I don't completely understand this. How can traditions become more oppressive as they lose relevance? Why didn't the growth of anti-traditionalist thinking from Kierkegaard, Marx, and Nietzche cause a break in tradition? I feel like these things would naturally follow.

It is interesting that through Kierkegaard's attempt to defend religion in a modern context, he goes against the tradition of relgion and opens the door for modern religious scrutiny.

I agree with this. If we policed work that break tradition, enforcing a singular traditionalist view, we support current and past oppressive structures for the future.

I have always been skeptical about whether privacy on social media is truly “private.” In many cases, so-called private messages are still accessible to platform developers or automated systems, which means users are trusting companies to protect their privacy rather than actually controlling it themselves. While this access can be helpful in situations like reporting threats or harassment, it also raises questions about who ultimately benefits from this arrangement. If only social media companies are able to see and manage my private data, I am not sure that this kind of “privacy” genuinely serves users’ interests rather than the platforms’ own priorities.

Private messaging creates’ an expectation of privacy, but there are situations where limited access is necessary to protect users from threats or harassment. The challenge is designing systems that allow reporting and evidence sharing in harmful situations without turning routine private communication into something routinely monitored.

It is interesting that using symbols, uppercase letters, and numbers does not significantly increase the difficulty of brute-force attacks, while increasing the length of a password dramatically raises the cost of cracking it. However, many social media platforms still emphasize “complex” password rules rather than encouraging longer passwords. This can create a false sense of security for users, who may believe their passwords are strong when they are not. Ironically, these complexity requirements can even make passwords harder to remember, leading users to reuse them or choose predictable patterns, which ultimately gives attackers more opportunities.

This statement shifts the focus of "protection" from the company level to the individual level, emphasizing that users still have actionable self-protection strategies. The significance of 2FA lies in the fact that even if a password is leaked, it is difficult for attackers to log in directly using only the password, thus reducing the probability of credential stuffing and account takeover. It also reminds us that security is layered – even the best platform security can be compromised by individual vulnerabilities such as phishing or weak passwords. However, this also highlights a limitation: 2FA can only reduce the risk of "account theft," but it cannot solve structural problems such as the platform itself leaking your private data.

This statement highlights the fragility of the "trust relationship" between users and the platform: we share information assuming the platform will protect it properly. It also implies that the problem is not an isolated incident, but "happens frequently," suggesting that security failures may be related to systems, processes, or business priorities. More importantly, the consequences of a single security breach often extend beyond the platform itself, affecting users' accounts on other websites and posing risks to their real lives. Readers will naturally ask: why did the platform fail—was it due to insufficient technical capabilities, management negligence, or prioritizing growth and convenience over security?

This shows how even major platforms can ignore basic security practices. How can platforms be held to stricter accountability standards when they fail to protect user data?

This suggests that exercise is an exceptionally reliable "first-line" intervention for the two most common complaints among healthcare workers: which is high stress and poor recovery.

This indicates that "being on your feet all day" at a hospital is not the same as purposeful exercise, and reinforces the need for structured interventions that happen outside of clinical duties.

This quote provides a powerful "business case" for hospitals and clinics to invest in staff exercise programs, framing them as a cost-saving measure rather than an expense.

By focusing solely on aerobic and resistance training, the review provides a more "clinical" look at how physical movement, independent of mindfulness, impacts the brain.

This shows that physical activity programs are a safety intervention, a healthier, more active workforce is statistically less likely to commit medical errors.

ince the significant gap between groups disappeared one month after the sessions ended, the researchers imply that physical activity must be a continuous lifestyle habit rather than a one-time "treatment" to keep burnout at bay.

This frames exercise and yoga programs as a "public health" measure for the hospital unit—by treating one nurse's burnout, managers may prevent it from spreading to others.

This provides a biological rationale for the results: yoga focuses on "rest and digest" (parasympathetic), which may more directly counteract the "fight or flight" (sympathetic) stress common in clinical settings.

This underscores that burnout isn't just a general workplace issue but a specific crisis within the nursing profession that requires specialized self-care strategies.

This suggests that for healthcare workers specifically suffering from the "drained" feeling of emotional exhaustion, yoga may be a more targeted intervention than traditional cardio.

This part stood out to me bcause it frames the systematic reviews as the most rigorous approach, especially in fields like medical science.

Before the reading, I was not really equipped with the idea of how differences between, systematic, semi-systematic, and integrative reviews. This helped me clarify how each approach serves as a different purpose.

This overall secrtion stood out to me because of the amount of clarity it provided on why literature reviews are essential across all research disciplines ,not just as background but aspart of the research process.

eLife Assessment

This valuable paper describes the regulation of the association of meiotic chromosome axis proteins on chromosome ends with sub-telomeric elements in budding yeast. The genome-wide analyses of binding of chromosome components as well as chromatin regulators, complemented with the mapping of meiotic DNA double-strand breaks on chromosome ends, provided incomplete evidence to support the authors' conclusion. The results in the paper are of interest to researchers in meiotic recombination and the structure of genomes and chromosomes.

Reviewer #1 (Public review):

Meiotic recombination at chromosome ends can be deleterious, and its initiation-the programmed formation of DSBs-has long been known to be suppressed. However, the underlying mechanisms of this suppression remained unclear. A bottleneck has been the repetitive sequences embedded within chromosome ends, which make them challenging to analyze using genomic approaches. The authors addressed this issue by developing a new computational pipeline that reliably maps ChIP-seq reads and other genomic data, enabling exploration of previously inaccessible yet biologically important regions of the genome.

In budding yeast, chromosome ends (~20 kb) show depletion of axis proteins (Red1 and Hop1) important for recruiting DSB-forming proteins. Using their newly developed pipeline, the authors reanalyzed previously published datasets and data generated in this study, revealing heretofore unseen details at chromosome ends. While axis proteins are depleted at chromosome ends, the meiotic cohesin component Rec8 is not. Y' elements play a crucial role in this suppression. The suppression does not depend on the physical chromosome ends but on cis-acting elements. Dot1 suppresses Red1 recruitment at chromosome ends but promotes it in interior regions. Sir complex renders subtelomeric chromatin inaccessible to the DSB-forming machinery.

The high-quality data and extensive analyses provide important insights into the mechanisms that suppress meiotic DSB formation at chromosome ends. To fully realise this value, several aspects of data presentation and interpretation should be clarified to ensure that the conclusions are stated with appropriate precision and that remaining future issues are clearly articulated.

(1) To assess the chromosome fusion effects on overall subtelomeric suppression, authors should guide how to look at the data presented in Figure 2b-c. Based on the authors' definition of the terminal 20 kb as the suppressed region, SK1 chrIV-R and S288c chrI-L would be affected by the chromosome fusion, if any. In addition, I find it somewhat challenging to draw clear conclusions from inspecting profiles to compare subtelomeric and internal regions. Perhaps, applying a quantitative approach - such as a bootstrap-based analysis similar to those presented earlier-would be easier to interpret.

(2) The relationship between coding density and Red1 signal needs clarification. An important conclusion from Figure 3 is that the subtelomeric depletion of Red1 primarily reflects suppression of the Rec8-dependent recruitment pathway, whereas Rec8-independent recruitment appears similar between ends and internal regions. Based on the authors' previous papers (referencess 13, 16), I thought coding (or nucleosome) density primarily influences the Rec8-independent pathway. However, the correlations presented in Figure 2d-e (also implied in Figure 3a) appear opposite to my expectation. Specifically, differences in axis protein binding between chromosome ends and internal regions (or within chromosome ends), where the Rec8-dependent pathway dominates, correlate with coding density. In contrast, no such correlation is evident in rec8Δ cells, where only the Rec8-independent pathway is active and end-specific depletion is absent. One possibility is that masking coding regions within Y' elements influences the correlation analysis. Additional analysis and a clearer explanation would be highly appreciated.

(3) The Dot1-Sir3 section staring from L266 should be clarified. I found this section particularly difficult to follow. It begins by stating that dot1∆ leads to Sir complex spreading, but then moves directly to an analysis of Red1 ChIP in sir3∆ without clearly articulating the underlying hypothesis. I wonder if this analysis is intended to explain the differences observed between dot1∆ and H3K79R mutants in the previous section. I also did not get the concluding statement - Dot1 counteracts Sir3 activity. As sir3Δ alone does not affect subtelomeric suppression, it is unclear what Dot1 counteracts. Perhaps, explicitly stating the authors' working model at the outset of this section would greatly clarify the rationale, results, and conclusions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Raghavan and his colleagues sought to identify cis-acting elements and/or protein factors that limit meiotic crossover at chromosome ends. This is important for avoiding chromosome rearrangements and preventing chromosome missegregation.

