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I've heard about this in designing multiple times. However, I don't think it is possible to create a platform or environment that have options for everyone. There must be someone that we could not empathize on. In Utilitarian approach, as long as our design maximize its potential of usage in groups of people, it should work.

Yes, I think in today's society designers do have a responsibility to ensure that the environment is suitable for everyone. This behavior is not very popular in China, so I rarely see disabled people on the street. I think they may have limited mobility and the environment around them is not suitable for them. In the United States, I can often see disabled people on the street, and they seem to be integrated into life like normal people. In hotel elevators, you can see points to help blind people understand, and in trains and buses, Tools to assist in getting in and out of wheelchairs.

After reading this passage, I understand that the strategy of people with disabilities "masking" their disabilities is very taxing. It also reveals that social environments often place the responsibility of adaptation on individuals, reflecting a lack of societal support.

As someone who doesn't have a disability that's visible on the surface, architectural designs are the designs that I notice almost instantly in public places. And by this I mean the addition of ramps, placement of braille on signs, bathroom rails, entry ways (bigger doors, glass windows etc.). These mostly categorize physical disabilities, but as our society has progressed and has started to acknowledge that people have mental disabilities/illnesses, I have noticed that more public spots have accommodated spaces for people to have mental/brain breaks. These are mostly populated in work offices, but outside of the U.S, you can see these spots in very public spaces, like malls and parks. I definitely think that architecture needs to be updated for all categorizations of people with disabilities, but it's nice to know that the disabilities that didn't once get attention, are now being factored into designs.

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Citizens

from : Gien LinkedIn post on Citizen

I remember in my learning of INFO360, our instructor mentioned the word, "empathy" in designs. One group of people that we should empathize is people with disability. I'm wondering the disability mentioned in this book would also count as a disability in design? Are they similar?

People with conditions like chronic pain, mental health issues, or sensory impairments that are not immediately apparent face significant barriers not only in accessing support but also in having their conditions recognized as legitimate. This calls for a broader understanding and sensitivity towards all forms of disability, emphasizing that not all disabilities are apparent and that all deserve equal recognition and accommodation.

One of the sources cited in the chapter discusses the principles of universal design in architecture. This source emphasizes that designing environments and products to be usable by all people, to the greatest extent possible, without the need for adaptation or specialized design, not only assists individuals with disabilities but also benefits the broader population.

I think we should take special care of people who are different from us. For example, for people with color blindness, we should acknowledge their difficulties and design specific functions that make things fair for them. We define what 'ordinary' is; as a result, we should also help other people who are not ordinary to be as fair as we can.

This makes me wonder, what about people who are prone to addiction? Research shows some people are more likely to become addicted to others, and addiction, after all, is a disease. What can be done as a accessibility feature to prevent these people from developing addictions through social media and the internet? Additionally, what resources are available for people who have a rare disability that impacts their use of social media? What could be done for them?

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eLife assessment

This important study uses a novel light sheet imaging technique to investigate how different TLR4 agonists regulate Myddosome formation. The data showing that LPS and A-beta can control the kinetics and size of Myddosome assembly are compelling. This paper should be of substantial interest to the innate immunity field.

Reviewer #1 (Public Review):

Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event triggering inflammation and overcoming Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event in triggering inflammation and overcoming infection by gram-negative bacteria. However, TLR4 has recently been found to respond to other endogenously derived ligands. This has implicated TLR4 signaling in the development of disease pathology, for example, Alzheimer's disease, through the recognition of amyloid-beta. Intriguingly, the signaling response to these non-bacterial-derived ligands differs from that of bacterial-derived LPS, suggesting mechanistic differences between endogenous and bacterial-derived agonists. In this work, the authors set out to characterize these mechanistic differences. TLR4 signals through two large macromolecular complexes that assemble at activated receptors: the Myddosome and Triffosome. One hypothesis the authors aimed to test was that different ligands alter these signaling complexes' kinetics and nano-scale features. The authors focused on testing this hypothesis by examining the formation of the Myddosome in live cells. A significant strength of the paper is that the authors developed technological innovations to address this problem. Using a nanopipette delivery mechanism combined with light sheet microscopy, the authors could observe Myddosome signaling in the whole cell volume of live macrophages. This allowed them to accurately quantify the Myddosome number, size, and kinetics of complex formation and compare cells stimulated with amyloid-beta and LPS. The authors discovered differences in Myddosomes formed under LPS versus amyloid-beta stimulation. In general, amyloid-beta TLR4 stimulation resulted in slower Myddosome formation with altered morphology. One limitation of the work, which the authors point out in the discussion, is that they could not distinguish signaling-competent Myddosomes. Future work will be needed to understand whether these amyloid beta induced Myddosomes assembly have a similar or altered complement of downstream signaling proteins (such as the IRAK4/1 and TRAF6). Secondly, the structural basis for how TLR4 would distinguish between different radically agonists remains speculative, and will need further investigation. Nonetheless, this paper is important for the technological innovation to look at the molecular dynamics of signal transduction, a technology that could be adapted to study other receptor signaling pathways.

It is already known that the subcellular location of intracellular TLRs is important for limiting the recognition of self-derived ligands and maintaining tolerance. This work hints at another possible layer of regulation: that a cell surface TLR (TLR4) generates diverse signaling outcomes to extrinsic or intrinsically derived agonists by changing the dynamic behavior of signaling proteins. If correct (and much further work is required to understand endogenous TLR ligands better), it might suggest that the innate immune system employs the same molecular hardware but with altered kinetics to distinguish between exogenous and endogenous inflammatory signals. Thus, pathological aggregates or markers of sterile inflammation might be recognized and responded to by a specific signaling program that is defined kinetically. It will be an interesting direction for future studies to investigate whether and how diverse pathogen and endogenous inflammatory signals modulate the dynamics of signaling complexes.

eLife assessment

This important study describes exhaustive deep mutational scanning (DMS) of the gonadotropin-releasing hormone wild-type receptor and for two single point mutations that impact its folding and structure, monitoring how plasma membrane expression levels are influenced by the introduced mutations. With solid evidence, the authors have pioneered an exploration of the interaction between mutations (epistasis) in a membrane protein, with a potential for explaining membrane protein evolution and genetic diseases.

Reviewer #1 (Public Review):

Summary:

The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influences TM insertion (V267T) and ii) influences protein stability (W107A) and then measures the effect of these mutants on correct plasma membrane expression (PME).

Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.

Strengths:

This is a novel and exhaustive approach to dissect how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.

Weaknesses:

The premise for the study requires an in-depth understanding of how the single point mutations analysed effect membrane protein folding in context of DMS, but the single point mutants used could do with further validation. The V267T mutant only reduced MP insertion by 10% and the effect of W107A on protein stability was not assessed. Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can effect trafficking and potential post-translational modifications, like glycosylation.

Reviewer #2 (Public Review):

Summary:

In this paper, Chamness and colleagues make a pioneering effort to map epistatic interactions among mutations in a membrane protein. They introduce thousands of mutations to the mouse GnRH Receptor (GnRHR), either under wild-type background or two mutant backgrounds, representing mutations that destabilize GnRHR by distinct mechanisms. The first mutant background is W107A, destabilizing the tertiary fold, and the second, V276T, perturbing the efficiency of cotranslational insertion of TM6 to the membrane, which is essential for proper folding. They then measure surface expression of these three mutant libraries, using it as a proxy for protein stability, since misfolded proteins do not typically make it to the plasma membrane. The resulting dataset is then used to shed light on how diverse mutations interact epistatically with the two genetic background mutations. Their main conclusion is that epistatic interactions vary depending on the degree of destabilization and the mechanism through which they perturb the protein. The mutation V276T forms primarily negative (aggravating) epistatic interactions with many mutations, as is common to destabilizing mutations in soluble proteins. Surprisingly, W107A forms many positive (alleviating) epistatic interactions with other mutations. They further show that the locations of secondary mutations correlate with the types of epistatic interactions they form with the above two mutants.

Strengths:

Such a high throughput study for epistasis in membrane proteins is pioneering, and the results are indeed illuminating. Examples of interesting findings are that: (1) No single mutation can dramatically rescue the destabilization introduced by W107A. (2) Epistasis with a secondary mutation is strongly influenced by the degree of destabilization introduced by the primary mutation. (3) Misfolding caused by mis-insertion tends to be aggravated by further mutations. The discussion of how protein folding energetics affects epistasis (Fig. 7) makes a lot of sense and lays out an interesting biophysical framework for the findings.

