate more general social interaction or motivation-related activities. Given the known role of POA in male mating behavior, a group-housed TRAP experiment in males with a female visitor is especially important for understanding the selectivity of the labeled cells.

Without proper controls, referring to the labeled neurons as "POAiso" neurons is potentially misleading. The data thus far suggests these neurons may predominantly reflect a "POA social behavior" population rather than a set of cells distinctly responsive to isolated housing.

Overall, this paper is well-written and provides valuable new data on the neural circuit for female social behaviors and the potentially complex role of POA in social behavior control.

eLife assessment

This useful study has identified a subset of neurons in the preoptic hypothalamus that promote social behavior in single-housed female mice. The approach is solid; however, due to a lack of significance in the key findings and competing outcomes between different manipulation methods, the evidence is incomplete. The authors have the potential to demonstrate evidence by either increasing the number of experimental animals represented in the study or by adjusting the language in the conclusions to reflect the findings.

Reviewer #1 (Public Review):

Summary:

Zhao et al. perform a series of experiments aimed at identifying the role of the preoptic area (POA) in controlling the impact of social isolation on same-sex female social behavior. They focus their manuscript on the effects of short-term (3d) isolation and females, both of which have been relatively understudied, making the overall topic of the manuscript exciting and important.

Strengths:

The work highlighted is well designed, the experiments original, and the manuscript is elegant and clearly written. The strengths of the manuscript lie in the attention to multiple facets of social behavior (investigation, mounting, USVs), sex differences, and the use of multiple loss- and gain-of-function approaches.

Weaknesses:

The main weaknesses of the paper are a lack of significance in key findings, and relatedly, concluding effects from insignificant findings. Additional elements could be improved to help strengthen this overall well-rounded and intriguing set of results.

Reviewer #3 (Public Review):

Summary:

How short-term isolation acts on the brain to promote social behavior remains incompletely understood. The authors found that social interactions after a period of acute isolation increased investigation promoted mounting, and increased the production of ultrasonic vocalizations (USVs). This was true for females during same-sex interactions as well as for males interacting with females. Concomitant with these increased behavioral readouts, cFos expression in the preoptic area of the hypothalamus (POA) was found to increase selectively in single-housed females. Chemogenetic silencing of these POA neurons attenuated all three behavioral measures in socially isolated females. Surprisingly, ablation of the same POA neurons decreased mounting duration without impacting social investigation or USV production. While optogenetic activation was sufficient to evoke USV production, it did not affect either mounting or social investigation. In males, chemogenetic silencing of POA neurons decreased mounting but not other behaviors. Together, these data point towards a role of POA neurons in mediating social behaviors after acute isolation but the exact nature of that control appears to depend on the choice of perturbation method, sex, and social context in complex ways that are hard to parse. This study is an essential first step; additional experiments will be needed to explain the apparent discrepancy between the various circuit perturbation results and to gain a more comprehensive understanding of the role of POA in social isolation.

Strengths:

The goal of understanding the neural circuit mechanisms underlying acute social isolation is clearly important and topical. Using a state-of-the-art technique to tag specific neurons that were active during certain behavioral epochs, the authors managed to identify the POA as a critical circuit locus for the effects of social isolation. The experimental design is perfectly reasonable and the quality of the data is good. The control experiments (Figures 2B-D) showing that chemogenetic inactivation of other hypothalamic regions (AH and VMH) do not affect social behavior is indeed quite satisfying and points towards a specific role of POA within the hypothalamus. Using a combination of behavioral assays, activity-dependent neural tagging, and circuit manipulation techniques, the authors present convincing evidence for the role of the preoptic area of the hypothalamus in mediating certain behaviors following social isolation. These data are likely to be a valuable resource for understanding how hypothalamic circuits adjust to the challenges of social isolation.

Weaknesses:

While the authors should be commended for performing and reporting multiple circuit perturbation experiments (e.g., chemogenetics, ablation), the conflicting effects on behavior are hard to interpret without additional experiments. For example, chemogenetic silencing of the POA neurons (using DREADDs) attenuated all three behavioral measures but the ablation of the same POA neurons (using CASPACE) decreased mounting duration without impacting social investigation or USV production. Similarly, optogenetic activation of POA neurons was sufficient to generate USV production as reported in earlier studies but mounting or social investigation remained unaffected. Do these discrepancies arise due to the efficiency differences between DREADD-mediated silencing vs. Casp3 ablation? Or does the chemogenetic result reflect off-manifold effects on downstream circuitry whereas a more permanent ablation strategy allows other brain regions to compensate due to redundancy? It is important to resolve whether these arise due to technical reasons or whether these reflect the underlying (perhaps messy) logic of neural circuitry. Therefore, while it is clear that POA neurons likely contribute to multiple behavioral readouts of social isolation, understanding their exact roles in any greater detail will require further experiments.

Reviewer #2 (Public Review):

The paper has two main merits. Firstly, it documents a new and important characteristic of the re-organization of the brains of the deaf, namely its variability. The search for a well-defined set of functions for the deprived auditory cortex of the deaf has been largely unsuccessful, with several task-based approaches failing to deliver unanimous results. Now, one can understand why this was the case: most likely there isn't a fixed one well-defined set of functions supported by an identical set of areas in every subject, but rather a variety of functions supported by various regions. In addition, the paper extends the authors' previous findings from blind subjects to the deaf population. It demonstrates that the heightened variability of connectivity in the deprived brain is not exclusive to blindness, but rather a general principle that applies to other forms of deprivation. On a more general level, this paper shows how sensory input is a driver of the brain's reproducible organization.

The method and the statistics are sound, the figures are clear, and the paper is well-written. The sample size is impressively large for this kind of study.

The main weakness of the paper is not a weakness, but rather a suggestion on how to provide a stronger basis for the authors' claims and conclusions. I believe this paper could be strengthened by including in the analysis at least one of the already published deaf/hearing resting-state fMRI datasets (e.g. Andin and Holmer, Bonna et al., Ding et al.) to see if the effects hold across different deaf populations. The addition of a second dataset could strengthen the evidence and convincingly resolve the issue of whether delayed sign language acquisition causes an increase in individual differences in functional connectivity to/from Broca's area. Currently, the authors may not have enough statistical power to support their findings.

Secondly, the authors could more explicitly discuss the broad implications of what their results mean for our understanding of how the architecture of the brain is determined by the genetic blueprint vs. how it is determined by learning (page 9). There is currently a wave of strong evidence favoring a more "nativist" view of brain architecture, for example, face- and object- sensitive regions seem to be in place practically from birth (see e.g. Kosakowski et al., Current Biology, 2022). The current results show what is the role played by experience.

eLife assessment

This study presents valuable data on the increase in individual differences in functional connectivity with the auditory cortex in individuals with congenital/early-onset hearing loss compared to individuals with normal hearing. The evidence supporting the study's claims is convincing, although additional analyses and a deeper conceptual framing would have strengthened the study. The work will be of interest to neuroscientists working on brain plasticity and may have implications for the design of interventions and compensatory strategies.

Reviewer #1 (Public Review):

This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest. I do have some concerns with the way that the project has been conceptualized, which I share below.

The authors should provide careful working definitions of what exactly they think is occurring in the brain following sensory deprivation. Characterizing these changes as 'large-scale neural reorganization' and 'compensatory adaptation' gives the impression that the authors believe that there is good evidence in support of significant structural changes in the pathways between brain areas - a viewpoint that is not broadly supported (see Makin and Krakauer, 2023). The authors report changes in connectivity that amount to differences in coordinated patterns of BOLD signal across voxels in the brain; accordingly, their data could just as easily (and more parsimoniously) be explained by the unmasking of connections to the auditory cortex that are present in typically hearing individuals, but which are more obvious via MR in the absence of auditory inputs.

I found the argument that the deaf use a single modality to compensate for hearing loss, and that this might predict a more confined pattern of differential connectivity than had been previously observed in the blind to be poorly grounded. The authors themselves suggest throughout that hearing loss, per se, is likely to be driving the differences observed between deaf and typically-hearing individuals; accordingly, the suggestion that the modality in which intentional behavioral compensation takes place would have such a large-scale effect on observed patterns of connectivity seems out of line.

The analyses highlighting the areas observed to be differentially connected to the auditory cortex and areas observed to be more variable in their connectivity to the auditory cortex seem somewhat circular. If the authors propose hearing loss as a mechanism that drives this variability in connectivity, then it is reasonable to propose hypotheses about the directionality of these changes. One would anticipate this directionality to be common across participants and thus, these areas would emerge as the ones that are differently connected when compared to typically hearing folks.

While the authors describe collecting data on the etiology of hearing loss, hearing thresholds, device use, and rehabilitative strategies, these data do not appear in the manuscript, nor do they appear to have been included in models during data analysis. Since many of these factors might reasonably explain differences in connectivity to the auditory cortex, this seems like an omission.

Reviewer #3 (Public Review):

Summary:

This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

- The manuscript is well written.

- The methods are clearly described and appropriate.

- Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes.

- The results are interesting and novel.

Weaknesses:

- Analyses were conducted for task-based data rather than resting-state data. It was unclear whether groups differed in task performance. If congenitally deaf individuals found the task more difficult this could lead to changes in FC.

- No differences in overall activation between groups were reported. Activation differences between groups could lead to differences in FC. For example, lower activation may be associated with more noise in the data, which could translate to reduced FC.

- Figure 2B shows higher FC for congenitally deaf individuals than normal-hearing individuals in the insula, supplementary motor area, and cingulate. These regions are all associated with task effort. If congenitally deaf individuals found the task harder (lower performance), then activation in these regions could be higher, in turn, leading to FC. A study using resting-state data could possibly have provided a clearer picture.

- The correlation between the FC map and the FC variability map is 0.3. While significant using permutation testing, the correlation is low, and it is not clear how great the overlap is.

evaporation is not given in answer. latent heat is given: 64 W/m2, thus evaporation is 64/rho*lambda [m/s].