By reanalyzing published ChIP datasets, the researchers identified a correlation between low levels of protein axis binding - which are known to modulate homologous recombination - and the presence of cis-acting elements such as the subtelomeric element Y' and low gene density. Genetic analyses coupled with ChIP experiments revealed that the differential binding of the Red1 protein in subtelomeric regions requires the methyltransferase Dot1. Interestingly, Red1 depletion in subtelomeric regions does not impact DSB formation. Another surprising finding is that deleting DOT1 has no effect on Red1 loading in the absence of the silencing factor Sir3. Unlike Dot1, Sir3 directly impacts DSB formation, probably by limiting promoter access to Spo11. However, this explains only a small part of the low levels of DSBs forming in subtelomeric regions.

Strengths:

(1) This work provides intriguing observations, such as the impact of Dot1 and Sir3 on Red1 loading and the uncoupling of Red1 loading and DSB induction in subtelomeric regions.

(2) The separation of axis protein deposition and DSB induction observed in the absence of Dot1 is interesting because it rules out the possibility that the binding pattern of these proteins is sufficient to explain the low level of DSB in subtelomeric regions.

(3) The demonstration that Sir3 suppresses the induction of DSBs by limiting the openness of promoters in subtelomeric regions is convincing.

Weaknesses:

(1) The impact of the cis-encoded signal is not demonstrated. Y' containing subtelomeres behave differently from X-only, but this is only correlative. No compelling manipulation has been performed to test the impact of these elements on protein axis recruitment or DSB formation.

(2) The mechanism by which Dot1 and Sir3 impact Red1 loading is missing.

(3) Sir3's impact on DSB induction is compelling, yet it only accounts for a small proportion of DSB depletion in subtelomeric regions. Thus, the main mechanisms suppressing crossover close to the ends of chromosomes remain to be deciphered.

Reviewer #3 (Public review):

Summary:

The paper by Raghavan et. al. describes pathways that suppress the formation of meiotic DNA double-strand breaks (DSBs) for interhomolog recombination at the end of chromosomes. Previously, the authors' group showed that meiotic DSB formation is suppressed in a ~20kb region of the telomeres in S. cerevisiae by suppressing the binding of meiosis-specific axis proteins such as Red1 and Hop1. In this study, by precise genome-wide analysis of binding sites of axis proteins, the authors showed that the binding of Red1 and Hop1 to sub-telomeric regions with X and Y' elements is dependent on Rec8 (cohesin) and/or Hop1's chromatin-binding region (CBR). Furthermore, Dot1 functions in a histone H3K79 trimethylation-independent manner, and the silencing proteins Sir2/3 also regulate the binding of Red1 and Hop1 and also the distribution of DSBs in sub-telomeres.

Strengths:

The experiments were conducted with high quality and included nice bioinformatic analyses, and the results were mostly convincing. The text is easy to read.

Weaknesses:

The paper did not provide any new mechanistic insights into how DSB formation is suppressed at sub-telomeres.

eLife Assessment

This important study reveals intriguing connections between chromosome breakage and DNA elimination during programmed genome rearrangement in the ciliate Tetrahymena thermophila. By developing a novel FISH approach that distinguishes germline and somatic telomeres, the authors provide compelling evidence that chromosome breakage removes germline telomeres along with hundreds of kilobases of germline-limited sequences. By disrupting a single chromosome breakage site, they further showed that DNA elimination was globally affected, which opens up a new direction for mechanistic studies. Thus, this work reveals additional similarity between the programmed DNA elimination in ciliates and nematodes that underlies the transition from germline to somatic telomeres.

Reviewer #1 (Public review):

Summary:

In this study, Nagao and Mochizuki examine the fate of germline chromosome ends during somatic genome differentiation in the ciliate Tetrahymena thermophila. During sexual reproduction, a new somatic genome is created from a zygotic, germline-derived genome by extensive programmed DNA elimination events. It has been known for some time that the termini of the germline chromosomes are eliminated, but the exact process and kinetics of the elimination events have not been thoroughly investigated. The authors first use germline-specific telomere probes to show that the loss of these chromosome ends occurs with similar timing as other DNA elimination events. By comparative analysis of the assembled germline and somatic genomes, the authors find that the ends of each of the germline chromosomes are composed of a few hundred kilobases of micronuclear limited sequences (MLS) that are removed starting around 14 hours after the start of conjugation, which initiates sexual development. They then develop an in situ hybridization assay to track the fate of one end of chromosome 4 while simultaneously following the adjacent macronuclear destined sequence (MDS) retained in the new somatic genome. This allows the authors to more clearly show that these adjacent chromosomal segments are initially amplified in the developing genome before the terminal MLS is eliminated. Finally, they mutate the chromosome breakage sequence (CBS) that normally separates the MLS terminus from the adjacent MDS region, to show that strains that develop with only one mutant chromosome can produce viable sexual progeny, but it appears that both the MLS and the MDS from the mutant chromosome are lost. If both chromosome copies have the CBS mutation, the cells arrest during development and do not eliminate many germline-limited sequences and fail to produce viable progeny. Overall, this study provides many new insights into the fate of germline chromosome ends during somatic genome remodeling and suggests extensive coordination of different DNA elimination events in Tetrahymena.

Strengths:

Overall, the experiments were well executed with appropriate controls. The findings are generally robust. Importantly, the study provides several novel findings. First, the authors provide a fairly comprehensive characterization of the size of the MLS at the end of each germline chromosome. I'm not sure whether this has been published elsewhere. Second, the authors develop a novel method to study the fate of chromosome termini during development and use it to conclusively track the elimination of these termini. Third, the authors show that the elimination of these termini appears to occur concurrently with most other DNA elimination events during somatic genome differentiation. And fourth, the authors show that failure to separate these eliminated sequences from the normally retained chromosome alters the fate of these adjacent MDS and the loss of the cells' ability to produce viable progeny.

Weaknesses:

It appears the authors did extensive analysis of the MLS chromosome ends, but did not provide too much information related to their composition. If this has not been published elsewhere, it would be useful to describe the proportion of unique and repetitive sequences and provide more information about the general composition of the chromosome ends. Such information would help the reader understand the nature of these MLS and how they may or may not differ from other eliminated sequences. Although the development of the novel FISH probes for large chromosome ends allowed for these novel discoveries, the signal in several images was visible, but often quite faint. I'm not sure there is anything the authors could do to improve the signal-to-noise ratio, but one needs to stare at the images carefully to understand the findings. One main weakness in the opinion of this reviewer is that the authors did very little to understand why, when a terminal MLS and the adjacent MDS fail to get separated because of failure in chromosome breakage, both segments are eliminated. The authors propose that possibly essential genes in the MDS get silenced, and the resulting lack of gene expression is the issue, but this and other possibilities were not tested. The study would provide more mechanistic insight if they had tried to assess whether the MDS on the CBS mutant chromosome becomes enriched in silencing modifications (e.g., H3K9me3). Alternatively, the authors could have examined changes in gene expression for some of the loci on the neighbouring MDS. The other main weakness is that since the authors only mutated the end of one germline chromosome, it is not clear whether the elimination of the MDS adjacent to the terminal MLS on chromosome 4 when the CBS is mutated is a general phenomenon, i.e., would happen at all chromosome ends, or is unique to the situation at Chromosome 4R. Knowing whether it is a general phenomenon or not would provide important insight into the authors' findings.

Reviewer #2 (Public review):

Summary:

Nagao and Mochizuki investigated how the germline (MIC) telomere was removed during programmed genome rearrangement in the developing somatic nucleus (MAC). Using an optimized oligo-FISH procedure, the authors demonstrated that MIC telomeres were co-eliminated with a large region of MIC-limited sequences (MLS) demarcated on the opposite side by a sub-telomeric chromosome breakage site (CBS). This conclusion was corroborated by the latest assembly of the Tetrahymena MIC genome. They further employed CRISPR-Cas9 mutagenesis to disrupt a specific sub-telomeric CBS (4R-CBS). In uniparental progeny (mutant X WT), DNA elimination of the sub-telomeric MLS was not affected, but the adjacent MAC-destined sequence (MDS) may be co-eliminated. However, in biparental progeny (mutant X mutant), global DNA elimination was arrested, revealing previously unrecognized connections between chromosome breakage and DNA elimination. It also paves the way for future studies into the underlying molecular mechanisms. The work is rigorous, well-controlled, and offers important insights into how eukaryotic genomes demarcate genic regions (retained DNA) and regions derived from transposable elements (TE; eliminated DNA) during differentiation. The identification of chromosome breakage sequences as barriers preventing the spread of silencing (and ultimately, DNA elimination) from TE-derived regions into functional somatic genes is a key conceptual contribution.