Weaknesses:

The major weakness comes from the potential limitations in the measurements of surface expression of severely misfolded mutants. It seems that only about 5% of the W107A makes it to the plasma membrane compared to wild-type. This point is discussed quite fairly in the paper. (Figures 2 and 3). This might be a low starting point from which to accurately measure the effects of secondary mutations. I am concerned about the extent to which surface expression can report on protein stability, especially when it comes to double mutants where each mutation alone severely decreases surface expression. It is possible that in these cases, both the single and double mutants are completely misfolded, beyond repair. The surface-expressed proteins in such mutants may not be stable, folded or active at all, and the authors do not provide any indication that the combined effects of the mutations are derived from effects on folding stability or misfolding. Therefore, the reason for the epistatic effects of these mutations is hard to interpret, leaving a notable gap in our understanding. However, I find that this point is discussed much more fairly in the current manuscript.

With that said, I believe that the results regarding the epistasis of V276T with other mutations are strong and very interesting on their own.

Another concern relates to the measurements of the epistatic effects of mutations in the background of the V107A mutation. I am concerned about their measurement accuracy. Firstly, the authors note that the surface immunostaining measurements of these mutants are on average only 2-fold above background, which is quite a low signal-to-noise regimen. Secondly, I believe that the authors still haven't demonstrated the reproducibility of their surface expression measurements. To showcase the reproducibility, the authors show the correlation of two biological replicates in Figure S3. However, these are shown only for the 251 mutations that passed a reproducibility filter, after the authors "discarded variant scores for which the difference in percentile rank across the two replicates was greater than 25%. " . this means that all mutations that showed irreproducible results were filtered out before the analysis in Figure S3. It is, therefore, no surprise that the remaining mutations are highly reproducible, and such an analysis cannot serve as an indication of the reproducibility. It remains possible that a large fraction of the surface immunostaining scores of the V107A variants are dominated by noise and that their correlation in these two replicates might be random and may not necessarily be reproduced in a third replicate, for example.

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eLife assessment

This important study provides solid evidence, both from biochemical analyses and in vivo mouse models, that soluble uric acid serves as an enzymatic inhibitor of the NADase CD38, thereby impacting inflammatory responses. By shedding light onto the intricate interplay between uric acid and CD38, the authors highlight potential therapeutic avenues for inflammatory and age-related conditions, which may be of interest to medical biologists, biochemists, and cell biologists. Further in vivo and in vitro validation suggested would be helpful to cement the significance and implications of these findings.

Reviewer #1 (Public Review):

This manuscript describes soluble Uric Acid (sUA) as an endogenous inhibitor of CD38, affecting CD38 activity and NAD+ levels both in vitro and in vivo. Importantly, the inhibition constants calculated support the claim that sUA inhibits CD38 under physiological conditions. These findings are of extreme importance to understanding the regulation of an enzyme that has been shown to be the main NAD+/NMN-degrading enzyme in mammals, which impacts several metabolic processes and has major implications for understanding aging diseases. The manuscript is well written, the figures are self-explanatory, and in the experiments presented, the data is very solid. The authors discuss the main limitations of the study, especially in regard to the in vivo results. As a whole, I believe that this is a very interesting manuscript that will be appreciated by the scientific community and that opens a lot of new questions in the field of metabolism and aging. I found some issues that I believe constitute a weakness in the manuscript, and although they do not require new experiments, they may be considered by the authors for discussion in the final version of the manuscript.

The authors acknowledge the existence of several previous papers involving pharmacological inhibition of CD38 and their impact on several models of metabolism and aging. However, they only cite reviews. Given the focus of the manuscript, I believe that the seminal original papers should be cited.

Related to the previous comment, the authors show that they have identified the functional group on sUA that inhibits CD38, 1,3-dihydroimidazol-2-one. How does this group relate with previous structures that were shown to inhibit CD38 and do not have this chemical structure? Is sUA inhibiting CD38 in a different site? A crystallographic structure of CD38-78c is available in PDB that could be used to study or model these interactions.

Although the mouse model used to manipulate sUA levels is not ideal, the authors discuss its limitations, and importantly, they have CD38 KO mice as control. However, all the experiments were performed in very young mice, where CD38 expression is low in most tissues (10.1016/j.cmet.2016.05.006). This point should be mentioned in the discussion and maybe put in the context of variations of sUA levels during aging.

Reviewer #2 (Public Review):

Summary:

This is an interesting work where Wen et al. aimed to shed light on the mechanisms driving the protective role of soluble uric acid (sUA) toward avoiding excessive inflammation. They present biochemical data to support that sUA inhibits the enzymatic activity of CD38 (Figures 1 and 2). In a mouse model of acute response to sUA and using mice deficient in CD38, they find evidence that sUA increases the plasma levels of nicotinamide nucleotides (NAD+ and NMN) (Figure 3) and that sUA reduces the plasma levels of inflammasome-driven cytokines IL-1b and IL-18 in response to endotoxin, both dependent on CD38 (Figure 4). Their work is an important advance in the understanding of the physiological role of sUA, with mechanistic insight that can have important clinical implications.

Strengths:

The authors present evidence from different approaches to support that sUA inhibits CD38, impacts NAD+ levels, and regulates inflammatory responses through CD38.

Weaknesses:

The authors investigate macrophages as the cells impacted by sUA to promote immunoregulation, proposing that inflammasome inhibition occurs through NAD+ accumulation and sirtuin activity due to sUA inhibition of CD38. Unfortunately, the study still lacks data to support this model, as they could not replicate their in vivo findings using murine bone marrow-derived macrophages, a standard model to assess inflammasome activation. Without an alternative approach, the study lacks data to establish in vitro that sUA inhibition of CD38 reduces inflammasome activation in macrophages - consequently, they cannot determine yet if both NAD+ accumulation and sirtuin activity in macrophages is a mechanism leading to sUA role in vivo.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors propose that soluble Uric acid (sUA) is an enzymatic inhibitor of the NADase CD38 and that it controls levels of NAD modulating inflammatory response. Although interesting the studies are at this stage preliminary and validation is needed.

Strengths:

The study characterizes the potential relevance of sUA in NAD metabolism.

Weaknesses:

(1) A full characterization of the effect of sUA in other NAD-consuming and synthesizing enzymes is needed to validate the statement that the mechanism of regulation of NAD by sUA is mediated by CD38, The CD38 KO may not serve as the ideal control since it may saturate NAD levels already. Analysis of multiple tissues is needed.

(2) The physiological role of sUA as an endogenous inhibitor of CD38 needs stronger validation (sUA deficient model?).

(3) Flux studies would also be necessary to make the conclusion stronger.

eLife assessment

The authors analyze the relationship between human mobility and genomic data of SARS-CoV-2 using mobile phone mobility data and sequence data and present a solid proof of concept. This useful work was conducted on a fine spatial scale and provides suggestions on how mobility-derived surveillance could be conducted, although these results are mixed. The primary significance of this work is the strong use of large datasets that were highly granular. The authors provide a rigorous study, but with less clear predictive power of mobility to inform transmission patterns.

Reviewer #1 (Public Review):

Summary:

In "1 Exploring the Spatial Distribution of Persistent SARS-CoV-2 Mutations -Leveraging mobility data for targeted sampling" Spott et al. combine SARS-CoV-2 genomic data alongside granular mobility data to retrospectively evaluate the spread of SARS-CoV-2 alpha lineages throughout Germany and specifically Thuringia. They further prospectively identified districts with strong mobility links to the first district in which BQ.1.1 was observed to direct additional surveillance efforts to these districts. The additional surveillance effort resulted in the earlier identification of BQ.1.1 in districts with strong links to the district in which BQ.1.1 was first observed.

Strengths:

There are two important strengths of this work. The first is the scale and detail in the data that has been generated and analyzed as part of this study. Specifically, the authors use 6,500 SARS-CoV-2 sequences and district-level mobility data within Thuringia. I applaud the authors for making a subset of their analyses public e.g. on the associated micro react page.

Further, the main focus of the article is on the potential utility of mobility-directed surveillance sequences. While I may certainly be mistaken, I have not seen this proposed elsewhere, at least in the context of SARS-CoV-2. The authors were further able to test this concept in a real-world setting during the emergence of BQ.1.1. This is a unique real-world evaluation of a novel surveillance sequencing strategy and there is considerable value in publishing this analysis.