Can this be added?

^2

Proper reference can be found on https://library.wur.nl/WebQuery/hydrotheek/2095238

This quote highlights a deep and relatable experience of poverty that goes beyond temporary financial challenges. It captures the struggle of working tirelessly yet barely making ends meet, a reality that many can empathize with but few truly understand firsthand.

Based on the situation illustrated here. I wonder how can we , as an educator, do to know more about a student’s family situation instead of asking them or their parents directly. I feel like you have to make a relation with the student first. But I don’t know what to do next.

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

Interestingly, I saw his name and immediately thought of Barbara Bel Geddes and Vertigo.

check superscrips

Express h in percentage. Thus h=33.7%

I felt so bad for this racism word. No one in this world should be treat unequally base on their wealth. Even we normally heard those kind of news happened on minority race, it doesn't means there are no such thing for white people

If some student fail at learning, the teacher will getting excuses to convince both themselves or the public that problem is come from the students but not them. In fact, the indifference of teacher to students motivation of study and biased treatment to the rich and poor students will make the thing worse

This is really affecting students` behavior. Without the guide by their parents and accompany, students could have some bad habits and being more treason

These behaviors are really just the stereotype impression to the poor students. Maybe the teacher is overwhelmingly state the fact, but I think there do have relationship between the motivation of study

for - book - Citizens - foreward - Brian Eno

for - stories - salience of adjacency- imagination - stories - futures - Ernest Becker - self - timebinding - symbolosphere - quote - Brian Eno - book - Citizens - Jon Alexander - Arian Conrad - citizens - not consumers

quote - Brian Eno

• The stories we tell
  • shape how we see ourselves, and
  • how we see the world.
• When we see the world differently,
  • we begin behaving differently,
  • living into the new story.
• When Martin Luther King said
  • “I have a dream,”
• he was
  • inviting others to dream it with him,
  • inviting them to step into his story.
• Once a story becomes shared in that way,
  • current reality gets measured against it and
  • then modified towards it.
• As soon as we sense the possibility of a more desirable world,
  • we begin behaving differently,
    • as though that world is starting to come into existence,
    • as though, in our minds at least, we’re already there.
• The new story becomes an invisible force which pulls us forward.
• By this process it starts to come true.

• Imagining the future makes it more possible.

• Sometimes this work of imagination and storytelling is about the future,

  • as in Dr King’s story.
• Art can play this role:
  • what is possible in art becomes thinkable in life.
• We become our new selves first in simulacrum, through
  • style and
  • fashion and
  • art,
• our deliberate immersions in virtual worlds.
• Through them we sense what it would like
  • to be another kind of person
  • with other kinds of values.
• We rehearse new
  • feelings and
  • sensitivities.
• We imagine other ways of thinking about
  • our world and
  • its future.
• We use art to model new worlds so that
  • we can see how we might feel about them.

comment - This is a really powerful writing from Brian Eno. - Storytelling is an exercise in - the imagination of alternative possibilities to our own reality. - Stories can become both - inspirational and - aspirational - They can paint a picture in our mind of - a fantasy - a world that does not yet exist - but that nonexistent but desirable reality can then serve as the goal for which we strive - Mapping Futures interventions is then, essentially an act of desirable, inspirational make believe, and mustering the resources to turn the fantasy into reality - Progress relies on design, the imagination of unrealities in vivid detail, - in order to turn them into realities - In doing this, it is not an act carried out in ivory towers, - but in the everyday life of every one of us - We are all engaged in desirable fantasies daily whenever - we decide what meal we will prepare or restaurant to dine at - which clothing outfit to wear today - what we plan to write or say next to another - Every decision we make as a choice between different future alternatives - When it comes to planning major future decisions, - we need to have as much detail as possible of the imagined future - The Town Anywhere project conceived by Ruth Ben-Tovin and employed in the Transition Town movement for many years fis an example of such a simulacrum - https://hyp.is/mqeCtAE_Ee-Yxleqg7GFww/docdrop.org/video/cRvhY4S94ic/ - It provides an artistic space for citizens to imagine a desirable fantasy that can be embodied, enacted and deeply remembered through the participatory and collective citizen act of creating a proxy of their future local habitat in the present, and exploring and momentarily inhabiting their simulacrum. - In this way, this compelling experience is like a branding iron, searing the memory deep into our memory, where it can help guide our actions to realize the desirable fantasy. - Couched within a citizen's FREEligion and FREElosophy we generically call Deep Humanity, an open source, open knowledge approach to universal raison d'etre for what it deeply means to be human, Town Anywhere can scale to fire up the imagination of citizens to co-create our collective future. - Town Anywhere, along with other citizen initiatives which I belong to that advocate healthy citizen power such as SONEC, Stop Reset Go, Deep Humanity, the Indyweb, Living Cities Earth and many, many others can emerge a human murmuration to drive the transition - https://hyp.is/go?url=https%3A%2F%2Fleemor.medium.com%2Fmesmerized-by-the-murmuration-on-human-potential-f4c9ffe06ffa&group=world - As Jon Alexander and Arian Conrad write here, we have to find the narratives that matter to us, where WE is the citizens. Other thinkers like Jose Ramos write along the same line: - https://hyp.is/go?url=https%3A%2F%2Foff-planet.medium.com%2Fdiscovering-the-narratives-that-matter-to-us-327958a2daec&group=world

M T-3

M L-1 T-2

M L-1 T-2

eLife assessment

This valuable work by Zheng and colleagues uses a large cohort database from Shanghai to identify that post-infection vaccination among previously vaccinated individuals provides significant low to moderate protection against re-infection. The evidence supporting the conclusion is solid with some limitations, e.g., lack of symptom severity as an outcome, no inclusion of time since infection as an independent variable, improper definitions of some key variables, difficult-to-interpret figures, and exclusion of key groups (infected and then vaccinated). This study will be of interest to vaccinologists, public health officials and clinicians.

Reviewer #1 (Public Review):

Summary:

Zheng and colleagues assessed the real-world efficacy of SARS-CoV-2 vaccination against re-infection following the large omicron wave in Shanghai in April 2022. The study was performed among previously vaccinated individuals. The study successfully documents a small but real added protective benefit of re-vaccination, though this diminishes in previously boosted individuals. Unsurprisingly, vaccine preventative efficacy was higher if the vaccine was given in the month before the 2nd large wave in Shanghai. The re-infection rate of 24% suggests that long-term anti-COVID immunity is very difficult to achieve. The conclusions are largely supported by the analyses. These results may be useful for planning the timing of subsequent vaccine rollouts.

Strengths:

The strengths of the study are a very large and unique cohort based on synchronously timed single infection among individuals with well-documented vaccine histories. Statistical analyses seem appropriate. As with any cohort study, there are potential confounders and the possibility of misclassification and the authors outline limitations nicely in the discussion.

Weaknesses:

(1) Partially and fully vaccinated are never defined and it is difficult to understand how this differs from single, and double, booster vaccines. The figures including all of these groups are a bit confusing for this reason.

(2) Figure 3 is a bit challenging to interpret because it is a bit atypical to compare each group to a different baseline (ie 2V-I-V vs 2V-I). I would label the y-axis 2V-I-V vs 2V-I (change all of the labels) to make this easier to understand.

(3) A 15% reduction in infection is quite low. It would be helpful to discuss if any quantitative or qualitative signals suggest at least a reduction in severe outcomes such as death, hospitalization, ER visits, or long COVID. I am not sure that a 15% reduction in cases supports extra vaccination without some other evidence of added benefit.

(4) Why exclude the 74962 unvaccinated from the analysis. it would be interesting to see if getting vaccinated post-infection provides benefits to this group

(5) Pudong should be defined for those who do not live in China.

(6) The discussion about healthcare utilization bias is welcomed and well done. It would be great to speculate on whether this bias might favor the null or alternative hypothesis.

Reviewer #2 (Public Review):

Summary:

This paper evaluates the effect of COVID-19 booster vaccination on reinfection in Shanghai, China among individuals who received primary COVID-19 vaccination followed by initial infection, during an Omicron wave.

Strengths:

A large database is collated from electronic vaccination and infection records. Nearly 200,000 individuals are included in the analysis and 24% became reinfected.

Weaknesses:

The article is difficult to follow in terms of the objectives and individuals included in various analyses. There appear to be important gaps in the analysis. The electronic data are limited in their ability to draw causal conclusions.

More detailed comments:

In multiple places (abstract, introduction), the authors frame the work in terms of understanding the benefit of booster vaccination among individuals with hybrid immunity (vaccination + infection). However, their analysis population does not completely align with this framing. As best as I can tell, only individuals who first received COVID-19 vaccination, and subsequently experienced infection, were included. Why the analysis does not also consider individuals who were infected and then vaccinated is not clear.

In vaccine effectiveness analyses, why was time since initial infection not examined as a modifier of the booster effect? Time since the onset of the Omicron wave is only loosely tied to the immune status of the individual.

The effect of booster vaccination on preventing symptomatic vs. asymptomatic reinfection does not appear to have been evaluated; this is a key gap in the analysis and it would seem the data would support it.

In lines 105-108, the demographic description of the analysis population is incomplete. Is sex or gender identity being described? Are any individuals non-binary? What is the age distribution? (Only the proportions 20-39 and under 6 are stated.)

Figure 1 consort diagram is confusing. In the last row, are the two boxes independent or overlapping sets of individuals? Are all included in secondary analyses?

eLife assessment

This study provides an important tool for predicting binding between immune cells receptors and antigens based on protein sequence data. The analysis convincingly showed the tool's effectiveness in both supervised TCR binding prediction and unsupervised clustering, surpassing existing methods in accuracy and reducing annotation costs. This study will be of interest to immunologists and computational biologists.