Strengths:

New method development: Oligo-FISH in Tetrahymena. This allows high-resolution visualization of critical genome rearrangement events during MIC-to-MAC differentiation. This method will be a very powerful tool in this area of study.

Integration of cytological and genomic data. The conclusion is strongly supported by both analyses.

Rigorous genetic analysis of the role played by 4R-CBS in separating the fate of sub-telomeric MLS (elimination) and MDS (retention). DNA elimination in ciliates has long been regarded as an extreme form of gene silencing. Now, chromosome breakage sequences can be viewed as an extreme form of gene insulators.

Weaknesses:

The finding of global disruption of DNA elimination in 4R-CBS mutant progeny is highly intriguing, but it's mostly presented as a hypothesis in the Discussion. The authors propose that the failure to separate MLS from MDS allows aberrant heterochromatin spreading from the former into the latter, potentially silencing genes required for DNA elimination itself. While supported by prior literature on heterochromatin feedback loops, the specific targets silenced are not identified. While results from ChIP-seq and small RNA-seq can greatly strengthen the paper, the reviewer understands that direct molecular characterization may be beyond the scope of the current work.

Reviewer #3 (Public review):

Programmed DNA elimination (PDE) is a process that removes a substantial amount of genomic DNA during development. While it contradicts the genome constancy rule, an increasing number of organisms have been found to undergo PDE, indicating its potential biological function. Single-cell ciliates have been used as a prominent model system for studying PDE, providing important mechanistic insights into this process. Many of those studies have focused on the excision of internally eliminated sequences (IES) and the subsequent repair using non-homologous end joining (NHEJ). These studies have led to the identification of small RNAs that mark retained or eliminated regions and the transposons that generate double-strand breaks.

In this manuscript, Nagao and Mochizuki examined the other type of breaks in ciliates that were healed with telomere addition. They specifically focused on the sequences at the ends of the germline (MIC) chromosomes, which have received relatively less attention due to the technical challenges associated with the highly repetitive nature of the sequences. The authors used the Tetrahymena model and developed a set of new tools. They used a novel FISH strategy that enables the distinction between germline and somatic telomeres, as well as the retained and eliminated DNA near the chromosome ends. This allows them to track these sequences at the cellular level throughout the development process, where PDE occurs. They also analyzed the more comprehensive germline and somatic genomes and determined at the sequence level the loss of subtelomeric and telomere sequences at all chromosome ends. Their result is reminiscent of the PDE observed in nematodes, where all germline chromosome ends are removed and remodeled. Thus, the finding connects two independent PDE systems, a protozoan and a metazoan, and suggests the convergent evolution of chromosome end removal and remodeling in PDE.

The majority of sites (8/10) at the junctions of retained and eliminated DNA at the chromosome ends contain a chromosome breakage sequence (CBS). The authors created a set of mutants that modify the CBS at the ends of chromosome 4R. CBS regions are challenging for CRISPR due to their AT-rich sequences, making the creation of the 4R-CBS mutants a significant breakthrough. They used the FISH assay to determine if PDE still occurs in these mutant strains with compromised CBS. Surprisingly, they found that instead of blocking PDE, its adjacent retained DNA is now eliminated, suggesting a co-elimination event when the breakage is impaired. Furthermore, in biparental mutant crosses, no PDE occurred, and no viable progeny were produced, indicating that the removal of chromosome ends is crucial for proper PDE and sexual progeny development. Overall, the work demonstrates a critical role for 4R-CBS in separating retained and eliminated DNA.

It is hard to ascertain whether Zhao cares about women's education genuinely (even though it is within a patriarchal context), or if this is a means to an end (which is to further cement men's power in a marriage).

I'm interested to know what Zhao would think of widows and remarriage, as well as adulterous husbands.

The author's original claim of being unable to do anything remarkable in life and struggling with her duties seems to show here. This comment on how women should always live in fear seems to be projection from her own time as a 'lowly subject'.

The gendering of strength and weakness is particularly interesting here because Zhao does not think that either one is inferior to the other; he just thinks that both must exist to complement the other. It is interesting to consider the possibility of this proverb hinting at an observation of men and women, particularly that there is a 'fear' that women may turn into tigers.

R0:

Reviewer #1:

Methodology: study design & datasource, heading overlap with data source and extraction. You may consider differentiating the study design+population from the data source+data extraction to improve clarity and avoid repetition.

The covariates are presented in a table. Consider adding a concise paragraph describing key variables and their categorization. Perhaps detailed coding and operational definitions should be presented in a supplementary table.

The analysis plan refers to Objective 2 and Objective 3 however, the study objectives are not stated anywhere earlier in the manuscript. They should be explicitly stated to improve coherence

Also, this contradicts “shared frailty at the individual level”. Please clarify the intended clustering structure. Did you model with the shared or Individual-level frailty?

Reviewer #2:

This manuscript addresses an important and under-researched area by examining long-term trends in attrition from HIV care and associated predictors among adolescent girls and young women (AGYW) with non-viral load suppression in Tanzania. The use of routine programmatic data is a major strength and enhances the relevance of the findings for the national program. The objectives are clear, and the results provide insights into patterns of disengagement from care in this vulnerable population. However, the manuscript would benefit from clearer operational definitions of key outcomes particularly inconsistent viral load suppression. Further discussion linking the findings to existing regional and global literature, as well as clearer description of programmatic and policy implications, would strengthen the paper. I recommend minor revisions to improve clarity and consistency.

R1:

Reviewer #1:

All comments have been addressed

Reviewer #2:

General review of the revised manuscript I have reviewed the revised manuscript and found that the authors have addressed all the comments; however, a few minor edits are still required before publication. Please see the table below.

Section: Introduction <br /> Comment: Correct spelling UANIDS <br /> Line number: 58

Section: Methods <br /> Comment: It is essential that the term inconsistent viral load suppression needs to be clearly defined in the manuscript. Note that two viral load results ≥1000 copies/ml at least six months after ART initiation is the standard definition for non-viral suppression or ART virological failure.<br /> Ideally, inconsistent viral load suppression would have been a situation whereby PLHIV achieves an undetectable viral load at least 6 months after ART initiation, but subsequently experience detectable viral load result/s ≥1000 copies/ml. To avoid confusion, refer to this sentence in conclusion: Notably, individuals with an initial unsuppressed viral load were more likely to disengage from care.

Section: Results <br /> Comment: Table 1: First line and second line not defined. Not every reader knows what Tanzanian ART regimens. Line number: 170-171

Section: Discussion Comment: A citation is not inserted <br /> Line number: 253

Section: Discussion Comment: Correct the word form to read from Line number: 254

Your ideas should be In order so that they make sense for yourself and the reader as well.

Great way to think about it!

If there is a pattern you should fix it to better your writing.

Take your time because at the end of the day your are trying to help improve a piece of writing

Read the whole thing first before giving feedback.

Be formal and respectful when giving feedback to others.

Always do the instructor questions first because that's what's most likely to be graded.

You will make mistakes so it's best to revise at least once.

We need to understand the evidence we use so that the reader understand as well.

eLife Assessment

This important study presents new insights into the post-transcriptional mechanisms that govern cortical development. Through state-of-the-art methodology to track neuronal birth order, the data provide compelling evidence that Imp1 (Igf2bp1/Zbp1) orchestrates radial glia fate transitions and cortical neurogenesis. The findings establish a new framework for understanding how post-transcriptional mechanisms integrate with transcriptional and epigenetic regulatory layers to control cortical temporal patterning.

Reviewer #1 (Public review):

Summary:

A hallmark of cortical development is the temporal progression of lineage programs in radial glia progenitors (RGs) that orderly generate a large set of glutamatergic projection neuron types, which are deployed to the cortex in a largely inside-out sequence. This process is thought to contribute to the formation of proper cortical circuitry, but the underlying cellular and molecular mechanisms remain poorly understood. To a large extent, this is due to technical limitations that can fate map RGs and their progeny with cell type resolution, and manipulate gene expression with proper cell and temporal resolution. Building on the TEMPO technique that Tsumin Lee group developed, here Azur et al show that the RNA binding protein Imp1 functions as a dosage- and stage-dependent post-transcriptional mechanism that orchestrates developmental stage transitions in radial glial progenitors, and controls neuronal fate decisions and spatial organization of neuronal and glial cell progeny. Their results suggest that while transcriptional regulators define available cellular states and gate major transitions, post-transcriptional mechanisms like Imp1 provide an additional layer of control by modulating stage-specific transcript stability. Imp1 thus acts as a temporal coordinator whose dosage and timing determine whether developmental transitions are temporarily delayed or blocked. These findings establish a new framework for understanding how post-transcriptional mechanisms integrate with transcriptional and epigenetic regulatory layers to control cortical temporal patterning.