Weaknesses:

The article is quite strong and I find the analyses to generally be rigorous. However, there are places where I believe the text should be modified to slightly weaken the conclusions drawn from the presented analyses. Specific examples include:

- It seems the mobility-guided increased surveillance included only districts with significant mobility links to the origin district and did not include any "control" districts (those without strong mobility links). As such, you can only conclude that increasing sampling depth increased the rate of detection for BQ.1.1., not necessarily that doing so in a mobility-guided fashion provided an additional benefit. I absolutely understand the challenges of doing this in a real-world setting and think that the work remains valuable even with this limitation, but I would like the lack of control districts to be more explicitly discussed.

- Line 313: While this work has reliably shown that the spread of Alpha was slower in Thuringia, I don't think there have been sufficient analyses to conclude that this is due to the lack of transportation hubs. My understanding is that only mobility within Thuringia has been evaluated here and not between Thuringia and other parts of Germany.

- Line 333 (and elsewhere): I'm not convinced, based on the results presented in Figure 2, that the authors have reliably identified a sampling bias here. This is only true if you assume (as in line 235) that the variant was in these districts, but that hasn't actually been demonstrated here. While I recognize that for high-prevalence variants there is a strong correlation between inflow and variant prevalence, low-prevalence variants by definition spread less and may genuinely be missing from some districts. To support this conclusion that they identified a bias, I'd like to see some type of statistical model that is based e.g. on the number of sequences, prevalence of a given variant in other districts, etc. Alternatively, the language can be softened ("putative sampling bias").

Reviewer #2 (Public Review):

In the manuscript, the authors combine SARS-CoV-2 sequence data from a state in Germany and mobility data to help in understanding the movement of the virus and the potential to help decide where to focus sequencing. The global expansion in sequencing capability is a key outcome of the public health response. However, there remains uncertainty about how to maximise the insights the sequence data can give. Improved ability to predict the movement of emergent variants would be a useful public health outcome. Also knowing where to focus sequencing to maximising insights is also key. The presented case study from one State in Germany is therefore a useful addition to the literature. Nevertheless, I have a few comments.

One of the key goals of the paper is to explore whether mobile phone data can help predict the spread of lineages. However, it appears unclear whether this was actually addressed in the analyses. To do this, the authors could hold out data from a period of time, and see whether they can predict where the variants end up being found.

The abstract presents the mobility-guided sampling as a success, however, the results provide a much more mixed result. Ultimately, it's unclear what having this strategy really achieved. In a quickly moving pandemic, it is unclear what hunting for extra sequences of a specific, already identified, variant really does. I'm not sure what public health action would result, especially given the variant has already been identified.

Relatedly, it is unclear to me whether simply relying on spatial distance would not be an alternative simpler approach than mobile phone data. From Figure 2, it seems clear that a simple proximity matrix would work well at reconstructing viral flow. The authors could compare the correlation of spatial, spatial proximity, and CDR data.

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Q&A with Typewriter Collector Steve Soboroff by [[American Writers Museum]]

Reviewer #1 (Public Review):

Summary:

Zanzibar archipelago is close to achieve malaria elimination, but despite the implementation of effective control measures there is still low level seasonal malaria transmission. This could be due to the frequent importation of malaria from the mainland Tanzania and Kenya, reservoir of asymptomatic infections and competent vectors. To investigate population structure and gene flow of P. falciparum in Zanzibar and mainland Tanzania, they used 178 samples from mainland Tanzania and 213 from Zanzibar that were previously sequenced using molecular inversion probes (MIPs) panels targeting single nucleotide polymorphisms (SNPs). They performed Principal Component Analysis (PCA) and identity by descent (IBD) analysis to assess genetic reladness between isolates. Parasites from coastal mainland Tanzania contribute for the genetic diversity in parasite population in Zanzibar. Despite this, there is a pattern of isolation by distance and microstructure within the achipelago, and evidence of local sharing of highly related strains sustaining malaria transmission in Zanzibar that are important targets for interventions such as mass drug administration and vector control, in addition to measures against imported malaria.

Strengths:

This study presents important samples to understand population structure and gene flow between mainland Tanzania and Zanzibar, especially from rural Bagamoyo District, where malaria transmission persists and there is a major port of entry to Zanzibar. In addition, this study includes a larger set of SNPs, providing more robustness for analyzes such as PCA and IBD. Therefore, the conclusions of this paper are well supported by data.

Comments on revised version:

The authors answered all my questions.

Not sure what to make of this, a combination of Solid Pods and AP, with Solid being the data storage. Meaningless refs to data ownership (you don't own your data on Fedi, you've spread it to 100s of servers around the globe with each message. You don't even have control over the database you use in your client, unless self-hosted. You can move, without your messages.) It's just that nobody afayk mines the stuff for adtech. And data ownership doesn't legally exist in most parts of the world. So what is the purpose of Solid here, if you store recipes and ephemeral socmed messages in it? Just that it's there so you can skip having to build the database part of an AP server / client combo? So that everyone can run their personal instance with something that can also do other things? It doesn't say but that would be a potential step up (assuming people know how to run a solid pod that is).

= HTML as a data exchange format

eLife assessment

This study reports an important mechanism through which the TGF-beta signaling pathway promotes contacts between oocytes and their surrounding somatic cells by regulating the number of transzonal projections (TZPs) in mice. Convincing data support the conclusions. The work will be of interest to biomedical researchers who work on ovarian biology and female fertility.

eLife assessment

This study presents useful findings regarding the role of formin-like 2 in mouse oocyte meiosis. Some of the data are supported by incomplete methodological details and analyses, and several conclusions are overstated. This paper would be of interest to reproductive biologists.

Reviewer #1 (Public Review):

Summary:

The presented study focuses on the role of formin-like 2 (FMNL2) in oocyte meiosis. The authors assessed FMNL2 expression and localization in different meiotic stages and subsequently, by using siRNA, investigated the role of FMNL2 in spindle migration, polar body extrusion, and distribution of mitochondria and endoplasmic reticulum (ER) in mouse oocytes.

Strengths:

Novelty in assessing the role of formin-like 2 in oocyte meiosis

Weaknesses:

Overstating some of the presented data

Unconvincing analysis of the endoplasmic reticulum and mitochondria distribution

The authors addressed all my comments. The section materials and methods was improved. However, some statements still need to be clarified, as they seem to be overstated. I'm still not convinced about the main findings. For example, the analysis of ER and mitochondria distribution was based on a subjective assessment of clustering in meiosis I oocytes, and it's missing objective parameters and timing of the analysis.

Comments on revised version:

The authors addressed all my comments. The section materials and methods was improved. However, some statements still need to be clarified, as they seem to be overstated.

Reviewer #2 (Public Review):

Summary:

This research involves conducting experiments to determine the role of Fmnl2 during oocyte meiosis I.

Strengths:

Identifying the role of Fmnl2 during oocyte meiosis I is significant.

Weaknesses:

The quantitative analysis and the used approach to perturb FMNL2 function would benefit from more confirmatory approaches and rigorous analysis.

Comments on revised version:

The authors addressed most of my comments. However, some comments were not addressed convincingly.

My concern is still valid. The authors used only one approach to knockdown FMNL2 which is "siRNA-mediated knockdown". Using an additional approach to inhibit FMNL2 (Trim-Away or morpholino,..) would be beneficial to confirm that the effect of siRNA-mediated knockdown of FMNL2 is specific.

Response 1: In the author's response, they mentioned that successful migration was quantified based on the contact between the spindle pole and the oocyte cortex.<br /> After spindle migration, it is very common for the spindle to be close to (but not in contact with) the cortex for a considerable time. The spindle pole comes in contact with the cortex later (just before anaphase onset and polar body extrusion). Fig. 3A shows an example where at 9 h, the spindle is already migrated but did not come in contact with the cortex until 9:30 h. Based on Fig. 3B,C, the authors assessed spindle migration in fixed oocytes, making it impossible to fix all oocytes at the time of spindle contact with the cortex. Also,<br /> the representative images in Fig. 3C do not show spindle staining to assess the contact between the spindle and the cortex.<br /> Overall, I still believe that the distance between the spindle and the cortex is more accurate for quantifying spindle migration.

Response 2: The authors mentioned, "we made appropriate modifications to the relevant descriptions of immunoprecipitation experiments". I can't find these modifications in the manuscript. The authors need to state clearly that the immunoprecipitation results do not necessarily reflect meiotic oocytes specifically because these experiments were done using the whole ovary which contains both somatic cells and oocytes.

Response 5: The authors mentioned that "Based on our observations, during the extrusion of the first polar body in oocytes, there is a temporary occurrence of cellular morphological fragmentation due to cortical reorganization". Unfortunately, this means that the live imaging system in the authors' laboratory is not ideal for oocyte maturation. Several publications show normal oocyte morphology during cytokinesis. Please delete or replace Fig. 2E.