Reviewer #2 (Public Review):

In the manuscript, the authors highlighted the importance of T-cell receptor (TCR) analysis and the lack of amino acid embedding methods specific to this domain. The authors proposed a novel bi-directional context-aware amino acid embedding method, catELMo, adapted from ELMo (Embeddings from Language Models), specifically designed for TCR analysis. The model is trained on TCR sequences from seven projects in the ImmunoSEQ database, instead of the generic protein sequences. They assessed the effectiveness of the proposed method in both TCR-epitope binding affinity prediction, a supervised task, and the unsupervised TCR clustering task. The results demonstrate significant performance improvements compared to existing embedding models. The authors also aimed to provide and discuss their observations on embedding model design for TCR analysis: 1) Models specifically trained on TCR sequences have better performance than models trained on general protein sequences for the TCR-related tasks; and 2) The proposed ELMo-based method outperforms TCR embedding models with BERT-based architecture. The authors also provided a comprehensive introduction and investigation of existing amino acid embedding methods. Overall, the paper is well-written and well-organized.

The work has originality and has potential prospects for immune response analysis and immunotherapy exploration. TCR-epitope pair binding plays a significant role in T cell regulation. Accurate prediction and analysis of TCR sequences are crucial for comprehending the biological foundations of binding mechanisms and advancing immunotherapy approaches. The proposed embedding method presents an efficient context-aware mathematical representation for TCR sequences, enabling the capture and analysis of their structural and functional characteristics. This method serves as a valuable tool for various downstream analyses and is essential for a wide range of applications.

Reviewer #3 (Public Review):

In this study, Zhang and colleagues proposed an ELMo-based embedding model (catELMo) for TCRβ CDR3 amino acid sequences. They showed the effectiveness of catELMo in both supervised TCR binding prediction and unsupervised clustering, surpassing existing methods in accuracy and reducing annotation costs. The study provides insights on the effect of model architectures to TCR specificity prediction and clustering tasks.

The authors have addressed our prior critiques of the manuscript.

eLife assessment

Franke et al. explore and characterize the color response properties in the mouse primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The data is solid; however, the evidence supporting some conclusions is incomplete. In its current form, the paper makes a useful contribution to how color is coded in mouse V1. Significance would be enhanced with some additional analyses and a clearer discussion of the limitations of the data presented.

Reviewer #1 (Public Review):

Summary:

In this study, Franke et al. explore and characterize color response properties across primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake 2P imaging to define the spectral response properties of visual interneurons in layer 2/3. They find that opponent responses are more pronounced at photopic light levels, and that diversity in color opponent responses exists across the visual field, with green ON/ UV OFF responses more strongly represented in the upper visual field. This is argued to be relevant for the detection of certain features that are more salient when using chromatic space, possibly due to noise reduction. In the revised version, Franke et al. have addressed the potential pitfalls in the discussion, which is an important point for the non-expert reader. Thus, this study provides a solid characterization of the color properties of V1 and is a valuable addition to visual neuroscience research.

My remaining concerns are based more on the interpretation. I'm still not convinced by the statement "This type of color-opponency in the receptive field center of V1 neurons was not present in the receptive field center of retinal ganglion cells and, therefore, is likely computed by integrating center and surround information downstream of the retina." and I would suggest rewording it in the abstract.

As discussed previously and now nicely added to the discussion, it is difficult to make a direct comparison given the different stimulus types used to characterize the retina and V1 recordings and the different levels of adaptation in both tissues. I will leave this point to the discussion, which allows for a more nuanced description of the phenomenon. Why do I think this is important? In the introduction, the authors argue that "the discrepancy [of previous studies] may be due to differences in stimulus design or light levels." However, while different light levels can be tested in V1, this cannot be done properly in the retina with 2P experiments. To address this, one would have to examine color-opponency in RGC terminals in vivo, which is beyond the scope of this study. Addressing these latter points directly in the discussion would, in my opinion, only strengthen the study.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

While the study presents convincing evidence about the asymmetric distribution of color-opponent neurons in V1, the paper would greatly benefit from a more in-depth discussion of the caveats related to the conclusions drawn about their origin. This is particularly relevant regarding the conclusion drawn about the contribution of color opponent neurons in the retina. The mismatch between retinal color opponency and V1 color opponency could imply that this feature is not solely inherited from the retina, however, there are other plausible explanations that are not discussed here. Direct evidence for this statement remains weak.

In addition, the paper would benefit from adding explicit neuron counts or percentages to the quadrants of each of the density plots in Figures 2-5. The variance explained by the principal components does not capture the percentage of color opponent cells. Additionally, there appear to be some remaining errors in the figure legend and labels that have not been addressed (e.g. '??' in Fig 2 legend).

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

Reviewer #3 (Public Review):

This paper studies chromatic coding in mouse primary visual cortex. Calcium responses of a large collection of cells are measured in response to a simple spot stimulus. These responses are used to estimate chromatic tuning properties - specifically sensitivity to UV and green stimuli presented in a large central spot or a larger still surrounding region. Cells are divided based on their responses to these stimuli into luminance or chromatic sensitive groups. The results are interesting and many aspects of the experiments and conclusions are well done; several technical concerns, however, limit the support for several main conclusions,

Limitations of stimulus choice<br /> The paper relies on responses to a large (37.5 degree diameter) modulated spot and surround region. This spot is considerably larger than the receptive fields of both V1 cells and retinal ganglion cells (it is twice the area of the average V1 receptive field). As a result, the spot itself is very likely to strongly activate both center and surround mechanisms, and responses of cells are likely to depend on where the receptive fields are located within the spot (and, e.g., how much of the true neural surround samples the center spot vs the surround region). Most importantly, the surrounds of most of the recorded cells will be strongly activated by the central spot. This brings into question statements in the paper about selective activation of center and surround (e.g. page 2, right column). This in turn raises questions about several subsequent analyses that rely on selective center and surround activation.

Comparison with retina<br /> A key conclusion of the paper is that the chromatic tuning in V1 is not inherited from retinal ganglion cells. This conclusion comes from comparing chromatic tuning in a previously-collected data set from retina with the present results. But the retina recordings were made using a considerably smaller spot, and hence it is not clear that the comparison made in the paper is accurate. For example, the stimulus used for the V1 experiments almost certainly strongly stimulates both center and surround of retinal ganglion cells. The text focuses on color opponency in the receptive field centers of retinal ganglion cells, but center-surround opponency seems at least as relevant for such large spots. This issue needs to be described more clearly and earlier in the paper.

Limitations associated with ETA analysis<br /> One of the reviewers in the previous round of reviews raised the concern that the ETA analysis may not accurately capture responses of cells with nonlinear receptive field properties such as On/Off cells. This possibility and whether it is a concern should be discussed.

Discrimination performance poor<br /> Discriminability of color or luminance is used as a measure of population coding. The discrimination performance appears to be quite poor - with 500-1000 neurons needed to reliably distinguish light from dark or green from UV. Intuitively I would expect that a single cell would provide such discrimination. Is this intuition wrong? If not, how do we interpret the discrimination analyses?

According to Ramírez's "Latinx Assimilation," assimilation refers to becoming more alike to the mainstream majority in society. As the minority absorbs the culture from the majority, a type of boundary crossing occurs (Ramírez, 2021, 1)

Ramírez, Catherine. “Latinx Assimilation.” Oxford Research Encyclopedia of American History, February 23, 2021. https://oxfordre.com/americanhistory/display/10.1093/acrefore/9780199329175.001.0001/acrefore-9780199329175-e-791.

eLife assessment

This study presents valuable findings on the relationship between prediction errors and brain activation in response to unexpected omissions of painful electric shock. The strengths are the research question posed, as it has remained unresolved if prediction errors in the context of biologically aversive outcomes resemble reward-based prediction errors. The evidence is solid but there are weaknesses in the experimental design, where verbal instructions do not align with experienced outcome probabilities. There is also disconnect between the introduction which focuses on the role of prediction error signaling for learning and the lack of analyses accounting for learning and updating of expectations. The work will be of interest to cognitive neuroscientists and psychologists studying appetitive and aversive learning.

Reviewer #1 (Public Review):

Summary:

Willems and colleagues test whether unexpected shock omissions are associated with reward-related prediction errors by using an axiomatic approach to investigate brain activation in response to unexpected shock omission. Using an elegant design that parametrically varies shock expectancy through verbal instructions, they see a variety of responses in reward-related networks, only some of which adhere to the axioms necessary for prediction error. In addition, there were associations between omission-related responses and subjective relief. They also use machine learning to predict relief-related pleasantness, and find that none of the a priori "reward" regions were predictive of relief, which is an interesting finding that can be validated and pursued in future work.

Strengths:

The authors pre-registered their approach and the analyses are sound. In particular, the axiomatic approach tests whether a given region can truly be called a reward prediction error. Although several a priori regions of interest satisfied a subset of axioms, no ROI satisfied all three axioms, and the authors were candid about this. A second strength was their use of machine learning to identify a relief-related classifier. Interestingly, none of the ROIs that have been traditionally implicated in reward prediction error reliably predicted relief, which opens important questions for future research.

Weaknesses:

To ensure that the number of omissions is similar across conditions, the task employs inaccurate verbal instructions; i.e. 25% of shocks are omitted, regardless of whether subjects are told that the probability is 100%, 75%, 50%, 25%, or 0%. Given previous findings on interactions between verbal instruction and experiential learning (Doll et al., 2009; Li et al., 2011; Atlas et al., 2016), it seems problematic a) to treat the instructions as veridical and b) average responses over time. Based on these prior work, it seems reasonable to assume that participants would learn to downweight the instructions over time through learning (particularly in the 100% and 0% cases); this would be the purpose of prediction errors as a teaching signal. The authors do recognize this and perform a subset analysis in the 21 participants who showed parametric increases in anticipatory SCR as a function of instructed shock probability, which strengthened findings in the VTA/SN; however given that one third of participants (n=10) did not show parametric SCR in response to instructions, it seems like some learning did occur. As prediction error is so important to such learning, a weakness of the paper is that conclusions about prediction error might differ if dynamic learning were taken into account.