Strengths:

The authors apply a novel genetic fate mapping and gene manipulation technology (TEMPO) with cellular resolution. This reveals a dosage- and stage-dependent post-transcriptional mechanism that orchestrates developmental stage transitions in radial glial progenitors, and controls neuronal fate decisions and spatial organization of neuronal and glial cell/astrocyte progeny.

Weaknesses:

The endogenous developmental expression pattern of Imp1 and TEMPO-mediated overexpression are not well described or characterized with cellular resolution (whether only in radial glial cells or also in post-mitotic neurons). Thus, the interpretations of the overexpression phenotypes are not always clear.

Reviewer #2 (Public review):

Summary:

In this manuscript, Azur et al seek to determine the role of Imp1/Igf2bp1 in regulating the temporal generation of cortical neuron types. The authors showed that overexpression of Imp1 changes the laminar distribution of cortical neurons and suggest that Imp1 plays a temporal role in specifying cell fates.

Strengths:

The study uniquely used TEMPO to investigate the temporal effects of Imp1/Igf2bp1 in cortical development. The disrupted laminar distribution and delayed fate transition are interesting. The results are presented with proper quantification, they are generally well interpreted, and suggest important roles for Imp1.

Weaknesses:

(1) While the results suggest Imp1 is important in regulating cortical neurogenesis, it remains unclear when and where it is expressed to execute such temporal functions. For instance, where is Imp1 expressed in the developing brain? Is it specific to the radial glial cells or ubiquitous in progenitors and neurons? Does it show temporal expression in RGCs?

(2) The advantage and interpretation of TEMPO need further clarification. TEMPO is an interesting method and appears useful in simultaneously labelling cells and controlling gene expression. Since the reporter, Cas9, and gRNA triggers are all driven by ubiquitous promoters and integrated into the genome using piggyBac, it appears logical that the color transition should happen in all cells over time. The color code appears to track the time when the plasmids got integrated instead of the birthday of neurons. Is this logically true? If the TEMPO system is introduced into postmitotic neurons and the CAG promoter is not silenced, would the tri-color transition happen?

(3) The accumulation of neurons at the subplate region would benefit from showing larger views of the affected hemisphere. IUE is invasive. The glass pipette may consistently introduce focal damages and truncate RGCs. It is important to examine slices covering the whole IUE region.

Reviewer #3 (Public review):

Summary:

The work by Azur and colleagues makes use of the TEMPO (Temporal Encoding and Manipulation in a Predefined Order) methodology to trace cortical neurogenesis in combination with overexpression of Imp1. Imp1 is a mammalian homologue of the Drosophila Imp, which has been shown to control temporal identity in a stem cell context. In their work, they show that overexpression of Imp1 in radial glia, which generate neurons and macroglia in a sequential manner during cortical development, leads to a disruption of faithful neuron/glia generation. They show that continuous overexpression leads to a distinct phenotypic outcome when compared to paradigms where Imp1 was specifically overexpressed in defined temporal windows, enabled by the unique TEMPO approach. Interestingly, the observed phenotype with 'ectopic' generation of mainly lower cortical layer neurons appears not to be due to migration deficits. Strikingly, the overexpression of Imp1 specifically at later stages also leads to ectopic glia-like foci throughout the developing cortical plate. Altogether, the new data provide new insights regarding the role of the post-transcriptional Imp1 regulator in controlling temporal fate in radial glia for the faithful generation of neurons and glia during cortical development.

Strengths:

The TEMPO approach provides excellent experimental access to probe Imp1 gene function at defined temporal windows. The data is very robust and convincing. The overexpression paradigm and its associated phenotypes match very well the expected outcome based on Imp1 loss-of-function. Overall, the study contributes significantly to our understanding of the molecular cues that are associated with the temporal progression of radial glia fate potential during cortical development.

Weaknesses:

The authors provide some experimental evidence, including live imaging, that deficits related to Imp1 overexpression and subsequent overabundance of lower-layer neurons, or accumulation at the subplate, appear to evolve independently of neuronal migration deficits. However, the analysis at the population level might not suffice to make the claim robust. To analyze neuronal migration in more depth, the authors could trace individual neurons and establish speed and directional parameters for comparison.

In their analysis, the authors mainly rely on temporal parameters/criteria to associate the generation of certain neuron fates. While two markers were used to identify the neuronal fate, the variance seems quite high. The authors could consider utilizing an antibody against Satb2, which would provide additional data points that could help to establish statistical significance in some of the analyses.

The analysis of glia was done at postnatal day 10, although gliogenesis and, in particular, astrocyte maturation last at least until postnatal day 28. The authors could consider extending their analysis to capture the full spectrum of their astrocyte phenotype.

R0:

Reviewer #1:

The article is relevant and timely. Please use COREQ guidelines/other community guidelines for qualitative research listed on the EQUATOR network to improve the rigour and flow of the article. Additional suggestions for your consideration are listed below. 1. The rationale for the article as well as research question needs to be made more explicit. Additionally, the novelty of the article is relatively unclear. 2. The use of a framework devised for infectious diseases needs to be examined- why was this framework chosen? 3. Line 52-54: It may be useful to examine the cause of death in context of age; additionally, the later use of terms like young-old-middle aged are ambiguous later in the article, please provide an operational definition. 4. Line 54-57: The reason for prioritising lived experience is unclear. The logical flow establishing the rationale for this inquiry needs strengthening. 5. The linkages of NCDs with urbanisation need more citation and can be explored better. The authors may consider reassessing the introduction section which seems to appear definitive without providing a research gap. 6. Why is this research needed? What makes it relevant for global health? This needs to be elucidated in the text. 7. The use of the word "drug peddler" should be in quotes at least at first use, to ensure that it is seen in its local context. 8. The narrative findings are rich and well presented- the global health impact of these findings/future paths based on these findings need to be be made explicit.

Reviewer #2:

This manuscript is well-written and engaging, and I would like to commend the authors for presenting such rich and dense information.

Here are my comments, which I believe will enhance the content of this manuscript.

1. Page 2, line 27 (abstract section): The term "oppression" appears quite strong, and I do not find any evidence supporting it in the results section. Could the authors clarify what they mean by "oppression"? Additionally, the sentence “as gender norms and experiences of masculinities and femininities reflected the division of roles and access to resources by men and women, which in turn shaped their ability to seek early and better healthcare interventions” seems to contradict the notion of oppression if participants are able to seek early and improved healthcare interventions.

2. Page 6, line 133: typo: a dot is in the middle of the sentence. Consider removing.

3. Page 7 on selection of study participants: How did researchers identify participants based on diabetes and hypertension criteria, given that these medical conditions require a confirmed diagnosis from health professionals? Additionally, were any participants identified using a snowball sampling technique?

4. It is mentioned that this study was conducted as part of the broad ARISE project. Can the authors clarify whether the study participants were enrolled into the main project and if so, to what extent this initial involvement of the main study had impacted the data collection?

5. An important section on data collection is missing. I would like authors to include this in order to explain what specific data were collected at each round, why was it necessary to split data collection over different timepoints, where and in which language were data collected, how long lasted interviews on average, and who collected the data, any bias that may result from the way the study participants perceived the data collection team during fieldwork?

6. The data analysis section requires further clarification. It would be beneficial if the authors could elaborate on their data coding strategy. It appears that a deductive approach was employed, yet they did not explain how the coding frame was initially developed before its application to the data, nor did they specify who conducted these procedures. Additionally, information regarding the software used to facilitate the data analysis is missing.

7. The use of relevant qualitative data reporting guideline is missing. This is important to include as it will help ensure that ensure that the methods and findings are clearly and transparently communicated in the manuscript.

8. In relation to adapting the framework for Infectious Diseases of Poverty Research, it would be beneficial if the authors could clarify the specific adaptations made to the framework and how it was applied during data analysis.

9. They mentioned that participants who faced distress during the interviews were allowed to grieve and were consoled. How many participants developed such condition during interviews at which round of data collection, and to what extent did this impact on data collected?