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

eLife assessment

This study presents valuable new insights into a HIV-associated nephropathy (HIVAN) kidney phenotype in the Tg26 transgenic mouse model, and delineates the kidney cell types that express HIV genes and are injured in these HIV-transgenic mice. A series of compelling experiments demonstrated that PKR inhibition can ameliorate HIVAN with reversal of mitochondrial dysfunction (mainly confined to endothelial cells), a prominent feature shared in other kidney diseases. The data support that inhibition of PKR and mitochondrial dysfunction has potential clinical significance for HIVAN.

Reviewer #1 (Public Review):

Summary:

HIV associated nephropathy (HIVAN) is a rapidly progressing form of kidney disease that manifests secondary to untreated HIV infection, and is predominantly seen in individuals of African descent. Tg26 mice carrying an HIV transgene lacking gag and pol exhibit high levels of albuminuria and rapid decline in renal function that recapitulates many features of HIVAN in humans. HIVAN is seen predominantly in individuals carrying two copies of missense variants in the APOL1 gene, and the authors have previously shown that APOL1 risk variant mRNA induces activity of the double strand RNA sensor kinase PKR. Because of the tight association between the APOL1 risk genotype and HIVAN, the authors hypothesized that PKR activation may mediate the renal injury in Tg26 mice, and tested this hypothesis by treating mice with a commonly used PKR inhibitory compound called C16. Treatment with C16 substantially attenuated renal damage in the Tg26 model as measured by urinary albumin/creatinine ratio, urinary NGAL/creatinine ratio and improvement in histology. The authors then performed bulk and single-nucleus RNAseq on kidneys from mice from different treatment groups to identify pathways and patterns of cell injury associated with HIV transgene expression as well as to determine the mechanistic basis for the effect of C16 treatment. They show that proximal tubule nuclei from Tg26 mice appear to have more mitochondrial transcripts which was reversed by C16 treatment and suggest that this may provide evidence of mitochondrial dysfunction in this model. They explore this hypothesis by showing there is a decrease in the expression of nuclear encoded genes and proteins involved in oxidative phosphorylation as well as a decrease in respiratory capacity via functional assessment of respiration in tubule and glomerular preparations from these mouse kidneys. All of these changes were reversed by C16 treatment. The authors propose the existence of a novel injured proximal tubule cell-type characterized by the leak of mitochondrial transcripts into the nucleus (PT-Mito). Analysis of HIV transgene expression showed high level expression in podocytes, consistent with the pronounced albuminuria that characterizes this model and HIVAN, but transcripts were also detected in tubular and endothelial cells. Because of the absence of mitochondrial transcripts in the podocytes, the authors speculate that glomerular mitochondrial dysfunction in this model is driven by damage to glomerular endothelial cells.

Strengths:

The strengths of this study include the comprehensive transcriptional analysis of the Tg26 model, including an evaluation of HIV transgene expression, which has not been previously reported. This data highlights that HIV transcripts are expressed in a subset of podocytes, consistent with the highly proteinuric disease seen in mouse and humans. However, transcripts were also seen in other tubular cells, notably intercalated cells, principal cells and injured proximal tubule cells. Though the podocyte expression makes sense, the relevance of the tubular expression to human disease is still an open question.

The data in support of mitochondrial dysfunction are also robust and rely on combined evidence from downregulation of transcripts involved in oxidative phosphorylation, decreases in complex I and II as determined by immunoblot, and assessments of respiratory capacity in tubular and glomerular preparations. These data are largely consistent with other preclinical renal injury model reported in the literature as well as previous, less thorough assessments in the Tg26 model.

Weaknesses:

The key weakness of the study lies in the use of a PKR inhibitor with questionable specificity. C16 has been reported to inhibit numerous other kinases including cyclin CDKs and GSK3α and -β, and this means that the conclusions of this study with respect to the role of PKR are highly questionable. The rationale for the dose used was not provided (and is lower than used in other publications with C16), and in the absence of drug exposure data and assessment of target engagement, it is difficult to ascertain whether substantial inhibition of PKR was achieved.

A second key weakness lies in the identification of the PT-Mito cell cluster. Though the authors provide some rationale for the identification of this specific cell type, it seems equally plausible the cells merely reflect a high background capture of mitochondria in a subset of droplets. The IHC analysis that was provided is not convincing enough to support the claim and more careful high resolution imaging and in situ hybridization (with appropriate quantitation) will be needed to provide substantive support for the presence of a proximal tubule cell type with mitochondrial transcript that are trafficked to the nucleus.

Revision summary:

The authors have revised the manuscript to acknowledge the potential limitations of the C16 tool compound used and have performed some additional analyses that suggest the PT-Mito population can be identified in samples from KPMP. The authors added some control images for the in situ hybridizations, which are helpful, though they don't get to the core issue of limited resolution to determine whether mitochondrial RNA is present in the nuclei of injured PT cells. Some additional work has been done to show that C16 treatment results in a decrease in phospho-PKR, a readout of PKR inhibition. These changes strengthen the manuscript by providing some evidence for the translatability of the PT-mito cluster to humans and some evidence for on-target activity for C16. It would be helpful if the authors could quantify the numbers of cells in IHC with nuclear transcripts as well as pointing out some specific examples in the images provided, as comparator data for the snRNAseq studies in which 3-6% of cortex cells had evidence of nuclear mitochondrial transcripts.

Reviewer #2 (Public Review):

Summary:

Numerous studies by the authors and other groups have demonstrated an important role for HIV gene expression kidney cells in promoting progressive chronic kidney disease, especially HIV associated nephropathy. The authors had previously demonstrated a role for protein kinase R (PKR) in a non-HIV transgenic model of kidney disease (Okamoto, Commun Bio, 2021). In this study, the authors used innovative techniques including bulk and single nuclear RNAseq to demonstrate that mice expressing a replication-incompetent HIV transgene have prominent dysregulation of mitochondrial gene expression and activation of PKR and that treatment of these mice with a small molecule PKR inhibitor ameliorated the kidney disease phenotype in HIV-transgenic mice. They also identified STAT3 as a key upstream regulator of kidney injury in this model, which is consistent with previously published studies. Other important advances include identifying the kidney cell types that express the HIV transgene and have dysregulation of cellular pathways.

Strengths:

Major strengths of the study include the use of a wide variety of state-of-the-art molecular techniques to generate important new data on the pathogenesis of kidney injury in this commonly used model of kidney disease and the identification of PKR as a potential druggable target for the treatment of HIV-induced kidney disease. The authors also identify a potential novel cell type within the kidney characterized by high expression of mitochondrial genes.

Weaknesses:

Though the HIV-transgenic model used in these studies results in a phenotype that is very similar to HIV-associated nephropathy in humans, the model has several limitations that may prevent direct translation to human disease, including the fact that mice lack several genetic factors that are important contributors to HIV and kidney pathogenesis in humans. Additional studies are therefore needed to confirm these findings in human kidney disease.

Reviewer #1 (Public Review):

Summary:

The authors constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the key coding regions. They showed that intranasal inoculation with the candidate vaccine-induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs and protected against a wild-type SARS-CoV-2 strain with D614G mutation and the latest Omicron subvariant (BA.5) strain. The neutralizing antibodies induced by BK2102 persisted for the long term (up to 364 days). Furthermore, they confirmed the safety of the proposed vaccine using transgenic (Tg) mice expressing human ACE2 (hACE2).

Strengths:

The authors followed a robust methodology to establish the proposed vaccine's protective effect and safety profile in the hamsters and transgenic mice expressing human ACE2.

Weaknesses:

(1) A comparative safety assessment of the available m-RNA and live attenuated vaccines will be necessary. The comparison should include details of the doses, neutralizing antibody titers with duration of protection, tissue damage in the various organs, and other risks, including virulence reversal.

(2) The vaccine's effect on primates is doubtful. The study fails to explain why only two of four monkeys developed neutralizing antibodies. Information about the vaccine's testing in monkeys is also missing: What was the level of protection and duration of the persistence of neutralizing antibodies in monkeys? Were the tissue damages and other risks assessed?

(3) The vaccine's safety in immunosuppressed individuals or individuals with chronic diseases should be assessed. Authors should make specific comments on this aspect.

(4) The candidate vaccine has been tested with a limited number of SARS-CoV-2 strains. Of note, the latest Omicron variants have lesser virulence than many early variants, such as the alfa, beta, and delta strains.

(5) Limitations of the study have not been discussed.