Reviewer #2 (Public Review):

The question of whether the neural mechanisms for reward and punishment learning are similar has been a constant debate over the last two decades. Numerous studies have shown that the midbrain dopamine neurons respond to both negative and salient stimuli, some of which can't be well accounted for by the classic RL theory (Delgado et al., 2007). Other research even proposed that aversive learning can be viewed as reward learning, by treating the omission of aversive stimuli as a negative PE (Seymour et al., 2004).

Although the current study took an axiomatic approach to search for the PE encoding brain regions, which I like, I have major concerns regarding their experimental design and hence the results they obtained. My biggest concern comes from the false description of their task to the participants. To increase the number of "valid" trials for data analysis, the instructed and actual probabilities were different. Under such a circumstance, testing axiom 2 seems completely artificial. How does the experimenter know that the participants truly believe that the 75% is more probable than, say, the 25% stimulation? The potential confusion of the subjects may explain why the SCR and relief report were rather flat across the instructed probability range, and some of the canonical PE encoding regions showed a rather mixed activity pattern across different probabilities. Also for the post-hoc selection criteria, why pick the larger SCR in the 75% compared to the 25% instructions? How would the results change if other criteria were used?

To test axiom 3, which was to compare the 100% stimulation to the 0% stimulation conditions, how did the actual shock delivery affect the fMRI contrast result? It would be more reasonable if this analysis could control for the shock delivery, which itself could contaminate the fMRI signal, with extra confound that subjects may engage certain behavioral strategies to "prepare for" the aversive outcome in the 100% stimulation condition. Therefore, I agree with the authors that this contrast may not be a good way to test axiom 3, not only because of the arguments made in the discussion but also the technical complexities involved in the contrast.

Comments on revised version:

I want to thank the authors for their thorough and comprehensive work in revising this manuscript. I agree with the authors that learning paradigms might not be a necessity when it comes to study the PE signals, but I don't particularly agree with some of the responses in the rebuttal letter ("Furthermore, conditioning paradigms generally only include one level of aversive outcome: the electrical stimulation is either delivered or omitted."). This is of course correct description for the conditioning paradigm, but the same can be said for an instructed design: the aversive outcome was either delivered or not. That being said, adopting the instructed design itself is legitimate in my opinion.

My main concern, which the authors spent quite some length in the rebuttal letter to address, still remains about the validity for different instructed probabilities. Although subjects were told that the trials were independent, the big difference between 75% and 25% would more than likely confuse the subjects, especially given that most of us would fall prey to the Gambler's fallacy (or the law of small numbers) to some degree. When the instruction and subjective experience collides, some form of inference or learning must have occurred, making the otherwise straightforward analysis more complex. Therefore, I believe that a more rigorous/quantitative learning modeling work can dramatically improve the validity of the results. Of course, I also realize how much extra work is needed to append the computational part but without it there is always a theoretical loophole in the current experimental design.

As the authors mentioned in the rebuttal letter, "selecting participants only if their anticipatory SCR monotonically increased with each increase in instructed probability 0% < 25% < 50% < 75% < 100%, N = 11 participants", only ~1/3 of the subjects actually showed strong evidence for the validity of the instructions. This further raises the question of whether the instructed design, due to the interference of false instruction and the dynamic learning among trials, is solid enough to test the hypothesis.

Reviewer #3 (Public Review):

Summary:

The authors conducted a human fMRI study investigating the omission of expected electrical shocks with varying probabilities. Participants were informed of the probability of shock and shock intensity trial-by-trial. The time point corresponding to the absence of the expected shock (with varying probability) was framed as a prediction error producing the cognitive state of relief/pleasure for the participant. fMRI activity in the VTA/SN and ventral putamen corresponded to the surprising omission of a high probability shock. Participants' subjective relief at having not been shocked correlated with activity in brain regions typically associated with reward-prediction errors. The overall conclusion of the manuscript was that the absence of an expected aversive outcome in human fMRI looks like a reward-prediction error seen in other studies that use positive outcomes.

Strengths:

Overall, I found this to be a well-written human neuroimaging study investigating an often overlooked question on the role of aversive prediction errors, and how they may differ from reward-related prediction errors. The paper is well-written and the fMRI methods seem mostly rigorous and solid.

Comments on revised version:

The authors were extremely responsive to the comments and provided a comprehensive rebuttal letter with a lot of detail to address the comments. The authors clarified their methodology, and rationale for their task design, which required some more explanation (at least for me) to understand. Some of the design elements were not clear to me in the original paper.

The initial framing for their study is still in the domain of learning. The paper starts off with a description of extinction as the prime example of when threat is omitted. This could lead a reader to think the paper would speak to the role of prediction errors in extinction learning processes. But this is not their goal, as they emphasize repeatedly in their rebuttal letter. The revision also now details how using a conditioning/extinction framework doesn't suit their experimental needs.

It is reasonable to develop a new task to answer their experimental questions. By no means is there a requirement to use a conditioning/extinction paradigm to address their questions. As they say, "it is not necessary to adopt a learning paradigm to study omission responses", which I agree with.

But the authors seem to want to have it both ways: they frame their paper around how important prediction errors are to extinction processes, but then go out of their way to say how they can't test their hypotheses with a learning paradigm.

Part of their argument that they needed to develop their own task "outside of a learning context" goes as follows:<br /> (1) "...conditioning paradigms generally only include one level of aversive outcome: the electrical stimulation is either delivered or omitted. As a result, the magnitude-related axiom cannot be tested."<br /> (2) "....in conditioning tasks people generally learn fast, rendering relatively few trials on which the prediction is violated. As a result, there is generally little intra-individual variability in the PE responses"<br /> (3) "...because of the relatively low signal to noise ratio in fMRI measures, fear extinction studies often pool across trials to compare omission-related activity between early and late extinction, which further reduces the necessary variability to properly evaluate the probability axiom"

These points seem to hinge on how tasks are "generally" constructed. However, there are many adaptations to learning tasks:<br /> (1) There is no rule that conditioning can't include different levels of aversive outcomes following different cues. In fact, their own design uses multiple cues that signal different intensities and probabilities. Saying that conditioning "generally only include one level of aversive outcome" is not an explanation for why "these paradigms are not tailored" for their research purposes. There are also several conditioning studies that have used different cues to signal different outcome probabilities. This is not uncommon, and in fact is what they use in their study, only with an instruction rather than through learning through experience, per se.<br /> (2) Conditioning/extinction doesn't have to occur fast. Just because people "generally learn fast" doesn't mean this has to be the case. Experiments can be designed to make learning more challenging or take longer (e.g., partial reinforcement). And there can be intra-individual differences in conditioning and extinction, especially if some cues have a lower probability of predicting the US than others. Again, because most conditioning tasks are usually constructed in a fairly simplistic manner doesn't negate the utility of learning paradigms to address PE-axioms.<br /> (3) Many studies have tracked trial-by-trial BOLD signal in learning studies (e.g., using parametric modulation). Again, just because other studies "often pool across trials" is not an explanation for these paradigms being ill-suited to study prediction errors. Indeed, most computational models used in fMRI are predicated on analyzing data at the trial level.

Again, the authors are free to develop their own task design that they think is best suited to address their experimental questions. For instance, if they truly believe that omission-related responses should be studied independent of updating. The question I'm still left puzzling is why the paper is so strongly framed around extinction (the word appears several times in the main body of the paper), which is a learning process, and yet the authors go out of their way to say that they can only test their hypotheses outside of a learning paradigm.

The authors did address other areas of concern, to varying extents. Some of these issues were somewhat glossed over in the rebuttal letter by noting them as limitations. For example, the issue with comparing 100% stimulation to 0% stimulation, when the shock contaminates the fMRI signal. This was noted as a limitation that should be addressed in future studies, bypassing the critical point.

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eLife assessment

This important study identifies the gustatory receptors for sugar sensing in the larval and adult forms of the cotton bollworm, which is responsible for the destruction of many food crops world-wide. The authors find that the larval and adult forms utilise different receptors to sense sugars. The data are convincing and will be of interest neuroscientists working in sensory coding of sugars and to the pest management field.

Reviewer #1 (Public Review):

Summary:

The process of taste perception is significantly more intricate and complex in Lepidopteran insects. This investigation provides valuable insights into the role of Gustatory receptors and their dynamics in the sensation of sucrose, which serves as a crucial feeding cue for insects. The article highlights the differential sensitivity of Grs to sucrose and their involvement in feeding and insect behavior.

Strengths:

To support the notion of the differential specificity of Gr to sucrose, this study employed electrophysiology, ectopic expression of Grs in Xenopus, genome editing, and behavioral studies on insects. This investigation offers a fundamental understanding of the gustation process in lepidopteran insects and its regulation of feeding and other gustation-related physiological responses. This study holds significant importance in advancing our comprehension of lepidopteran insect biology, gustation, and feeding behavior.

Weaknesses:

While this manuscript demonstrates technical proficiency, there exists an opportunity for additional refinement to optimize comprehensibility for the intended audience. Several crucial sugars have been overlooked in the context of electrophysiology studies and should be incorporated. Furthermore, it is imperative to consider the potential off-target effects of Gr knock-out on other Gr expressions. This investigation focuses exclusively on Gr6 and Gr10, while neglecting a comprehensive narrative regarding other Grs involved in sucrose sensation.

Reviewer #2 (Public Review):

Summary:

To identify sugar receptors and assess the capacity of these genes the authors first set out to identify behavioral responses in larva and adult as well as physiological response. They used phylogenetics and gene expression (RNAseq) to identify candidates for sugar reception. Using first an in vitro oocyte system they assess the responses to distinct sugars. A subsequent genetic analysis shows that the Gr10 and Gr6 genes provide stage specific functions in sugar perception.