10. This study seeks to explore the lived experiences of individuals affected by NCDs in informal settlements. However, the findings presented in the results section, particularly the initial part titled "lived experience of NCD," fall short of adequately conveying the participants' current experiences with these health conditions. Firstly, the authors include excerpts from three informants but fail to demonstrate how these accounts are representative of all study participants. Secondly, the discussion of informants' lives prior to developing their current health conditions renders the information less pertinent to the study's objectives. It would be beneficial if the authors could focus on participants current lives to discuss the intersectionality of NCDs with social, economic, and gender factors within the participants' living environments, and how these elements contribute to shaping the management of their health conditions. This way of presenting the findings restricts our understanding, as the same observation is reiterated throughout the discussion section.

11. In the context of chronic disease, it can be assumed that participants with diabetes, hypertension, or comorbidities might encounter similar yet distinct challenges. It would be beneficial for the authors to discuss the findings across these cases, highlighting both similarities and differences, and to establish a hierarchy within the data.

12. The use of terms like "people," "women," or "men" in the results section can be misleading, as they might suggest that the findings apply to the general population. Instead, it is advisable to use terms such as "informants," "study participants," or "men" or "women in this study" to ensure that the information reported is limited to those individuals who participated in this data collection.

The "semi-hidden" file like .git in a git repository implies the existence of a higher order of hidden-ness.

I searched this up and eventually ended up on the Wikipedia article linked below. The explanation bifurcates the idea of a hidden file into stuff hidden for convenience and super important stuff that the OS uses, which "remain hidden unless another setting, called "Hide protected operating system files (Recommended)," is cleared." This fully hidden-ness for sys files likely is to prevent a PEBKAC.

https://en.wikipedia.org/wiki/Hidden_file_and_hidden_directory#:~:text=or%20clear%20attributes.-,File%20Explorer,of%20hidden%20items%20semi%2Dtransparently.

I agree with this, and this limitation was exemplified by when I was going through a command line maze, but the directories used spaces instead of dashes or underscores, which initially led to a bit of friction in me figuring out how to proceed to the next terminal (I used quotes).

The issue with spaces is a good example of how a normal human habit like just using spaces becomes a technical problem. Something that comes so naturally to us when naming a file turns into extra work in command-line environments, which j shows a big difference between human language and systems.

The goal of this piece is defintiely more practical than academic. It works like informal documentation meant to help readers toward better file-naming practices.

The author baiscally supports his point by describing his experience working across many operating systems. This shows that the issues he mentions come from actual technical experience.

The author’s main argument is that file naming is not just a personal preference. Poor file names create like actual technical problems, especially when files are shared or moved between different systems.

eLife Assessment

This is a well-executed intrathecal MRI tracer study that provides valuable early in vivo evidence for CSF drainage into human skull bone marrow and explores clinically relevant associations using robust imaging methodology and regional analyses. However, the evidence supporting the interpretation of early (4 h) tracer signal as impaired clearance is incomplete, and appears difficult to reconcile with established CSF tracer kinetics. They also note that the reported links to sleep and cognitive performance are weakened by reliance on subjective, retrospective questionnaires rather than objective physiological measurements.

Reviewer #1 (Public review):

Summary:

This manuscript examines the passage of an intrathecal CSF tracer into skull bone marrow, cortex, and venous compartments using serial MRI at multiple time points. The study builds on recent anatomical and imaging work suggesting direct communication between CSF spaces and bone marrow in the skull. It extends these observations to a larger, clinically heterogeneous human cohort. The imaging methodology is carefully executed, and the dataset is rich. The findings are potentially important for understanding CSF drainage pathways and their associations with inflammation, sleep quality, and cognition. However, key aspects of the interpretation - particularly regarding tracer kinetics and the definition of "clearance" - require clarification and, in my view, reconsideration.

Strengths:

(1) The study employs a well-established intrathecal contrast-enhanced MRI approach with multiple post-injection time points, enabling the assessment of regional tracer dynamics.

(2) The analysis of skull bone marrow in distinct anatomical regions (near the superior sagittal sinus, lateral fissure, and cisterna magna) is novel and informative.

(3) The cohort size is relatively large for an intrathecal tracer study in humans, and the authors make commendable efforts to relate imaging findings to clinical variables such as inflammation, sleep quality, and cognitive performance.

(4) The manuscript is clearly written, the figures are informative, and the discussion is well grounded in recent anatomical and experimental literature on skull-meningeal connections.

Weaknesses:

The central interpretation that a higher percentage increase in skull bone marrow tracer signal at 4.5 hours reflects reduced clearance is not convincingly justified. Based on the existing CSF tracer literature, the 4-6 hour time window is generally considered an enrichment or inflow phase rather than a clearance phase. Later time points (15 and 39 hours) are more likely to reflect clearance or washout. An alternative interpretation - that a higher signal at 4.5 hours reflects more pronounced tracer entry - should be considered and discussed.

Relatedly, the manuscript lacks a clear conceptual separation between tracer enrichment and clearance phases across time points. If 4.5 hours is intended to represent clearance, this assumption requires more vigorous justification and alignment with prior work.

CSF passage via the nasal/olfactory pathway is insufficiently discussed. Previous human imaging studies have questioned the importance of peri-olfactory CSF clearance, yet the present findings suggest delayed enrichment in the nasal turbinates. This discrepancy should be explicitly addressed, including a discussion of potential methodological limitations (e.g., timing of acquisitions, ROI definition, or sensitivity to slow drainage pathways).

More generally, given the descriptive nature of the study and the limited temporal sampling, some conclusions regarding directionality and efficiency of "drainage" may be overstated and would benefit from more cautious framing.

Reviewer #2 (Public review):

Summary

Zhou et al. utilize longitudinal, intrathecal contrast-enhanced MRI to investigate a novel physiological pathway: the drainage of cerebrospinal fluid (CSF) into the human skull bone marrow. By mapping tracer enrichment across 87 patients at multiple time points, the authors identify regional variations in drainage speed and link these dynamics to systemic factors like aging, hypertension, and diabetes. Most notably, the study suggests that this drainage function serves as a significant mediator between sleep quality and cognitive performance.

Strengths

(1) The study provides a significant transition from murine models to human subjects, showing that CSF-to-marrow communication is a broader phenomenon in clinical cohorts.

(2) The use of four imaging time points (0h to 39h) allows for a precise characterization of tracer kinetics, revealing that the parietal region near the superior sagittal sinus (SSS) is a rapid exit route.

(3) The statistical finding that skull bone marrow drainage accounts for approximately 38% of the link between sleep and cognition provides a provocative new target for neurodegenerative research.

Weaknesses

(1) Figure 1: The figure relies on a single representative brain to illustrate a process that likely varies significantly across different skull anatomies and disease states. In the provided grayscale MRI scans, the tracer enrichment is essentially imperceptible to the naked eye. Without heatmaps or digital subtraction maps (Post-injection minus Baseline) for the entire cohort, it is difficult to substantiate the quantitative "percentage change" data visually.

Reliance on a single, manually placed circular Region of Interest (ROI) is susceptible to sampling bias. A more robust approach would involve averaging multiple ROIs per region (multi-sampling) to ensure the signal is representative of the whole marrow compartment.

(2) Methodological Rigor of Sleep Analysis: The study relies exclusively on the self-reported Pittsburgh Sleep Quality Index (PSQI), which is retrospective and highly prone to recall bias, particularly in a cohort with cognitive impairment. There is no objective verification of sleep (e.g., actigraphy or polysomnography). Since waste clearance is physiologically tied to specific stages, such as Slow-Wave Sleep, subjective scores cannot determine whether drainage is linked to sleep physiology or reflects a higher general disease burden. The MRI captures an acute state during hospitalization, whereas the sleep quality reported covers the month preceding admission. This mismatch complicates the claim that the current drainage function directly reflects historical sleep quality.

Appraisal and Impact

The authors demonstrate the feasibility of monitoring CSF-to-skull marrow drainage in humans. However, the strength of the associations with sleep and cognition is currently attenuated by a lack of visual "proof" in the raw data and a reliance on subjective behavioral metrics. If these technical gaps are explicitly addressed through the use of population heatmaps and more rigorous multi-ROI sampling, this work will significantly advance our understanding of the brain's waste-clearance systems and their role in systemic health.

Reviewer #3 (Public review):

Summary:

In this study, the authors injected a contrast agent into patients and followed the induced signal change with MRI. Doing so, they observed cerebrospinal fluid (CSF) drainage whose magnitude and dynamics varied by anatomical location and scaled with a range of cognitive and socio-demographic metrics, including sleep scores and sex.

Strengths:

I would first like to stress that I am not a specialist in the topic of that paper; so my comments should be taken with a grain of salt, and feedback from the other reviewers should also be carefully considered.