Reviewer #2 (Public Review):

Summary:

In this manuscript "Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms" by Suzuki-Okutani et al., the authors evaluate the attenuation, immunogenicity, and protection efficacy of a live-attenuated SARS-CoV-2 vaccine candidate (BK2102) against SARS-CoV-2.

Strengths:

The authors demonstrate that intranasal inoculation of BK2102 is safe and able to induce humoral and cellular immune responses in hamsters, without apparent signs of damage in the lungs, that protects against homologous SARS-CoV-2 and Omicron BA.5 challenge. Safety of BK2102 was further confirmed in a new hACE2 transgenic mouse model generated by the authors.

Weaknesses:

No major weaknesses were identified, however, this reviewer notes the following:

The authors missed the opportunity to include a mRNA vaccine to demonstrate that the immunity and protection efficacy of their live attenuated vaccine BK2102 is better than a mRNA vaccine.

One of the potential advantages of live-attenuated vaccines is their ability to induce mucosal immunity. It would be great if the authors included experiments to assess the mucosal immunity of their live-attenuated vaccine BK2102.

Reviewer #3 (Public Review):

Summary:

Suzuki-Okutani and collogues reported a new live-attenuated SARS-CoV-2 vaccine (BK2102) containing multiple deletion/substitution mutations. They show that the vaccine candidate is highly attenuated and demonstrates a great safety profile in multiple animal models (hamsters and Tg-Mice). Importantly, their data show that single intranasal immunization with BK2102 leads to strong protection of hamsters against D614G and BA.5 challenge in both lungs and URT (nasal wash). Both humoral and cellular responses were induced, and neutralization activity remained for >360 after a single inoculation.

Strengths:

The manuscript describes a comprehensive study that evaluates the safety, immunogenicity, and efficacy of a new live-attenuated vaccine. Strengths of the study include (1) strong protection against immune evasive variant BA.5 in both lungs and NW; (2) durability of immunity for >360 days; (3) confirmation of URT protection through a transmission experiment.

While first-generation COVID-19 vaccines have achieved much success, new vaccines that provide mucosal and durable protection remain needed. Thus, the study is significant.

Weaknesses:

Lack of a more detailed discussion of this new vaccine approach in the context of reported live-attenuated SARS-CoV-2 vaccines in terms of its advantages and/or weaknesses.

Antibody endpoint titers could be presented.

Lack of elaboration on immune mechanisms of protection at the upper respiratory tract (URT) against an immune evasive variant in the absence of detectable neutralizing antibodies.

eLife assessment

This fundamental study uses a creative experimental system to directly test Ohno's hypothesis, which describes how and why new genes might evolve by duplication of existing ones. In agreement with existing criticism of Ohno's original idea, the authors present compelling evidence that having two gene copies does not speed up the evolution of a new function as posited by Ohno, but instead leads to the rapid inactivation of one of the copies through the accumulation of mostly deleterious mutations. These findings will be of broad interest to evolutionary biologists and geneticists.

Reviewer #1 (Public Review):

Overview:

The authors construct a pair of E. coli populations that differ by a single gene duplication in a selectable fluorescent protein. They then evolve the two populations under differing selective regimes to assess whether the end result of the selective process is a "better" phenotype when starting with duplicated copies. Importantly, their starting duplicated population is structured to avoid the duplication-amplification process often seen in bacterial artificial evolution experiments. They find that while duplication increases robustness and speed of adaptation, it does not result in more highly adapted final states, in contrast to Ohno's hypothesis.

Major comments:

This is an excellent study with a very elegant experimental setup that allows a precise examination of the role of duplication in functional evolution, exclusive of other potential mechanisms. My main concern is to clarify some of the arguments relating to Ohno's hypothesis.

I think my main confusion on first reading the manuscript was in the precise definition of Ohno's hypothesis. I think this confusion was mine and not the authors, but it is likely common and could be addressed.

Most evolutionary biologists think of gene duplication as making neofunctionalization "easier" by providing functional redundancy and a larger mutational target, such that the evolutionary process of neofunctionalization is faster (as the authors observed). In this framework, the final evolved state might not differ when selection is applied to duplicated copies or a single-copy gene. Ohno's hypothesis, by contrast, argues that there generally exist adaptive conflicts between the ancestral function and the "desired" novel function, such that strong selection on a single-copy gene cannot produce the evolutionary optima that selection on two copies would. This idea is hinted at in the quotation from Ohno in paragraph 2 of the introduction. However, the sentences that follow I don't think reinforce this concept well enough and lead to some confusion.

With that definition in mind, I agree with the authors' conclusion that these data do not support Ohno's hypothesis. My quibble would be that what is actually shown here is that adaptive conflict in function is not universal: there are cases where a single gene can be optimized for multiple functions just as well as duplicated copies. I do not think the authors have, however, refuted the possibility that such adaptive conflicts are nonetheless a significant barrier to evolutionary innovation in the absence of gene duplication generally. Perhaps just a sentence or two to this effect might be appropriate.

I also think the authors need to clarify their approach to normalizing fluorescence between the two populations to control for the higher relative protein expression of the population with a duplicated gene. Since each population was independently selected with the highest fluorescing 60% (or less) of the cells selected, I think this normalization is appropriate. Of course, if the two populations were to compete against each other, this dosage advantage of the duplicates would itself be a selective benefit. Even as it is, the dosage advantage should be a source of purifying selection on the duplication, and perhaps this should be noted.

Finally, I am slightly curious about the nature of the adaptations that are evolving. The authors primarily discuss a few amino-acid changing mutations that seem to fix early in the experiment. Looking at Figure 3, it however, appears that the populations are still evolving late in the experiment, and so presumably other changes are occurring later on. Do the authors believe that perhaps expression changes to increase protein levels are driving these later changes?

Reviewer #2 (Public Review):

Summary:

Drawing from tools of synthetic biology, Mihajlovic et al. use a cleverly designed experimental system to dissect Ohno's hypothesis, which describes the evolution of functional novelty on the gene-level through the process of duplication & divergence.

Ohno's original idea posits that the redundancy gained from having two copies of the same gene allows one of them to freely evolve a new function. To directly test this, the authors make use of a fluorescent protein with two emission maxima, which allows them to apply different selection regimes (e.g. selection for green AND blue, or, for green NOT blue). To achieve this feat without being distracted by more complex evolutionary dynamics caused by the frequent recombination between duplicates, the authors employ a well-controlled synthetic system to prevent recombination: Duplicates are placed on a plasmid as indirect repeats in a recombination-deficient strain of E.coli. The authors implement their directed evolution approach through in vitro mutagenesis and selection using fluorescent-activated cell sorting. Their in-depth analysis of evolved mutants in single-copy versus double-copy genotypes provides clear evidence for Ohno's postulate that redundant copies experience relaxed purifying selection. In contrast to Ohno's original postulate, however, the authors go on to show that this does not in fact lead to more rapid phenotypic evolution, but rather, the rapid inactivation of one of the copies.

Strengths:

This paper contributes with great experimental detail to an area where the literature predominantly leans on genomics data. Through the use of a carefully designed, well-controlled synthetic system the authors are able to directly determine the phenotype & genotype of all individuals in their evolving populations and compare differences between genotypes with a single or double copy of coGFP. With it they find clear evidence for what critics of Ohno's original model have termed "Ohno's dilemma", the rapid non-functionalization by predominantly deleterious mutations.

Including an expressed but non-functional coGFP in (phenotypically) single copy genotypes provides an especially thoughtful control that allows determining a baseline dN/dS ratio in the absence of selection. All in all the study is an exciting example of how the clever use of synthetic biology can lead to new insights.

Weaknesses:

The major weakness of the study is tied to its biggest strength (as often in experimental biology there is a trade-off between 'resolution' and 'realism').

The paper ignores an important component of the evolutionary process in favour of an in-depth characterization of how two vs one copy evolve. Specifically, by employing a recombination-deficient strain and constructing their duplicates as inverted repeats their experimental design completely abolishes recombination between the two copies.

This is problematic for two reasons:

i) In nature, new duplicates do not arise as inverted, but rather as direct (tandem) repeats and - as the authors correctly point out - these are very unstable, due to the fact that repeated DNA is prone to recA-dependent homologous recombination (which arise orders of magnitude more frequently than point mutations).

ii) This instability often leads to further amplification of the duplicates under dosage selection both in the lab and in the wild (e.g. Andersson & Hughes, Annu. Rev. Genet. 2009), and would presumably also be an outcome under the current experimental set-up if it was not prevented from happening?