Strengths:

A clear strength of the manuscript is the breadth of techniques employed allowing a comprehensive study in a non-canonical model species.

Weaknesses:

There are no major weaknesses in the study for the current state of knowledge in this species. Since it is much basic work to establish a broader knowledge, context with other modalities remain unknown. It might have been possible to probe certain context known from the fruit fly, which would have strengthened the manuscript.

for - Town Anywhere - Transition Town - town anywhere - Ruth Ben-Tovim - Deep Humanity BEing journey - TPF - Town Anywhere BEing journey - LCE - Town Anywhere - adjacency - rapid whole system change - futures - town anywhere - SONEC

summary - Town Anywhere provides a simulacrum, as Brian Eno talks about in the forward to Jon Anderson & Arian Conrad's book Citizens - https://hyp.is/m_HuigEvEe--6UdGv2HVDA/www.jonalexander.net/the-foreword

Summary - This is a

eLife assessment

This important study identifies candidate mitochondrial metabolite carriers in stramenopile protists that may allow these divergent eukaryotes to maintain a compartmentalized glycolytic pathway. This study fills a gap in our understanding of glycolysis evolution and opens avenues for drug design to combat stramenopile parasites. The evidence, based on phylogenetic analysis, thermostability shift assays, and in vitro reconstitution of transport reactions, is convincing, albeit lacking direct in vivo confirmation of the physiological function of these candidates.

Reviewer #1 (Public Review):

Summary

This study identifies a family of solute transports in the enteric protist, Blastocystis, that may mediate the transport of glycolytic intermediates across the mitochondrial membrane. The study builds on previous observations suggesting that Blastocystis (and other Stramenopiles) are unusual in having a compartmentalized glycolytic pathway with enzymes involved in upper and lower glycolysis being located in the cytosol and mitochondria, respectively. In this study, the authors identified two putative Stamenopile metabolite transporters that are related to plant di/tricarboxylic acid transporters that might mediate the transport of glycolytic intermediates across the mitochondrial membrane. These GIC-transporters were localized to the Blastocystis mitochondrion using specific rabbit antibodies and shown to bind several glycolytic intermediates (including GAP, DHAP and PEP) based on thermostability shift assays. Direct evidence for transport activity was obtained by reconstituting native proteins in proteoliposomes and measuring uptake of 14C-malate or 35S-sulphate against unlabelled substrates. This assay showed that GIC-2 transported DHAP, GAP and PEP. However, significant transport activity was not observed for bGIC-2. Overall, the study provides strong, but not conclusive evidence that bGIC-2 is involved in transporting glycolytic intermediates across the inner membrane of the mitochondria, while the function of GIC-1 remains unclear, despite exhibiting the same metabolite binding properties as bGIC-2 n thermostability assays.

Strengths:

Overall, the findings are of interest in the context of understanding the diversity of core metabolic pathways in evolutionarily diverse eukaryotes, as well as the process by which cytosolic glycolysis evolved in most eukaryotes. The experiments are carefully performed and clearly described.

Weaknesses:

The main weakness of the study is the lack of direct evidence that either bGIC-1 and/or bGIC2 are active in vivo. While it is appreciated that the genetic tools for disrupting GIC genes in Blastocystis are limited/lacking, are there opportunities to ectopically express or delete these genes in other genetically tractable Stamenopiles, such as Phaeodactylum triconuteum?

The authors demonstrate that both bGIC-1 and bGIC-2 are targeted to the mitochondrion, based on immunofluorescence studies. However, the precise localization and topology of these carriers in the inner or outer membrane is not defined. The conclusions of the study would be strengthened if the authors could show that one/both transporters are present in the inner membrane using protease protection experiments following differential solubilization of the outer and inner mitochondrial membranes.

It is not clear why hetero-exchange reactions were not performed for bGIC-1 (only for bGIC-2).

In both their previous study (Bartulos et al (2018) and the current study, the authors have shown that Blastocystis express a TPI-GAPDH fusion protein which is located to the mitochondrion. The presence of the TPI domain in the mitochondrial matrix would obviate the need for bGIC-1/2 triose transporters and decrease their value as drug targets. It is noted that Blastocystis still retains some TPI activity in the cytosol, presumably due to expression of a second cytoplasmic isoform, which could account for the presence of the bGIC transporters. However, some discussion on the role of this mitochondrial TPI-GAPDG fusion protein in Blastocystis and other Stramenopiles would be useful.

The summary slide (Fig 7) in the revised manuscript no longer shows PEP being used as a countersolute for the import of G3P and DHAP. Although it complicates the story, the role of PEP as a counter solute should be shown for completeness and also to make sense of some of the statements in the discussion. In particular, as noted by the authors, mitochondrial PEP could be exported back to the cytsol and converted to pyruvate and/or lactate to generate ATP and NAD, although at the expense of ATP synthesis in the mitochondria.

Reviewer #2 (Public Review):

In this manuscript, the authors set out to identify transporters that must exist in Stramenophiles due to the fact that the second half of glycolysis appears to be conducted in the mitochondria. They hypothesize that a Stramenophile-specific clade of transporters related to the dicarboxylate carriers are likely the relevant family and then go on to test two proteins from Blastocystis due to the infectious disease relevance of this organism. They show rather convincingly that these two proteins are expressed and are localized to the mitochondria in the native organism. The purified proteins bind to glycolytic intermediates and one of them, GIC-2, transports several glycolytic intermediates in vitro. This is a very solid and well-executed study that clearly demonstrates that bCIC-2 can transport glycolytic intermediates.

(1) The major weakness is that the authors aren't able to show that this protein actually has this function in the native organism. This could be impossible due to the lack of genetic tools in Blastocystis, but it leaves us without absolute confidence that bGIC-2 is the important glycolytic intermediate mitochondrial transporter (or even that it has this function in vivo).

(2) My impression is that the authors under-emphasize the fact that the hDIC also binds (and is stabilized by) glycolytic intermediates (G3P and 3PG). In the opinion of this reviewer, this might change my interpretation about the uniqueness of the bGIC proteins. They act on additional glycolytic intermediates, but it's not unique.

Reviewer #3 (Public Review):

Summary:

Unlike most eukaryotes Blastocystis has a branched glycolysis pathway, which is split between the cytoplasm and the mitochondrial matrix. An outstanding question was how the glycolytic intermediates generated in the 'preparatory' phase' are transported into the mitochondrial matrix for the 'pay off' phase. Here, the authors use bioinformatic analysis to identify two candidate solute carrier genes, bGIC-1 and bGIC-2, and use biochemical and biophysical methods to characterise their substrate specificity and transport properties. The authors demonstrate that bGIC-2 can transport dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and phosphoenolpyruvate, establishing this protein as the 'missing link' connecting the two split branches of glycolysis in this branch of single celled eukaryotes. The authors also present their data on bGIC-1, which suggests a role in anion transport and bOGC, which is a close functional homologue of the human oxoglutarate carrier (hOGC, SLC25A11) and human dicarboxylate carrier (hDIC, SLC25A10).

Strengths:

The results are presented in a clear and logical arrangement, which nicely leads the reader through the process of gene identification and subsequent ligand screening and functional reconstitution. The results are compelling and well supported - the thermal stabilisation data is supported by the exchange studies. Caveats, where apparent, are discussed and rational explanations given.

Weaknesses:

The study does not contain any significant weaknesses in my view. I would like to see the authors include the initial rate plots used in the main figures (possibly as insets), so we can observe the data points used for these calculations. It would also have been interesting to include the AlphaFold models for bGIC-1 and bGIC-2 and a discussion/rationalisation for the substrate specificity discussed in the study.

eLife assessment

This valuable study explores the neural basis for a well-known auditory illusion, often utilized in movie soundtracks, in which a sequence of two complex tones can be perceived as either rising or falling in pitch depending on the context in which they are presented. Solid single-neuron data and analyses are presented to show that correlates of these pitch-direction changes are found in the ferret primary auditory cortex. The manuscript is, however, difficult to assess in places and would benefit from greater consideration of how the results fit more broadly into models of auditory coding.

Reviewer #1 (Public Review):

Summary:

Previous work demonstrated a strong bias in the percept of an ambiguous Shepard tone as either ascending or descending in pitch, depending on the preceding contextual stimulus. The authors recorded human MEG and ferret A1 single-unit activity during presentation of stimuli identical to those used in the behavioral studies. They used multiple neural decoding methods to test if context-dependent neural responses to ambiguous stimulus replicated the behavioral results. Strikingly, a decoder trained to report stimulus pitch produced biases opposite to the perceptual reports. These biases could be explained robustly by a feed-forward adaptation model. Instead, a decoder that took into account direction selectivity of neurons in the population was able to replicate the change in perceptual bias.

Strengths:

This study explores an interesting and important link between neural activity and sensory percepts, and it demonstrates convincingly that traditional neural decoding models cannot explain percepts. Experimental design and data collection appear to have been executed carefully. Subsequent analysis and modeling appear rigorous. The conclusion that traditional decoding models cannot explain the contextual effects on percepts is quite strong.

Weaknesses:

Beyond the very convincing negative results, it is less clear exactly what the conclusion is or what readers should take away from this study. The presentation of the alternative, "direction aware" models is unclear, making it difficult to determine if they are presented as realistic possibilities or simply novel concepts. Does this study make predictions about how information from auditory cortex must be read out by downstream areas? There are several places where the thinking of the authors should be clarified, in particular, around how this idea of specialized readout of direction-selective neurons should be integrated with a broader understanding of auditory cortex.

Reviewer #2 (Public Review):

The authors aim to better understand the neural responses to Shepard tones in auditory cortex. This is an interesting question as Shepard tones can evoke an ambiguous pitch that is manipulated by a proceeding adapting stimulus, therefore it nicely disentangles pitch perception from simple stimulus acoustics.