I found the text concise and the figures straightforward to understand. Although they are manually defined, the authors compared drainage across different anatomical locations, which is a positive feature. Albeit purely correlative, the attempt to connect these otherwise 'peripheral' measures to cognitive variables is quite interesting. I also particularly liked the last paragraph of the discussion, which listed the main limitations of the study.

Weaknesses:

In the paragraph starting at line 446, the authors interpret poor sleep quality as being a cause and a consequence of impaired CSF clearance, but their approach is purely correlational. In other words, a third variable could be driving both of these parameters (correct?), thereby explaining their correlation. Later, they also proposed that therapeutically altering CSF clearance could improve cognitive symptoms, but, again, if there's a hidden cause of the correlation, that does not seem like a valid possibility. I believe there were other instances of this sort of inferential problem in the Discussion. It seems essential, particularly in clinical research, to precisely identify what the available evidence supports (correlation) and what is speculation (causation).

Assuming I did not miss it, the approach for testing and reporting correlations is not specified. In particular, the authors report correlation with CSF drainage and a variety of other metrics. But how many tests did the authors perform? They solely mention that they used the Benjamini-Hochberg method to correct for multiple comparisons. How were the decisions to test for this or that effect determined? Or did they test all the metrics they had? Also, that particular correction method is limited when statistics are negatively correlated. It would be helpful to validate findings with another approach.

I assume many of the metrics the authors use are also correlated with one another. Is it possible that a single principal component is driving the different correlations they see? Performing dimensionality reduction across available metrics and relating the resulting principal components to CSF drainage would help clarify the forces at play here.

In their interpretations, the authors claim that the CSF drainage they observe occurs through the bone marrow of the skull. How confident can we be in that claim? Is it that there are no other likely possibilities? It might be an unnecessary question, but given there seems to be no causal intervention (which is fine), and no consideration of alternatives, I am wondering whether this is because other possibilities are improbable or whether they were not adequately considered.

R0:

Reviewer #1: Title: Probabilistic Forecasting of Monthly Dengue Cases Using Epidemiological and Climate Signals: A BiLSTM–Naive Bayes Model Versus Mechanistic and Count-Model Baselines. Manuscript Number: PGPH-D-25-03170

This manuscript presents a rigorous comparative study of probabilistic forecasting models for monthly dengue incidence in Freetown, Sierra Leone, covering the period 2015–2025. It evaluates four major model classes—NB-GLM, INGARCH-NB, Renewal-NB, and BiLSTM-NB—under a leakage-safe rolling-origin evaluation. The article demonstrates strong methodological maturity, careful control of data leakage, and thorough probabilistic evaluation using proper scoring rules, interval coverage, sharpness metrics, PIT diagnostics, and Diebold–Mariano tests. The manuscript is generally well-written, technically sound, and addresses an important operational public health problem. It positions itself as one of the few works offering aligned comparisons of mechanistic, statistical, and deep-learning models under realistic constraints for West African dengue surveillance. This article presents a methodologically rigorous comparison of four probabilistic forecasting approaches—NB-GLM, INGARCH-NB, Renewal-NB, and BiLSTM-NB—applied to monthly dengue case data from Freetown, Sierra Leone (2015–2025). The study addresses an important gap by evaluating mechanistic, statistical, and deep-learning models under aligned, leakage-safe conditions. While the work is comprehensive and technically strong, several critical issues affect its accessibility, interpretability, and broader applicability.

Strengths The study excels in methodological rigor. Its strict leakage safeguards, careful feature-timing rules, and use of expanding-window rolling-origin evaluation significantly strengthen reliability. The inclusion of proper scoring rules, interval coverage, sharpness metrics, PIT histograms, and Diebold–Mariano tests provides a complete probabilistic evaluation rarely seen in dengue forecasting studies. The horizon-specific findings—INGARCH-NB outperforming at 1–2 months and BiLSTM-NB excelling at 3 months—are well supported by aligned comparisons and statistical significance tests. The transparency of data, code, and alignment artefacts enhances reproducibility and credibility. Additionally, the manuscript offers practical guidance for operational forecasting, including a realistic “light climate” input strategy suitable for resource-limited settings.

Limitations Despite its strengths, the manuscript is heavily technical, with extensive mathematical exposition in the main text. This may limit accessibility for public-health practitioners who are likely part of the target audience. The mechanistic renewal model is presented as a baseline but is arguably underspecified; the use of a short, fixed 3-month kernel may not realistically capture dengue’s generation interval dynamics, likely contributing to its poor performance. This limits the interpretive value of the mechanistic comparison. This limitation should be addressed. The study’s climate treatment, while intentionally conservative, may underexploit important environmental drivers. Although justified operationally, this constraint restricts exploration of potentially meaningful lag structures or seasonal climate anomalies. The analysis is limited to a single city and monthly data frequency, raising questions about generalizability across geographies with different climate patterns and dengue transmission dynamics. Moreover, the monthly temporal resolution may obscure rapid outbreak shifts, possibly disadvantaging mechanistic and hybrid models that rely on finer-grained dynamics. This should be addressed. The manuscript makes a valuable and original contribution to dengue forecasting, offering robust methodological innovations and practical insights for real-time surveillance systems. However, improved clarity, stronger justification for mechanistic assumptions, and expanded discussion of generalizability would enhance its usefulness and scholarly impact. With revisions to improve accessibility and contextual depth, the study is well positioned for publication and for informing operational forecasting practice in similar settings.

Reviewer #2: 1. What is PIT in the abstract stand for? The authors should avoid using abbreviations in the abstract. 2. The authors should providing some additional analysis, such as experimenting with alternative or longer serial-interval kernels, or simple sensitivity checks (e.g., different window lengths, or, if possible, finer temporal resolution). 3. Please, justifies the small climate feature set, mentioning any exploratory work with larger sets. 4. The authors should add a clearly labelled missing-data handling subsection that specifies: The imputation method, the number of imputed months, and how they were used in training/evaluation, plus any sensitivity. 5. While the architecture, optimization, and calibration steps are described, the process for choosing hyperparameters is not fully audit-ready. 6. I recommend that the authors conduct an additional experiment to demonstrate the generalizability of the proposed model.

eLife Assessment

This valuable work describes a computational and experimental workflow that turns a moderately stable α-helical bundle into a very stable fold. The authors advance our understanding of α-helix stabilization and provide a convenient framework with implications for the protein design field. The main claims are supported by convincing evidence through sound and well-validated methods, yet further characterization would strengthen specific conclusions for the design of mechanically, thermally, and chemically stable α-helical bundles.

Reviewer #1 (Public review):

Summary:

In the work from Qiu et al., a workflow aimed at obtaining the stabilization of a simple small protein against mechanical and chemical stressors is presented.

Strengths:

The workflow makes use of state-of-the-art AI-driven structure generation and couples it with more classical computational and experimental characterizations in order to measure its efficacy. The work is well presented, and the results are thorough and convincing.

Weaknesses:

I will comment mostly on the MD results due to my expertise.

The Methods description is quite precise, but is missing some important details:

(1) Version of GROMACS used.

(2) The barostat used.

(3) pH at which the system is simulated.

(4) The pulling is quite fast (but maybe it is not a problem)

(5) What was the value for the harmonic restraint potential? 1000 is mentioned for the pulling potential, but it is not clear if the same value is used for the restraint, too, during pulling.

(6) The box dimensions.

From this last point, a possible criticism arises: Do the unfolded proteins really still stay far enough away from themselves to not influence the result? This might not be the major influence, but for correctness, I would indicate the dimensions of the box in all directions and plot the minimum distance of the protein from copies of itself across the boundary conditions over time.

Additionally, no time series are shown for the equilibration phases (e.g., RMSD evolution over time), which would empower the reader to judge the equilibration of the system before either steered MD or annealing MD is performed.

Reviewer #2 (Public review):

Summary:

Qiu, Jun et. al., developed and validated a computational pipeline aimed at stabilizing α-helical bundles into very stable folds. The computational pipeline is a hierarchical computational methodology tasked to generate and filter a pool of candidates, ultimately producing a manageable number of high-confidence candidates for experimental evaluation. The pipeline is split into two stages. In stage I, a large pool of candidate designs is generated by RFdiffusion and ProteinMPNN, filtered down by a series of filters (hydropathy score, foldability assessed by ESMFold and AlphaFold). The final set is chosen by running a series of steered MD simulations. This stage reached unfolding forces above 100pN. In stage II, targeted tweaks are introduced - such as salt bridges and metal ion coordination - to further enhance the stability of the α-helical bundle. The constructs undergo validation through a series of biophysical experiments. Thermal stability is assessed by CD, chemical stability by chemical denaturation, and mechanical stability by AFM.