So in sum, recombination between duplicate genes is not merely a nuisance in experiments, but occurring at extremely high frequencies in nature (such that the authors needed to devise a clever engineering solution to abolish it), and is often observed in evolving populations, be it in the laboratory or the wild.

The manuscript sells controlling of copy number as a strength. And clearly, without it, the same insights could not be gained. However, if the basis for the very process of what Ohno's model describes is prevented from happening for the process to be studied, then, for reasons of clarity and context this needs pointing out, especially, to readers less familiar with the principles of molecular evolution.

Connected to this, there are several places in the introduction and the discussion where I feel that the existing literature, in particular models put forward since Ohno that invoke dosage selection (such as IAD) end up being slightly misrepresented.

My point is best exemplified in line 1 of Discussion: "To test Ohno's hypothesis and to distinguish its predictions from those of competing hypotheses, it is necessary to maintain a constant and stable copy number of duplicated genes during experimental evolution."

I think this statement is simply not true and might be misleading. To take the exaggerated position of a devil's advocate, the goal of evolutionary biology should be to find out how evolution actually proceeds in nature most of the time, rather than creating laboratory systems that manage to recapitulate influential ideas.

While fixing copy number may be a necessary step to understand how one copy evolves if a second one is present, it seems that if Ohno's hypothesis only works out in recA-deficient bacterial strains and on engineered inverted repeats, that Ohno might have missed one crucial aspect of how paralogs evolve. The mentioned competing hypotheses have been put forward to (a) address Ohno's dilemma (which the present study beautifully demonstrates exists under their experimental conditions) and (b) to reflect a commonly observed evolutionary process in bacteria (dosage gain in response to selection, e.g. a classic way of gaining antibiotic resistance). Fixing the copy number allowed the authors to show which predictions of Ohno's model hold up and which don't (under these specific conditions). But they do so without even preventing the processes described by alternative models from happening, so the experimental system is hardly appropriate to distinguish between Ohno & alternatives. Therefore, I think it could be made clearer that the experimental system is great to look at certain aspects Ohno's hypothesis in detail, but it can only inform us about a universe without recombination.

(1) Citing the works by ref 8, 26, 27 to merely state that "in some copies were gained and some were lost (ref 6, ref 25)" makes it seem as if fixing at 2 copies is some sort of sensible average. Yet ref 6 (Dhar et al.) specifically states that dosage is the most important response. Moreover, in this study gene copies are lost, but plasmid copies are gained instead. In Holloway et al. 2007 (ref 25), the 2 copies resided on different plasmids, so entirely different underlying molecular genetics might be at work (high cost of plasmid maintenance, and competitive binding on both proteins onto the respective (off)-target, where either way selection favored a single copy, so a different situation altogether). In both cited studies, fixing the copy would have prohibited learning something about the process of duplication & divergence.

Hence this statement seems to distract the readers from the main message, which seems that preventing recombination experimentally allows to follow the divergence of each copy and studying a response that does not involve dosage-increase.

(2) "These studies highlighted the importance of gene duplication in providing fast adaptation under changing environmental conditions but they focused on the importance of gene dosage." I think this constructs a false dichotomy. Instead, these studies pointed out that dosage (and with it, selection for dosage) is an important part of the equation that might have been missed by Ohno.

(3) "Such models are also easier to test experimentally, because they do not require precise control of gene copy number. The necessary tests can even benefit from massive gene amplifications8. Although Ohno's hypothesis is more difficult to test experimentally (...)" - again, I feel the wording is slightly misleading. The point is not that IAD is easier to test and Ohno's model is harder to test in laboratory experiments, rather, experiments (and some more limited observations of naturally evolving populations) seem to suggest that in reality evolution proceeds (more often?) according to IAD rather than Ohno's neofunctionalization hypothesis. However, as the authors point out, it will be exciting to see their clever experimental system used to test other genes and conditions to get a more comprehensive understanding of what gene- and selection- parameter values would overcome Ohno's dilemma.

This statement reflects the speaker's frustration with the reality that the speaker's parents are reluctant to interact with the speaker's teachers or participate in school activities due to work commitments or limitations in understanding how to participate in the educational system. The challenges faced by families under economic pressures are numerous, financial pressures and life burdens prevent parents from investing a lot of energy into their children's education and lives, it is not that they don't want to be involved in their children's upbringing it is that the burdens of life don't give them a chance to breathe. Lack of parental support and encouragement can lead to low self-esteem due to anxiety caused by family financial pressure and lack of love. The cyclical pattern of economic hardship impeding educational support perpetuates the disconnect between poor families and educational institutions.

This paragraph provides an insightful description of the psychology and impact of poverty on students in the school environment. It conveys deep-rooted societal attitudes that equate economic status with personal worth, leading to feelings of inferiority among the economically disadvantaged, as well as affecting social interactions and academic recognition. This experience of social exclusion based on economic disparity highlights the wider social problem of economic prejudice and its profound impact on children's self-esteem and social development. We need to be more informed and aware of how sensitive groups recognize how socio-economic factors affect interactions and perceptions in educational settings.

This segment discusses the complexities that can arise in educational settings regarding parental involvement. Act reflects on the various challenges that students face due to lack of parental support. Examples include parents not attending school events or not signing academic records, which are often interpreted as a lack of commitment to a child's academic life. It may not be that parents are irresponsible or do not keep their promises, but rather that heavy workloads or other constraints limit their involvement in their children's educational activities. Do not make hasty judgments about families and emphasize the need for empathy in assessing students.

It reflects the warmth that families give to their children and the positive attitudes towards life, despite economic difficulties. The strength of family bonds and the emotional support of family members is crucial. The speaker appreciates the love and relationships with parents and siblings as providing the basis for happiness and stability. The main drawback was the financial burden of hard-working parents, which emphasized a common aspect of poverty, namely that hard work is a necessity and not an option.

The sense of powerlessness that economic stability remains elusive despite constant effort and hard work is expressed here. This severe poverty is contrasted with the state of poverty that everyone reviews and flirts with. I believe that many people can empathize with the state of not being able to make ends meet through repetitive work day in and day out, but few people truly understand the harshness of reality.

This is what we have to face to improve the quality of teachers. Graduating from a prestigious school does not necessarily mean a responsible and quality educator. What we need to see more is the feedback of students. To see what students need rather than what we hope they need.

However, it is becoming more and more difficult to realize the American dream. For poor local families, I don’t see any way for them to rise up in class other than studying and having innate talents. So if everyone can succeed, why are there so many billions of people still struggling with poverty? I believe that learning is the only way to jump across classes.

It is true that sometimes blindly being fair will only affect students, and teaching students in accordance with their aptitude is the best solution. Sometimes what students need is not absolute fairness but mutual respect for people and equality of human rights. This does not mean that class will suppress people, but class does have an impact.

Therefore, some people think that the success of private schools is deserved. This is also a very difficult part of public schools, and it is also a great thing. Students who have been abandoned by private schools, students whose families cannot provide help, and students who come from poor backgrounds. All these negative factors will affect the ratings of public schools. But this is also the school that society needs most. Most people cannot afford to go to private schools, and studying is their only way out.

Recent reports show that more children are living in severe poverty than before, which affects their education and well-being. There are significant overlaps between race and poverty, with a high percentage of Black and Latino children living in poor conditions.

This class gap will indeed make students feel inequality and disparity. However, in the study of knowledge, the students decide what can be learned. In this aspect, it is fair. It does not mean that he can learn more just because he is rich, nor does he learn more because he is poor. I cannot know what the teacher taught me. No matter how much effort you put into learning this aspect, there will be equal rewards. Therefore, learning is the simplest and fairest way to make a positive leap.

Social safety net reduces need to be competitive which reduces drive to submit to market

I believe that teaching students in accordance with their aptitude is the highest realm of education. It is indeed very difficult but we can work harder to get closer to this aspect. On a smaller scale, you can pay attention to why students failed to complete their Homework several times. Generally speaking, why should we choose the major we like? These are very meaningful things for students.

I think this is also relative. The average level varies depending on the region. We have no way to use economic level to comment on the happiness of a family. Moreover, the economic level in the suburbs is lower than the average level in the city, and life in the suburbs may even be more nourishing and happy.

I think the homeless people on the street should be regarded as absolutely poor. They have no clothes, no food to eat, and no place to clean. Survival is a problem every day, and there is no time to think about how to achieve class advancement.

As I wrote in a book a few days ago, there are only three ways to turn around in this situation. Reading, learning and increasing knowledge. Only with certain success in the academic field can you have the opportunity to break out of the class. Second, just as divorce above can lead to poverty, marriage can also escape poverty and achieve class advancement. In China, this often occurs among traditional women. Marrying into a good family is still the wish of some Chinese families for their daughters. The third type is that if you are reincarnated well, your class will be determined at birth.