The authors use a combination of computational modelling, ferret A1 recordings of single neurons, and human EEG measurements.

Their results provide new insights into neural correlates of these stimuli. However, the manuscript submitted is poorly organized, to the point where it is near impossible to review. We have provided Major Concerns below. We will only be able to understand and critique the manuscript fully after these issues have been addressed to improve the readability of the manuscript. Therefore, we have not yet reviewed the Discussion section.

Major concerns

Organization/presentation<br /> The manuscript is disorganized and therefore difficult to follow. The biggest issue is that in many figures, the figure subpanels often do not correspond to the legend, the main body, or both. Subpanels described in the text are missing in several cases. Many figure axes are unlabelled. There is an inconsistent style of in-text citation between figures and the main text. The manuscript contains typos and grammatical errors. My suggestions for edits below therefore should not be taken as an exhaustive list. I ask the authors to consider the following only a "first pass" review, and I will hopefully be able to think more deeply about the science in the second round of revisions after the manuscript is better organized.

Frequency and pitch<br /> The terms "frequency" and "pitch" seem to be used interchangeably at times, which can lead to major misconceptions in a manuscript on Shepard tones. It is possible that the authors confuse these concepts themselves at times (e.g. Fig 5), although this would be surprising given their expertise in this field. Please check through every use of "frequency" and "pitch" in this manuscript and make sure you are using the right term in the right place. In many places, "frequency" should actually be "fundamental frequency" to avoid misunderstanding.

Insufficient detail or lack of clarity in descriptions<br /> There seems to be insufficient information provided to evaluate parts of these analysis, most critically the final pitch-direction decoder (Fig 6), which is a major finding. Please clarify.

Reviewer #3 (Public Review):

Summary:

This is an elegant study investigating possible mechanisms underlying the hysteresis effect in the perception of perceptually ambiguous Shepard tones. The authors make a fairly convincing case that the adaptation of pitch direction sensitive cells in auditory cortex is likely responsible for this phenomenon.

Strengths:

The manuscript is overall well written. My only slight criticism is that, in places, particularly for non-expert readers, it might be helpful to work a little bit more methods detail into the results section, so readers don't have to work quite so hard jumping from results to methods and back.

The methods seem sound and the conclusions warranted and carefully stated. Overall I would rate the quality of this study as very high, and I do not have any major issues to raise.

Weaknesses:

I think this study is about as good as it can be with the current state of the art. Generally speaking, one has to bear in mind that this is an observational, rather than an interventional study, and therefore only able to identify plausible candidate mechanisms rather than making definitive identifications. However, the study nevertheless represents a significant advance over the current state of knowledge, and about as good as it can be with the techniques that are currently widely available.

Wethouder Lauriks v Arnhem: schuldhulpverlening kost 17milj/jr, terwijl het om 3,5milj/jr aan schulden gaat. Een factor 5! Bron? Tegelijkertijd gaat dat geld natuurlijk ook in banen zitten. Kans op herhaling lijkt me key. Vgl daklozen experimenten en UBI.

eLife assessment

Peng et al. reported important findings that 36THz high-frequency terahertz stimulation (HFTS) could suppress the activity of pyramidal neurons by enhancing the conductance of voltage-gated potassium channels. The significance of the findings in this paper is that chronic pain remains a significant medical problem, and there is a need to find non-pharmacological interventions for treatment. The authors present convincing evidence that high-frequency stimulation of the anterior cingulate cortex can alter neuronal activity and improve sensory pain behaviors in mice.

Reviewer #1 (Public Review):

In this manuscript, by using simulation, in vitro and in vivo electrophysiology, and behavioral tests, Peng et al. nicely showed a new approach for the treatment of neuropathic pain in mice. They found that terahertz (THz) waves increased Kv conductance and decreased the frequency of action potentials in pyramidal neurons in the ACC region. Behaviorally, terahertz (THz) waves alleviated neuropathic pain in the mouse model. Overall, this is an interesting study. The experimental design is clear, the data is presented well, and the paper is well-written. I have a few suggestions.

(1) The authors provide strong theoretical and experimental evidence for the impact of voltage-gated potassium channels by terahertz wave frequency. However, the modulation of action potential also relies on non-voltage-dependent ion channels. For example, I noticed that the RMP was affected by THz application (Figure 3F) as well. As the RMP is largely regulated by the leak potassium channels (Tandem-pore potassium channels), I would suggest testing whether terahertz wave photons have also any impact on the Kleak channels as well.

(2) The activation curves of the Kv currents in Figure 2h seem to be not well-fitted. I would suggest testing a higher voltage (>100 mV) to collect more data to achieve a better fitting.

(3) In the part of behavior tests, the pain threshold increased after THz application and lasted within 60 mins. I suggest conducting prolonged tests to determine the end of the analgesic effect of terahertz waves.

(4) Regarding in vivo electrophysiological recordings, the post-HFTS recordings were acquired from a time window of up to 20 min. It seems that the HFTS effect lasted for minutes, but this was not tested in vitro where they looked at potassium currents. This long-lasting effect of HFTS is interesting. Can the authors discuss it and its possible mechanisms, or test it in slice electrophysiological experiments?

(5) How did the authors arrange the fiber for HFTS delivery and the electrode for in vivo multi-channel recordings? Providing a schematic illustration in Figure 4 would be useful.

(6) Some grammatical errors should be corrected.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Peng et al., reported that 36THz high-frequency terahertz stimulation (HFTS) can suppress the activity of pyramidal neurons by enhancing the conductance of voltage-gated potassium channel. The authors also demonstrated the effectiveness of using 36THz HFTS for treating neuropathic pain.

Strengths:

The manuscript is well written and the conclusions are supported by robust results. This study highlighted the potential of using 36THz HFTS for neuromodulation.

Weaknesses:

More characterization of HFTS is needed, so the readers can have a better assessment of the potential usage of HFTS in their own applications.

(1) It would be very helpful to estimate the volume of tissue that can be influenced by HFTS. It is not clear how 15 mins HFTS was chosen for this functional study. Does a longer time have a stronger effect? A better characterization of the relationship between the stimulus duration of HFTS and its beneficial effects would be very useful.

(2) How long does the behavioral effect last after 15 minutes of HFTS? Figure 5b only presents the behavioral effect for one hour, but the pain level is still effectively reduced at this time point. The behavioral measurement should last until pain sensitization drops back to pre-stim level.

(3) Although the manuscript only tested in ACC, it will also be useful to demonstrate the neural modulation effect on other brain regions. Would 36THz HFTS also robustly modulate activities in other brain regions? Or are different frequencies needed for different brain regions?

Reviewer #3 (Public Review):

Summary:

This manuscript by Peng et al. presents intriguing data indicating that high-frequency terahertz stimulation (HFTS) of the anterior cingulate cortex (ACC) can alleviate neuropathic pain behaviors in mice. Specifically, the investigators report that terahertz (THz) frequency stimulation widens the selectivity filter of potassium channels thereby increasing potassium conductance and leading to a reduction in the excitability of cortical neurons. In voltage clamp recordings from layer 5 ACC pyramidal neurons in acute brain slice, Peng et al. show that HFTS enhances K current while showing minimal effects on Na current. Current clamp recording analyses show that the spared nerve injury model of neuropathic pain decreases the current threshold for action potential (AP) generation and increases evoked AP frequency in layer 5 ACC pyramidal neurons, which is consistent with previous studies. Data are presented showing that ex-vivo treatment with HFTS in slice reduces these SNI-induced changes to excitability in layer 5 ACC pyramidal neurons. The authors also confirm that HFTS reduces the excitability of layer 5 ACC pyramidal neurons via in vivo multi-channel recordings from SNI mice. Lastly, the authors show that HFTS is effective at reducing mechanical allodynia in SNI using both the von Frey and Catwalk analyses. Overall, there is considerable enthusiasm for the findings presented in this manuscript given the need for non-pharmacological treatments for pain in the clinical setting.

Strengths:

The authors use a multifaceted approach that includes modeling, ex-vivo and in-vivo electrophysiological recordings, and behavioral analyses. Interpretation of the findings is consistent with the data presented. This preclinical work in mice provides new insight into the potential use of directed high-frequency stimulation to the cortex as a primary or adjunctive treatment for chronic pain.

Weaknesses:

There are a few concerns noted that if addressed, would significantly increase enthusiasm for the study.

(1) The left Na current trace for SNI + HFTS in Figure 2B looks to have a significant series resistance error. Time constants (tau) for the rate of activation and inactivation for Na currents would be informative.

(2) It is unclear why an unpaired t-test was performed for paired data in Figure 2. Also, statistical methods and values for non-significant data should be presented.

(3) It would seem logical to perform HFTS on ACC-Pyr neurons in acute slices from sham mice (i.e. Figure 3 scenario). These experiments would be informative given the data presented in Figure 4.

(4) As the data are presented in Figure 4g, it does not seem as if SNI significantly increased the mean firing rate for ACC-Pyr neurons, which is observed in the slice. The data were analyzed using a paired t-test within each group (sham and SNI), but there is no indication that statistical comparisons across groups were performed. If the argument is that HFTS can restore normal activity of ACC-Pyr neurons following SNI, this is a bit concerning if no significant increase in ACC-Pyr activity is observed in in-vivo recordings from SNI mice.

(5) The authors indicate that the effects of HFTS are due to changes in Kv1.2. However, they do not directly test this. A blocking peptide or dendrotoxin could be used in voltage clamp recordings to eliminate Kv1.2 current and then test if this eliminates the effects of HFTS. If K current is completely blocked in VC recordings then the authors can claim that currents they are recording are Kv1.1 or 1.2.