Strengths:

A hierarchical computational approach that begins with high-throughput generation of candidates, followed by a series of filters based on specific goal-oriented constraints, is a powerful approach for a rapid exploration of the sequence space. This type of approach breaks down the multi-objective optimization into manageable chunks and has been successfully applied for protein design purposes (e.g., the design of protein binders). Here, the authors nicely demonstrate how this design strategy can be applied to successfully redesign a moderately stable α-helical bundle into an ultrastable fold. This approach is highly modular, allowing the filtering methods to be easily swapped based on the specific optimization goals or the desired level of filtering.

Weaknesses:

Assessing the change in stability relative to the WT α-helical bundle is challenging because an additional helix has been introduced, resulting in a comparison between a three-helix bundle and a four-helix bundle. Consequently, the appropriate reference point for comparison is unclear. A more direct and informative approach would have been to redesign the original α-helical bundle of the human spectrin repeat R15, allowing for a more straightforward stability comparison.

While the authors have shown experimentally that stage II constructs have increased the mechanical stability by AFM, they did not show that these same constructs have increased the thermal and chemical stabilities. Since the effects of salt bridges on stability are highly context dependent (orientation, local environment, exposed vs buried, etc.), it is difficult to assess the magnitude of the effect that this change had on other types of stabilities.

The three constructs chosen are 60-70% identical to each other, either suggesting overconstrained optimization of the sequence or a physical constraint inherent to designing ultrastable α-helical bundles. It would be interesting to explore these possible design principles further.

While the use of steered MD is an elegant approach to picking the top N most stable designs, its computational cost may become prohibitive as the number of designs increases or as the protein size grows, especially since it requires simulating a water box that can accommodate a fully denatured protein.

Reviewer #3 (Public review):

Summary:

Qiu et al. present a hierarchical framework that combines AI and molecular dynamics simulation to design an α-helical protein with enhanced thermal, chemical, and mechanical stability. Strategically, chemical modification by incorporating additional α-helix, site-specific salt bridges, and metal coordination further enhanced the stability. The experimental validation using single-molecule force spectroscopy and CD melting measurements provides fundamental physical chemical insights into the stabilization of α-helices. Together with the group's prior work on super-stable β strands (https://www.nature.com/articles/s41557-025-01998-3), this research provides a comprehensive toolkit for protein stabilization. This framework has broad implications for designing stable proteins capable of functioning under extreme conditions.

Strengths:

The study represents a complete framework for stabilizing the fundamental protein elements, α-helices. A key strength of this work is the integration of AI tools with chemical knowledge of protein stability.<br /> The experimental validation in this study is exceptional. The single-molecule AFM analysis provided a high-resolution look at the energy landscape of these designed scaffolds. This approach allows for the direct observation of mechanical unfolding forces (exceeding 200 pN) and the precise contribution of individual chemical modifications to global stability. These measurements offer new, fundamental insights into the physicochemical principles that govern α-helix stabilization.

Weaknesses:

(1) The authors report that appending an additional helix increases the overcall stability of the α-helical protein. Could the author provide a more detailed structural explanation for this? Why does the mechanical stability increase as the number of helixes increase? Is there a reported correlation between the number of helices (or the extent of the hydrophobic core) and the stability?

(2) The author analyzed both thermal stability and mechanical stability. It would be helpful for the author to discuss the relationship between these two parameters in the context of their design. Since thermal melting probes equilibrium stability (ΔG), while mechanical stability probes the unfolding energy barriers along the pulling coordinate.

(3) While the current study demonstrates a dramatic increase in global stability, the analysis focuses almost exclusively on the unfolding (melting) process. However, thermodynamic stability is a function of both folding (kf) and unfolding (ku) rates. It remains unclear whether the observed ultrastability is primarily driven by a drastic decrease in the unfolding rate (ku) or if the design also maintains or improves the folding rate (kf)?

(4) The authors chose the spectrin repeat R15 as the starting scaffold for their design. R15 is a well-established model known for its "ultra-fast" folding kinetics, with folding rates (kf ~105s), near three orders of magnitude faster than its homologues like R17 (Scott et.al., Journal of molecular biology 344.1 (2004): 195-205). Does the newly designed protein, with its additional fourth helix and site-specific chemical modifications, retain the exceptionally high folding rate of the parent R15?

Round off to 2 decimal places

I enjoy this part because it showcases that despite the implied negatives of tourism, the accessibility of Mt. Fuji creates an environment where there are several ways to successfully climb the mountain. Someone does not have to be in peak physical condition to make the trek.

The author's inclusion of the monetary price even in common rituals on the mountain shows how deeply embedded the tourism culture is within a hike that used to be about religion.

The use of the word melting ties the piece together well as is fits with the previous weather mentions. For example, the author's highlight of both the extreme heatwave and two typhoons connect to the connotation of the description melting.

The emphasis on 9th century building adds to the foreign feeling. A general example of an old American building are colonial style buildings from the 17th century, which is nearly 800 years later. Additionally, the dedication to the Chinese scholar who died in 852 AD, implies the existence of preserved records from that time period which feels alien to me.

I like this visual. Having wet clothes drying on a line outside is something everyone recognizes, whether it is something they grew up doing, or they simply saw in multiple pieces of media. Thus it is very easy to picture that scene in your head. At the same time, the author manages to make it hint at the location by specifying that kimonos are drying as opposed to just leaving it at a general statement of clothes are drying.

1. How can workplaces better adapt policies, expectations, and environments to support neurodivergent employees rather than expecting them to conform to traditional norms?
   1. What responsibility do employers have to recognize neurodiversity as a strength while still addressing productivity and performance standards?

Connection: Mental disorders causing distress or disability really shows up for me in how hard it can be to balance school and staying physically active at the same time. When my mental health is struggling, even things I want to do—like going to class prepared, studying, or working out—start to feel overwhelming. Some days my body feels heavy, my motivation drops, and it becomes harder to stay consistent, even though I know movement usually helps my mental health.

This connection helps me see that these challenges aren’t about laziness or lack of discipline, but about how mental health directly affects functioning. Trying to meet academic expectations while pushing myself to stay active can create a lot of internal pressure, especially when I feel like I should be able to “handle it all.” Recognizing this helps me give myself more grace and reminds me that needing balance, flexibility, and rest is part of taking care of both my mental and physical well-being.

In this quote it shows how Islam adapted, similarly to Christianity, differently across groups of people instead of a uniform belief system. This shows how they were able to blend everyday religious practice with other older traditions. Again showing how religion was shaped by local culture or things like social context, as it highlights the flexibility Islam had as it spread similarly to Christianity.

This sentece shows how Christianity in Mexico became and adaptation of fitting existing religious frameworks in the area. The blending of beleifs as stated in one of my previous highlights the reshaping of a foreign religion to reflect their traditions, wihtout outright adopting Christianity. This way they were still able to perserve spiritual meanings of their own society.

This included quote is really striking and comparative to what we were talking about in class, with how indigenous people reacted to the conquest of Christian reform. Leading to ideological developments like apocalyptic ideas or incorporation of Christian God into their own beliefs. As stated in class they sometimes took on these reformations as a way of furthering their own colonies or interests.

What I find interesting with this quote is, it shows that a religious movement like this had broader implications like the skepticism toward authority. As the reformation challenged the Catholic Church's power, it established the inability of perfection within all higher ranking institutions. This I see as paving the way for independent thinking, which challenged the ongoing traditions or authority within society.

How did Chinese elites selective acceptance of European scientific knowledge reflects broader attitudes toward cultural superiority?

a shift from the Enlightenment faith in pure reason and logic toward ideas that emphasize emotion and nature. Rousseau and the Romantic movement challenged the belief that rational progress alone could improve society

deep internal divisors weaken unity between Western Catholic and Eastern Orthodox regions. At the same time, the Christendom faced military, political, and religious pressure from the expanding Islamic world.

Shows how Confucianism and Neo-Confucianism provided continuity in Chinese political and intellectual life while also allowing adaptation under different rulers

shows how religious movement gained political power and became the foundation of a state, demonstrating the close alliance between religious ideology and political authority in the islamic world

Protestant Reformation had broad social consequences beyond theology. It reshaped attitudes toward work and social status, and helped legitimized the rising middle class. However, it fueled unrest among peasants.

After the reformation, it led to the rise of multiple Protestant denominations, prolonged religious conflicts, and shifts in political power away from the church

Began with slaves brought over from the west coast of Africa in regards to Brazil. Islam was spread by traders, merchants, and through warfare just as Christianity was. After muhammeds death, armies conquered areas in the Byzantine and Sassanid empires.