The divorce was indeed unexpected, but when I think about it, it makes sense because it is a huge blow to my property and spiritual world. But compared with divorce, I feel that environmental disasters and health problems are more sudden and unprepared.

This article introduces a book called Justice by Design: Community-Led Practices to Build the World We Need. This book is about design, power and social justice and what the relationship between these three is. The design method of "design justice" is used to challenge structural inequality. At the same time the book shows how universalist design principles and practices can eliminate certain groups.

Color blindness is a hereditary condition where people lack certain types of cones needed to distinguish colors. We should take special care of these individuals because they perceive things differently from us. It's important to be aware of this potential difference. As an example, I enjoy playing League of Legends, and I believe the color blindness mode has made significant efforts to improve the overall experience for those with color blindness.

I strongly disagree with this post, and believe that the author is ignorant. I have an autistic brother, and have worked with many autistic children throughout my life. Austism significantly impacts their life in many ways. The vast majority of autistic kids have symptoms that impact their day-to-day activities, and it is foolish to write an article arguing that all forms of autism are not disabilities when there are so many different symptoms and impacts that autism can have on a person's life that are not accounted for in this article.

I think this is such a cool empowering product. It lets people with parkinson's disease to regain a sense of normality, which can be really important. One question I have is if there is a way to make this product without the tech, such as having the rubber part be filled with liquid or a weight. Possibly this way this product can be cheaper/more accessible to people.

I think Cynthia Bennett's personal website is a great resource that shows her research in human-computer interaction. Her achievements in accessibility and inclusive design offer important perspectives for discussing disability and technology.

From what we see on the internet of parents posting their children with autism having meltdowns, these videos create false narratives of what it means to have autism. The article talks about how autism isn’t actually a disability itself but can cause disability in certain aspects. This is a good way of explaining what autism is like and how all the meltdown videos misguide us.

The company under the name "Liftware", designed two models of spoons to assist individuals who have physical disabilities, like hand tremors or other mobility issues. Both of the spoons have stabilizing labels that are triggered by sensors that are translated through a user's hands. The stabilizing levels either stop the spoon from shaking, or prevent the spoon from moving in level -- keeping itself flat and level. One spoon is specifically designed for hand tremors and the other is designed for individuals who have trouble with arm mobility/range of motion. The product was designed with the intention of helping these people eat more confidently, but also easily.

All I can think of is that the Native Americans are always left out of the conversation.

I wanted to highlight this as a wider idea that admitting one is wrong or that one's stance is incorrect is a large part of these supremacist beliefs. It can also be related with understanding that not everyone has the same problems or needs the same solution.

It is always interesting to look back at these prior issues and realize they are 100% still current issues. Language and policies have always been and still are to keep white people in power despite the overwhelming majority knowing that we are all human beings.

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Dans le projet d'éducation à la sexualité en France, certaines recommandations ne sont pas pleinement mises en œuvre. Voici quelques-unes d'entre elles :

1. Allocation des Heures : Bien que le programme énonce des recommandations, la question du nombre d'heures allouées à cet enseignement interdisciplinaire reste non résolue. Il est essentiel de déterminer un temps adéquat pour couvrir les contenus de manière approfondie.

2. Collaboration avec les Partenaires : Le programme mentionne la collaboration avec des partenaires extérieurs agréés, mais il n'est pas toujours clair comment cette collaboration est effectivement mise en place. Une meilleure coordination avec ces partenaires pourrait renforcer l'efficacité de l'éducation à la sexualité.

3. Formation des Enseignants : Bien que le programme souligne l'importance de la formation des enseignants, il est essentiel de veiller à ce que tous les enseignants reçoivent une formation adéquate et continue pour dispenser cet enseignement de manière efficace.

4. Évaluation et Suivi : Le suivi de la mise en œuvre du programme et l'évaluation de son impact sont essentiels pour apporter des ajustements et améliorations. Cependant, ces aspects ne sont pas toujours systématiquement mis en place.

Il est important de continuer à travailler sur ces points afin d'améliorer l'efficacité et la pertinence de l'éducation à la sexualité dans les écoles françaises.

I will move it to the languaculture part in order to let "what is culture" stand out in the beginning.

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1. The first box on this page is author's perspective. We will probably want to keep it with heading of "author's perspective.
2. The heading, what does it have to do with language can be moved below in languaculture section to let "what is culture:stand out in the beginning.

https://web.archive.org/web/20240424051714/https://www.rabbit.tech/research

Seems the basic idea is: - Have a person perform a task (much like Automation on a laptop) and then the Rabbit (from taskrabbit no doubt) will be able to do variations on it. Automating in the style of [[Standard operating procedures met parameters 20200820202042]] - the device is a relatively simple edge , with most of the compute depending on data centers. This runs counter to smart edges, and interestingly counter to how they market the device (as no subscriptions or APIs needed). The simple edge should make it affordable and the centralised compute should make it scalable.

выбор

база

напоминать себе о лишениях

https://web.archive.org/web/20240424050235/https://www.rabbit.tech/rabbit-os

RabbitR1 claims about their workings allowing user actions not just information.

The Rabbit people say their LAM is a new type of foundation model, to be able to deduce user intention and decided on actions. Sounds like the cli tool I tried, but cutting human out of the loop to approve certain steps. Need to see their research what they mean by 'new foundation model'

Rabbit R1 is a personal AI assistant in a retro box. Supposedly without subscription fees, but with access to AI services and with internet connection. Designed to be able to take action (kind of like the promptchaining cli tool I tried out?). Says it has a LAM next to LLM, a 'large action model' which sounds like marketing rather than tech.

According to #dgcnect this page will track all EDICs set up by MS through implementing decisions by the EC. They also said every EDIC is expected to have their own website with contact details and composition. So no company register style set-up with UBO type info.

Здесь юзается брокер сообщений, по сути робот берёт ссылку кидает в брокер и потом обработчики, кто свободен берёт эту ссылку и обрабатывает её. если он не смог обработать, это ссылка опять в брокере. она выходит из таймаута видимости. время пока ссылка не будет видна другим обработчикам

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comment

this is comment

Honestly for me I can get it that you need to take a break from staring at a peice of paper for a long time. It is always good to take a break to be able to catch more mistakes and just have a fresh mind on what you are reading to decide if something needs more and or less in certain areas in writing.

short superior to long?

This article does not include any statements from Megan or anyone representing her side of the situtation.

The lawsuit is not over the incident but instead the overall harassment he faced while working for Megan.

The story is also odd which can increase peoples interest in the story.

People care about this story and it is being written because it is a famous person people know.

Title includes all pieces. A name people are drawn to and the crazy story to interest people.

The wood provides physical labor and rough work for these people

remnants of the surrounding town

Just enough. Just as it need be. Like everything else.

The northside has been growing longer, the south side of the street was houses that I would assume have been since deconstructed

Planted in 1870 by the queen. Interestingly enough, those trees have started to rot and are being chopped down:https://www.fallenandfelled.co.uk/blogs/news/kensington-palace-garden-planes

On the former grounds of Kensington Palace

Couple things: Elio ties the transition between immature to mature as the acceptance of a elongated, convoluted, and contradictory identity that cannot be condensed into words. Elio also displays this immaturity through one key behaviour: His "smuggling"of as much information into the fewest possible words, indicating his desire to condense his identity. Thirdly, in the next line, what that gives him in terms of appearance, he is unconfident and that juxtaposes him with Oliver

"Forced to call I want", implies societal pressure to put labels on feelings... What does Elio think of this? Assigning definitions based on symptoms. Based on others telling you -- this is the transition that Elio takes to become Oliver.

Yet again the skin motif -- his duality is what Elio had been searching for -- and it appears in a sexual manner but it really connects to the matter of Oliver's security of identity and of being a whole, even when he recognises that he cannot be one.

Oliver wrote a book on Heraclitus, the main connector between his ideology, characterization, and the theory of universal flux, that one may not necessarily be one's past temporal part -- but one who continues it, like an illusion of movement.

An assumption, like many others (such as the bathing suit situation) about Oliver's identity that is quickly refuted, because identities never make sense. A person as a whole cannot be summarized in rules or statements or if.. then.. conditions.

How does this structure, without the quotations, deviate from other dialogue. What does it imply about these listing of hobbies, or listing of identity, and what is its effect on us, reading? How does this tie into Aciman's exploration of what identity really is? How does it connect to what WORDS mean?