(6) The ACC is implicated in modulating the aversive aspect of pain. It would be interesting to know whether HFTS could induce conditioned place preference in SNI mice via negative reinforcement (i.e. alleviation of spontaneous pain due to the injury). This would strengthen the clinical relevance of using HFTS in treating pain.

eLife assessment

Hou and colleagues describe the the use of a previously characterized FRET sensor for use in determining gamma secretase activity in the brain of living mice. In an approach that targeted the sensor to neurons, they observe patterns of fluorescent sensor readout suggesting clustered regions of secretase activity. These results once validated would be valuable in the field of Alzheimer's Disease research, yet further validation of the approach is required, as the current evidence provided is inadequate to support the conclusions.

Reviewer #1 (Public Review):

Summary:

In their paper, Hou and co-workers explored the use of a FRET sensor for endogenous g-sec activity in vivo in the mouse brain. They used AAV to deliver the sensor to the brain for neuron specific expression and applied NIR in cranial windows to assess FRET activity; optimizing as well an imaging and segmentation protocol. In brief they observe clustered g-sec activity in neighboring cells arguing for a cell non-autonomous regulation of endogenous g-sec activity in vivo.

Weaknesses:

Overall the authors provide a very limited data set and in fact only a proof of concept that their sensor can be applied in vivo. This is not really a research paper, but a technical note. With respect to their observation of clustered activity, the images do not convince me as they show only limited areas of interest: from these examples (for instance fig 5) one sees that merely all neurons in the field show variable activity and a clustering is not really evident from these examples. Even within a cluster, there is variability. With r values between 0.23 to .36, the correlation is not that striking. The authors herein do not control for expression levels of the sensor: for instance, can they show that in all neurons in the field, the sensor is equally expressed, but FRET activity is correlated in sets of neurons? Or are the FRET activities that are measured only in positively transduced neurons, while neighboring neurons are not expressing the sensor? Without such validation, it is difficult to make this conclusion.

Secondly, I am lacking some more physiological relevance for this observation. The experiments are performed in wild-type mice, but it would be more relevant to compare this with a fadPSEN1 KI or a PSEN1cKO model to investigate the contribution of a gain of toxic function or LOF to the claimed cell non-autonomous activations. Or what would be the outcome if the sensor was targeted to glial cells?

For this reviewer it is not clear what resolution they are measuring activity, at cellular or subcellular level? In other words are the intensity spots neuronal cell bodies? Given g-sec activity are in all endosomal compartments and at the cell surface, including in the synapse, does NIR imaging have the resolution to distinguish subcellular or surface localized activities? If cells 'communicate' g-sec activities, I would expect to see hot spots of activity at synapses between neurons: is this possible to assess with the current setup?

Without some more validation and physiological relevant studies, it remains a single observation and rather a technical note paper, instead of a true research paper.

Reviewer #2 (Public Review):

Summary:

The manuscript by Hou et al is a short technical report which details the potential use of a recently developed FRET based biosensor for gamma-secretase activity (Houser et al 2020) for in vivo imaging in the mouse brain. Gamma-secretase plays a crucial role in Alzheimer disease pathology and therefore developing methodologies for precise in vivo measurements would be highly valuable to better understand AD pathophysiology in animal models.

The current version of the sensor utilizes a pair of far-red fluorescent proteins fused to a substrate of the enzyme. Using live imaging, it was previously demonstrated it is possible to monitor gamma-secretase activity in cultured cells. Notably, this is a variant of a biosensor that was previously described using CFP-YFP variants FRET pair (Maesako et al, iScience. 2020). The main claim and hypothesis for the MS is that IR excitation and emission has considerable advantages in terms of depth of penetration, as well as reduction in autofluorescence. These properties would make this approach potentially suitable to monitor cellular level dynamics of Gama-secretase in vivo.

The authors use confocal microscopy and show it is possible to detect fluorescence from single cortical cells. The paper described in detail technical information regarding imaging and analysis. The data presented in figures 5-8 details analysis of FRET ratio (FR) measurements within populations of cells. The authors claim it is possible to obtain reliable measurements at the level of individual cells. They compare the FR values across cells and mice and find a spatial correlation among neighboring cells. This is compared with data obtained after inhibition of endogenous gamma-secretase activity, which abolishes this correlation.

Strengths:

The authors describe in detail their experimental design and analysis for in vivo imaging of the reporter. The idea of using a far-red FRET sensor for in vivo imaging is novel and potentially useful to circumvent many of the pitfalls associated with intensity-based FRET imaging in complex biological environments (such as autofluorescence and scattering).

Weaknesses:

There are several critical points regarding validation of this approach and concerns with the data presented that must be addressed:

(1) Regarding the variability and spatial correlation- the dynamic range of the sensor previously reported in vitro is in the range of 20-30% change (Houser et al 2020) whereas the range of FR detected in vivo is between cells is significantly larger (Fig. 3). This raises considerable doubts for specific detection of cellular activity (see point 3).<br /> (2) One direct way to test the dynamic range of the sensor in vivo, is to increase or decrease endogenous gamma-secretase activity and to ensure this experimental design allows to accurately monitor gamma-secretase activity. In the previous characterization of the reporter (Hauser et al 2020), DAPT application and inhibition of gamma-secretase activity results in increased FR (Figures 2 and 3 of Houser et al). This is in agreement with the design of the biosensor, since FR should be inversely correlated with enzymatic activity. Here, while the authors repeat the same manipulation and apply DAPT to block gamma-secretase activity, it seems to induce the opposite effect and reduces FR (comparing figures 8 with figures 5,6,7). First, there is no quantification comparing FR with and without DAPT. Moreover, it is possible to conduct this experiment in the same animals, meaning comparing FR before and after DAPT in the same mouse and cell populations. This point is absolutely critical- if indeed FR is reduced following DAPT application, this needs to be explained since this contradicts the basic design and interpretation of the biosensor.<br /> (3) For further validation, I would suggest including in vivo measurements with a sensor version with no biological activity as a negative control, for example, a mutation that prevents enzymatic cleavage and FRET changes. This should be used to showcase instrumental variability and would help to validate the variability of FR is indeed biological in origin. This would significantly strengthen the claims regarding spatial correlation within population of cells.<br /> (4) In general, confocal microcopy is not ideal for in vivo imaging. Although the authors demonstrate data collected using IR imaging increases penetration depth, out of focus fluorescence is still evident (Figure 4). Many previous papers have primarily used FLIM based analysis in combination with 2p microscopy for in vivo FRET imaging (Some examples: Ma et al, Neuron, 2018; Massengil et al, Nature methods, 2022; DIaz-Garcia et al, Cell Metabolism, 2017; Laviv et al, Neuron, 2020). This technique does not rely on absolute photon number and therefore has several advantage sin terms of quantification of FRET signals in vivo.<br /> It is therefore likely that use of previously developed sensors of gamma-secretase with conventional FRET pairs, might be better suited for in vivo imaging. This point should be at least discussed as an alternative.

Reviewer #3 (Public Review):

This paper builds on the authors' original development of a near infrared (NIR) FRET sensor by reporting in vivo real-time measurements for gamma-secretase activity in the mouse cortex. The in vivo application of the sensor using state of the art techniques is supported by a clear description and straightforward data, and the project represents significant progress because so few biosensors work in vivo. Notably, the NIR biosensor is detectable to ~ 100 µm depth in the cortex. A minor limitation is that this sensor has a relatively modest ΔF as reported in Houser et al, which is an additional challenge for its use in vivo. Thus, the data is fully dependent on post-capture processing and computational analyses. This can unintentionally introduce biases but is not an insurmountable issue with the proper controls that the authors have performed here.

The observation of gamma-secretase signaling that spreads across cells is potentially quite interesting, but it can be better supported. An alternative interpretation is that there exist pre-formed and clustered hubs of high gamma-secretase activity, and that DAPT has stochastic or differential accessibility to cells within the cluster. This could be resolved by an experiment of induction, for example, if gamma-secretase activity is induced or activated at a specific locale and there was observed coordinated spreading to neighboring neurons with their sensor.

Furthermore, to rule out the possibility that uneven viral transduction was not simply responsible for the observed clustering, it would be helpful to see an analysis of 670nm fluorescence alone.

https://taz.de/CO2-Abscheidung-bei-der-Hannover-Messe/!6003357/

reply to u/bastugubbar at https://www.reddit.com/r/typewriters/comments/1ca8nwk/i_for_one_welcome_our_new_taylor_swift_overlords/

Let's be honest here, the most recent typewriter reference (presumably to that of an ex-boyfriend) is certainly not her first. I'm a modest Swiftie at best (from a trivia perspective), preferring to think of her work as poetry rather than musical pop-culture, so I imagine her more as a quill pen sort of writer, though my notes indicate she does take some of her notes for composition using her cell phone.

This being said, a few years back she did feature a red Sears Cutlass in All Too Well (10 Minute Version) (Taylor's Version) at the 8:28 mark, which hasn't driven the cost of these through the roof, though I have seen one listed for $1,000 (it unsurprisingly didn't sell for that.) For more here see Robert Messenger at OzTypewriter and Ryan Schocket for Buzzfeed. It's not listed anymore, but this past Christmas, she also had a red typewriter Christmas tree ornament in her online store.

Those who were privileged to attend the recent Eras Tour (or see it on Disney+) saw groups of typewriters in the background during several songs.

She's been featuring typewriters for a bit now and it hasn't driven prices through the roof any more than the typewriter renaissance that's been going on for the last few years or so. I suspect that this new round of references isn't going to shift things significantly.

If she does go full-typewriter, which model(s) do you suspect she'd be using amidst the pantheon of other writers? I'd suggest she may be romantic enough to do a late 40's Smith-Corona Clipper... or perhaps while jet-setting a Skyriter?

Type on!

The social model of disability is an approach to disability. The model identifies systemic barriers as the main issues facing people with disabilities. This contrasts with the medical model of disability. The medical model of disability identifies disability as an impairment of the individual.

In this source, the fundamental concept of inclusive design is discussed, including topics such as identifying the issue, addressing inclusive design, history, and other related topics. This site is intriguing to me since it presents numerous instances of inclusive design in our everyday lives that we often overlook, such as playgrounds and parks, text fonts on our phones, and other similar examples.

Hypothesis

Results

I think a very novel point is being made here. The concept of disability is one that is defined by the majority of people in society. If the people we consider disabled were in another society, would they then become ordinary people. We need to think about whether we are putting too much pressure on disabled people.

You are on the verge or falling asleep but you keep yourself awake. Is this a good example?

It seems to me that we have more control over our shaping of behaviors and habits than we have in our actions (free will). So, since we can only process one thought / perform one action at a time, we are training our habitual state of being with each sample.

It might also be the case that this is a way of saving cognitive load and saving energy for something more important at the time. I'd like to use survival as an explanation here.

"we could have made alternative decisions or taken different paths than the ones we actually took."

"we believe that we are the ones actively generating and controlling them. It suggests a sense of agency over our current mental processes and behaviors."

Transparent Peer Review

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

want the links

It is intriguing to consider the possibility that individuals may inadvertently conceal their disability by engaging in masking activities, which may have a negative impact on both their emotional and physical well-being. The fact that individuals modify their conduct in order to conform to the standards and expectations of society, often while hiding their own challenges, is something that is seen and accepted on a regular basis in everyday life. One concern that comes as a result of this conversation is how we may facilitate the creation of surroundings that are more inclusive and do not lay the responsibility of adaptation only on people who have impairments. In order to do this, it is necessary to take into account the ways in which the institutions and attitudes of society may either help or hinder persons with disabilities in terms of their coping mechanism and general well-being.

similar to - how nature works - physiosphere - the way people think - symbolosphere

reference - https://hyp.is/go?url=https%3A%2F%2Fwww.physics.utoronto.ca%2Fpeople%2Fprofiles%2Frobert-k-logan%2Fsymbolosphere-conceptualiztion-language-and-neo-dualism%2F&group=world

follow up - book - The Turning Point - author Fritjof Capra

from https://hyp.is/rKawpAEpEe-8BWfaUKjlmA/www.linkedin.com/posts/daniel-christian-wahl-51a54616_transformative-education-in-a-crisis-of-activity-7187710848671379456-gCqc/

for - symbolosphere - origins - definition - symbolosphere - definition - physiosphere - definition - neo-dualism - Robert K. Logan - John H. Schumann

origins - symbolosphere - John H. Schumann introduces the complimentary notions of

definition - symbolopshere - the non-physical world of symbolic relationships that includes all its thoughts and communication processes such as language

definition - physiosphere - the physical world, including the human brain.

• This paper introduces these terms in the context of a concept they developed called "neo-dualism",

definition - neo-dualism - a way pragmatic form of dualism that distinguishes mind and brain in the current understanding of neuroscience that is unable to provide an adequate explanation connecting the two.

SOURCE:

Councillors Linda Annis of Surrey, Daniel Fontaine of New Westminster, and Alexa Loo of Richmond, say they will bring motions to their councils that would call on the B.C. NDP government to scrap the three-year experiment that started on Jan. 31, 2023.

My story overlaps a bit with this reading, Its fascinating what stories people have to say about the places they grew up in and lived there.

The first thing that comes to mind is the trading of goods between people and a place where people could offer their services.

for - transition - rapid whole system change - Daniel Christian Wahl

to - https://medium.com/age-of-awareness/transformative-education-in-a-crisis-of-perception-a846075f57e0

for - rapid whole system change - Speed & Scale

summary - hmmm....what's mssing? - They don't explicitly promote citizen led action - They are still using the net zero by 2050 story, - which in many critics eyes is actually far too little and too late - See Kevin Anderson's critique of net zero - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=net%2Bzero - They don't address inequality, decolonialization or climate justice issues - They don't identify meta or polycrisis

from - https://hyp.is/J7oIeAEpEe-J1kuOInb20A/www.linkedin.com/posts/colinleduc_we-are-launching-our-speed-scale-2024-global-activity-7188309472837021696-SxSf/

for - rapid whole system change - Speed & Scale - Ryan Panchadsaram

to - https://hyp.is/6DUsiAEnEe-Yr5Ojo_wTSg/speedandscale.com/

Sharing one's experiences with others is a way to help people learn more about you and your background and also lets you reflect on yourself.

Banding students from different parts of the world with different backgrounds helps students learn different perspective.

for - Adverse Childhood Experiences - ACE - Aces Too High - childhood trauma - Intergenerational harm

Summary - A great resource that examines how adverse childhood experiences turns into harmful adult behavior that perpetuates the cycle - Most of the most famous pathological leaders of the modern era, including contemporary ones are examined from the perspective of their ACE

an

?

knew

I wonder if this is true because of the at home audience of the show. I would assume that most viewers are women who have the ability to stay home during the day and watch a daytime television show. This indicates that they likely have the resources to "work on themselves" and make different choices and try new things. The audience is likely not, say, a single, uneducated mother who is working to support her children at one of the jobs that she can get without a degree.

This notion (not just on this particular topic) likely harmed the show and the discussions that could be had on it. I would assume that this meant that only people from marginalized groups who were willing to play this game, and not more radical ones who would really defend themselves as being different and not wanting to fit into 'the norm' would go on the show.

There is something interesting about this inclusion in this article.. It seems as if the authors also can't put themselves in the shoes of the mother, who likely is not proud of what she had to do, but had to get her husband to work so that they could bring in money to live. I understand where they're coming from, but I think that this is unnecessary and sort of goes against the whole point and piles on the already attacked mother.

This seems like the show was using the allusion of being understanding and pushing boundaries to simply parrot the popular belief back to the public while patting themselves on the back for addressing the topic.

It's interesting that the studio audience (I'd assume) has the ability to travel to (likely) another city in the middle of a weekday to attend this taping. It seems as if they have the freedom of either not having children to look after or the financial ability/support system to have other look after them while they go to the studio. This shows how this setup does not work in terms of creating an empathetic space for people to share their stories, as the audience can't put themselves in the shoes of a family where there is one car and a parent who works nights.

I think this is true and I think often times now, theyre just for comedy rather than being forreal.

I wonder if this has changed as we see the government changing and currently what is going on in the world.

I see this as I grew up watching Oprah with my own African American family, and I see that Oprah has influenced many other people from marginalized communities to tune in to her show at the time.

This is especially true considering what is currently going on in America and how other people in other countries would rather deal with what America has going on than whats going on here.

I think that this is especially true considering the fact of how television is a unique way of storytelling that can connect people.

This whole analysis has been quite eye opening to the nature of the show. While the intentions of themes were positive, the reality is that in the process of trying to achieve great, it failed to do good. I think there is no doubt that Oprah Winfrey meant well, however, the way topics were addressed and tackled ended up reinforcing the core problems rather than helping solve them.

This is interesting to read after also watching the "Talk Show Murder" episode. Despite ethics always being a hot point in media, its interesting to see that ethics can technically be violated without much repercussions when the "violation" is done within the perspective of "helping others". It's another case where media can be too intrusive on someone's life and causes people to forget that the people on the show are human too.

I feel like this is a double-edged sword. I think it is a fantastic window for people to view therapy in a less stigmatized way, however, on that same token, it can easily be viewed as a substitute for professional help. On top of that, comparing this version of "therapy" to professional therapy, the cost difference also leans into this idea of "why pay a lot of money for a therapist, when I could get some therapy through my television?"

через api gateway - можно делать, те же запросы к backend. условно он уже отыграет роль proxy. он просто перенаправит (forwarding) на другой url

api gateway - даёт возможность, создавать уже своё api, для взаимодействия со внутреними сервисами yandec cloud

можно просто через это api gateway, создать запрос к другому сервису. apigateWay можно сказать играет роль swagger, он некоторый слой, который помогает именно отправлять запросы куда-то.

Limitations: no consideration of precedence constraints, shared resources, multiprocessors, overhead caused in a mode change

picking the best values ig

oh ok so we're actually investigating this

maybe he's doing averages...

still kinda weird idk

still weird tf

This is why energy is set to 1

Again assuming the WCET is like 5x the actual execution time

This makes sense as you'd expect the utilization of all HI-criticality tasks using C_HI values to be higher than the utilization of all HI-criticality tasks using C_LO values

utilization of all HI-criticality tasks using C_LO values

Ok so we're differing little thingies

Ok but this seems much more accurate so what's tea...

biggest difference along with slowing down speed in urgent mode...why is using actual execution times valid in urgent mode again...?

so randomly choosing a task period in between 1000 and 5000 inclusive

Maybe EAU does this more efficiently than Algorithm A idk

so no power-saving in the urgency mode

So the speeds we just discussed in the example before are not possible aurrrrkkayyy

Ok let's go with that

ok work let's go with that

This seems fake lol

post comment - Linked In SBTi controversy

public support

LOSE-LOSE FOR EVERYONE.

creates hard-to-break cycles and permanently ruins families, with small chance of redemption.

racism

making more things illegal rises crime rates instead of preventing people from doing those things.

proof of racial prejudice

corrupt motives

Nixon blows up these issues to get people on his side

widespread misinformation

beginning of regulation

beginning of drug use

по сути - это спецификая, которая помогает создавать единый стиль restApi. по сути спецификая, как и до этого было написано, удобный элемент для того, чтоб программам, работать с api в написаний документаций

possible evidence of effectiveness

end goal is to help stop drug use

provides home and resources for those in need

proof of efficiency

testimony to the helpfulness

introduction of first hand source

the blind leading the blind honey

So DVFS can't do anything to help save power dissipated by I/O and memory operations

rho is processor speed methinks

Phusion polymerase. Tested on ZymoBIOMICS Microbial Community DNA Standard

Figure out how much this is better by

why ecosystem specific databases vs general ones?

As opposed to a taxonomic assignment

government support

counterargument

evidence

first site

explanation

definition