Does this also foreshadow or symbolize his desire for Oliver?

Motif of skin introduced in CMBYN, where Oliver's duality of skin, tanned, and pink and untouched represents the multidimensionality of identity, and the contradictions that exist within him -- which is what fascinates Elio. The coexistence of both contradictions in such a beautiful, whole, masterpiece who has affinities leaping out of him is enlightening for Elio. Elio may see Oliver as an Elio who he wishes to mature into.

Figure out the implications of this

Does this foreshadow the duality and complexity of their relationship? Because there is a period of time when Elio is in an internal conflict with his desire and lack of desire for Oliver.

Can this characterise Oliver as someone who doesn't believe in the constructed identities of individuals, seeing as he says to all, "Later!" without naming? Or characterise him as someone who has no respect for societal obligations and is simply true to himself in such way?

Billowy blue shirt will eventually develop into a symbol, and his skin will be explored as a motif

Figure out the significance of "Later!"

:( please come for her

oh she traveled the waters alright

aww the rosemary

huh her and valentina??

HELP WHAT I WAS JUST EATING DINNER THINKING OF WHY MARTIN LUTHER ISNT MENTIONED

oh WOW

she's the one who returned...to the kitchen with the bread

good job

these poor girls

she's just a baby :(

shes back at the beginning

hualit was only able to help someone in death, through only material possesions....

WHAT did i just read?? the way albatross is playing rn

just like luzia's neighbor

NO NO NOOO

i'm so sad, she was so close to getting what she suffered all those years for

NOOO

their realtionship is so complex..i love it

i kinda get it...

i'm scared...

NOOO YOU BITCH

poor girls

AWW IM GONNA CRY

aww valentina

Used to make basic solution, part of the complex solution to form a high enough pH solution for Luminol to oxidize, but not high enough so that Copper does not dissolve. The Ammonium carbonate ensures that the pH remains stable.

Used to make basic solution, part of the complex solution to form a high enough pH solution for Luminol to oxidize, but not high enough so that Copper does not dissolve.

Used to make basic solution, part of the complex solution to form a high enough pH solution for Luminol to oxidize, but not high enough so that Copper does not dissolve.

I feel like this is a really important aspect when analyzing digital tools and apps to use in a classroom. I agree with the report that few preschool apps would be developmentally appropriate, because I generally feel that at the preschool age, children don't need their learning to be changed by adding digital tools and apps. I know that I want to go into early childhood education so I understand that digital tools and apps in the classroom will look very different than from a secondary/upper elementary perspective.

This table was really helpful to see direct connections and comparisons between Bloom's Taxonomy, which has been around and we all know very well, and new ideas for digital learning. I feel like it bridges the gap between old ways of teaching and new ways, by showing how we can use digital tools to enhance learning, but by doing so in similar traditional ways.

Threads vs Twitter is the narrative here. The idea that these two social media sites/apps with similar designs behind them are to be pit against each other. Twitter has seen heavy criticism ever since Elon Musk took over, and it feels like Threads it being suggested as the replacement people may be looking for.

A comparison to Twitter gives off the implication that it's meant to rival or maybe even surpass the site. That certainly could happen, considering the level of growth it has had, but the comparison to Twitter isn't really important in that regard.

Wow!

With how high the numbers got in such a short period of time, this feels like it was a necessary statement that needed to be made. It also makes the app's rise seem impressive when considering how new it was at the time of writing (and still is now).

Meta itself also putting major focus on the number of users. Could maybe be seen as prideful in a way.

Further hammering in the rate at which the app as been growing.

Putting an emphasis on both the rate of the growth and comparing it to its competitors.

At least they know how to sell a product. CV is what I want to see.

Not sure what this means exactly

Biofilms have a diverse chemical and biological gradient allowing for any type pollutants to stick on it which is why there is no correlation of Dow and Kow to Biofilm samples.

78

199

DOI: 10.1016/j.celrep.2021.108889

Resource: (Molecular Probes Cat# A-21203, RRID:AB_141633)

Curator: @Naa003

SciCrunch record: RRID:AB_141633

What is this?

DOI: 10.1016/j.celrep.2021.108889

Resource: (Molecular Probes Cat# A-21203, RRID:AB_141633)

Curator: @Naa003

SciCrunch record: RRID:AB_141633

What is this?

DOI: 10.1093/narcan/zcae018

Resource: (NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)

Curator: @evieth

SciCrunch record: RRID:CVCL_0060

What is this?

DOI: 10.1093/narcan/zcae018

Resource: (ATCC Cat# CRL-2806, RRID:CVCL_8787)

Curator: @evieth

SciCrunch record: RRID:CVCL_8787

What is this?

DOI: 10.1093/narcan/zcae018

Resource: (Coriell Cat# WC00048, RRID:CVCL_6808)

Curator: @evieth

SciCrunch record: RRID:CVCL_6808

What is this?

DOI: 10.1093/narcan/zcae018

Resource: (BCRJ Cat# 0278, RRID:CVCL_0132)

Curator: @evieth

SciCrunch record: RRID:CVCL_0132

What is this?

DOI: 10.1016/j.isci.2024.109652

Resource: RRID:Addgene_50465

Curator: @evieth

SciCrunch record: RRID:Addgene_50465

What is this?

DOI: 10.1016/j.isci.2024.109652

Resource: RRID:Addgene_26972

Curator: @evieth

SciCrunch record: RRID:Addgene_26972

What is this?

DOI: 10.1016/j.isci.2024.109652

Resource: RRID:Addgene_26973

Curator: @evieth

SciCrunch record: RRID:Addgene_26973

What is this?

DOI: 10.1016/j.jneumeth.2024.110079

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @evieth

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1016/j.jneumeth.2024.110079

Resource: (BDSC Cat# 35836,RRID:BDSC_35836)

Curator: @evieth

SciCrunch record: RRID:BDSC_35836

What is this?

DOI: 10.1016/j.celrep.2024.114151

Resource: RRID:Addgene_105553

Curator: @abever99

SciCrunch record: RRID:Addgene_105553

What is this?

DOI: 10.1016/j.celrep.2024.114151

Resource: RRID:Addgene_114472

Curator: @abever99

SciCrunch record: RRID:Addgene_114472

What is this?

DOI: 10.1016/j.celrep.2024.114151

Resource: RRID:Addgene_50475

Curator: @abever99

SciCrunch record: RRID:Addgene_50475

What is this?

DOI: 10.1016/j.celrep.2024.114151

Resource: RRID:Addgene_50459

Curator: @abever99

SciCrunch record: RRID:Addgene_50459

What is this?

DOI: 10.1016/j.celrep.2024.114151

Resource: RRID:Addgene_44362

Curator: @abever99

SciCrunch record: RRID:Addgene_44362

What is this?

DOI: 10.1016/j.celrep.2024.114144

Resource: (KCB Cat# KCB 90029YJ, RRID:CVCL_0004)

Curator: @abever99

SciCrunch record: RRID:CVCL_0004

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: Addgene_113724

Curator: @abever99

SciCrunch record: RRID:Addgene_113724

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: RRID:Addgene_71242

Curator: @abever99

SciCrunch record: RRID:Addgene_71242

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: RRID:Addgene_71243

Curator: @abever99

SciCrunch record: RRID:Addgene_71243

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: RRID:Addgene_64542

Curator: @abever99

SciCrunch record: RRID:Addgene_64542

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: IMSR_JAX:017490

Curator: @abever99

SciCrunch record: RRID:IMSR_JAX:017490

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: RRID:IMSR_JAX:018980

Curator: @abever99

SciCrunch record: RRID:IMSR_JAX:018980

What is this?

DOI: 10.1016/j.devcel.2024.04.002

Resource: (Proteintech Cat# 10068-1-AP, RRID:AB_2303998)

Curator: @abever99

SciCrunch record: RRID:AB_2303998

What is this?

DOI: 10.1016/j.celrep.2024.114132

Resource: (Millipore Cat# 04-401, RRID:AB_673111)

Curator: @abever99

SciCrunch record: RRID:AB_673111

What is this?

DOI: 10.1016/j.celrep.2024.114132

Resource: (Millipore Cat# 06-248, RRID:AB_2127890)

Curator: @abever99

SciCrunch record: RRID:AB_2127890

What is this?

DOI: 10.1016/j.theriogenology.2024.04.012

Resource: (Thermo Fisher Scientific Cat# A-11012, RRID:AB_2534079)

Curator: @abever99

SciCrunch record: RRID:AB_2534079

What is this?