Sir Isaac Newton unified early modern astronomy and physics by formulating the law of motion, which really took over science until the 20th century. What he thought and discovered was the same natural law that govern the heavens and the earth, linking them like falling apples and orbiting planets.

The lasting success of these missionaries is still shown in present day Philippines. With nearly 90% of the country apart of the Christian faith, it's the largest Christian nation on the Asian continent. The vast majority of these Christians are Roman Catholic, just like the Spaniards who settled in Philippines in the 1500's https://blogs.loc.gov/international-collections/2018/07/catholicism-in-the-philippines-during-the-spanish-colonial-period-1521-1898/

This goes to show that Europe developed legally independent institutions, especially universities, scholars gained greater freedom from church and state control. The independence allowed the study of nature to separate from philosophy and theology. This allowed universities and later scientific academic became key centers for producing scientific knowledge.

The Spaniard inflexibility to local religion makes sense, as Catholicism during this time was very much “Respect authority! Don’t question!” It’s clear that these religious traits appeared into the demeanors of the Spanish missionaries in Mesoamerica.

A war that I have not learned much about. This is considered one of the first modern wars due to its widespread violence and impact, large armies, and economic impact. It shows how Christianity, like other religions, have internal conflict and yet still remain relatively stable. This war also gave birth to Switzerland and the Dutch Republic after its conclusion.

In all my years, I haven't learned much about the Quaker community and have never heard of Anabaptist! After looking into more, it was created in Switzerland. It shows that even with numerous denominations in numerous areas of the world, Christianity still has a very strong presence and hold on Europe and other areas.

The printing press was a big factor in the split of the church. I think that without it, the Catholic Church may have been able to fight back quicker against the ideas presented by Martin Luther.

Brought to Japan by a Spanish priest named Francis Xavier in the 1500's, and to China in the 7th century, one could argue that it already was a genuine world religion being in the Middle East, Africa, and then Europe.

I find it very interesting how they were able to incorporate Christianity into ancient traditions that they had been practicing for generations. It must have been challenging for them to make such a drastic transition.

I think it is very interesting to see all the different forms of Christianity across the world. They all worshiped the same God just a different way.

I thought it was interesting that the Catholic Church sought out to “correct” the corruption within the church, but increased censorship.

Apparently, a big part of this censorship was banning a Bible in 1546 that contained commentary which “could be read as critical authority.”

https://hob.gseis.ucla.edu/HoBCoursebook_Ch_6.html

It is interesting that we still make distinctions between the advancements "western" and "eastern" science, technology, and medicine when both "fields" emerged at a similar time, but used for different purposes.

This is such an interesting definition of science. The idea that knowledge is acquired through "the product of human minds alone" is a definition I would associate with philosophy, religion, and theology more so than science.

I wonder if the gentler method of introducing religion that Islam used was more effective for spreading the religion to more people than that of Christianity and Catholicism. Does allowing your beliefs to adjust with the culture strengthen or weaken the collective faith of the people?

It is interesting to see how the problems Martin Luther had with the Catholic Church, excluding criticisms of doctrines, were, in a roundabout way, fixed by his ninety-five theses. By unintentionally creating a rift in Christianity, it conversely restrengthened the Catholic Church and corrected the problems that had arisen.

I think this is quite interesting. Obviously China was very powerful and had many strong thoughts and political stance. So it was very different from other countries and nations interacting with Christianity. the Jesuits had to convert to learn the culture of Chinese. They did this by learning the language, wearing the clothes, and getting "acquainted" with their text. They basically just exchanged ideas and thoughts. Then that was how they were aloud to spear their religion with respect and honor.

The reformation and the spread of religion boomed over the past years. The main help of this fact spreading religion was the printing press and how it would transform texts to different languages. This is what English protestant said was the word or what "God has appointed" to help give the voice of religion and God voice through the Pope. it most boomed through German but it also spread through France, Switzerland, and England.

This stood out to me because it is the first place I have read where they are saying women have the same equal chances as men in this religion.

This id interesting to me because the last chapter spoke about how the Europeans brought over diseases and wiped out civilizations and had majority control.

I find it interesting that though all of these other religions were growing and expanding, Christianity seems to be the one that motivated and influenced most of everything.

why were the convents closed and where did the women who were living in them end up? We they allowed to break their oath and get married or were they to abide by the rules even when they had no place to be?

I've never really considered how the cultural changes that came from the Scientific Revolution actually could've potentially led to difference in class such as hierarchies and political systems. I've only really thought of it as a cultural change and never really considered how the changes in culture can impact government decisions.

I think it's interesting that Africa had a lot of strong Christian empires in ancient times, I always have viewed Africa as a continent that has been a predominantly Islamic continent with many different countries so the idea of unified Christian empires is fascinating to me.

Even though I am religious and Christian, I think it's interesting to learn throughout history how Christian beliefs have impacted the way that empires view everything that they are doing, even if it maybe morally wrong. God says not to kill, but here their success in wars shows them how powerful their God is.

I think it's fascinating to see how Christianity was able to spread so fast from the Middle East all the way to Europe and Asia and how long it was able to retain it's power in those areas.

I already use on of these apps and I'm willing to try out more to see which one best fits my needs & learning style.

Taking ownership of your learning is one of the many keys to success.

This is one of my favorite parts of learning, having that "a-ha!" moment. Having a positive life & mind-changing experience and being able to share that with others is a blessing.

Living proof here. I've worked many many jobs without a college degree and also missed out on promotional opportunities due to lack of further education.

Do you recommend that referrals and workshops be the main driver to client acquisition?

How best do you suggest I create these? I have done DTC and they don't convert well...probably because they look amature.

Any AI strategies to make them high quality while also being time effective?

I never knew that an annotated bibliography can demonstrate quality in my research. To be honest, before this class I didn't know what a bibliography was so I am glad I got to learn about it.

Why is it difficult to do your research on your secondary source without writing any field notes? I am still confused on what exactly we can research. I was thinking of doing something related to the feeling people get when watching scary movies, but I wonder how it can relate to culture.

As a person who procrastinates, I want to know the deeper reasons and causes of it.

I read this article and what I took from it was that, yes, the internet is the leading cause for distractions at work, mostly with then millennial generation.

Las Casas point out that greed and disrespect drove the Spanish to there action against the natives.

This highlights the destruction and the depopulation caused by the Spanish, emphasizing the cruelty of colonization.

The number show the massive scale of death caused by Spanish actions, almost an entire population wiped out.

Las Casas emphasizes how peaceful and loyal the Native Americans were showing that they’re suffering was undeserved.

Describing the killings as treachery shows the betrayal felt by the Aztecs.

Montezuma‘s protest emphasizes how unfair and brutal the Spanish actions were.

The attack on the musicians highlights of the extreme violence of the conquest.

Montezuma‘s awe shows the impact the Spaniards had on the Aztecs.

Encountering the Skrelling must have been tense and shows how uncertain life in a new land can be.

I think building a proper shelter, shows how serious they were about surviving the winter.

The harsh landscape must have made exploring really challenging for the people.

It’s impressive that Leif stepped up to lead such a huge journey at a young age.

Disease from the Spanish wiped out most of the people in the south, even before European settled there. This shows how big and impact disease had on native communities.

It’s interesting how history can be viewed differently depending on who is telling the story.

This shows that Native American societies were organized and capable of building large cities, not just small villages.

This show that the Maya were very advanced because they created their own writing system to manage their society.

This is something I will definitely take note of. By making sure my sources are relevant I can fully utilize them and make sure that they fit and make sense in my paper. I wonder how I can effectively use my secondary source to strengthen my primary source.

I often wonder how early hominids figured out which animals were safe to eat and which were not, as well as how different animals were hunted. While much of this knowledge likely came from trial and error, how was it communicated among group members to help everyone avoid danger? How did they learn to hunt animals like rhinos? A rhino is massive and certainly not a friendly creature. These hominids must have learned through deliberate, calculated attempts rather than random attacks. Of course, scavenging from already deceased animals was an option, but I imagine their curiosity and ingenuity eventually led them taking on such formidable beasts as a challenge. I find that idea incredibly fascinating.

There is no one-size-fits-all learning system, everyone learns differently and that's the beauty of it all. There are so many learning strategies this book reviews, one or many are bound to fit.

à mettre à jour pour les trois sources

ça donne l'impression qu'on peut éditer ces fichiers. Mais ils sont protégé par un checksum et il faut modifier les Safety settings dans le UI.

So is this simple substitution Cipher uncrackable? Well, let's wait a minute here. It's not, for example, one of the pieces of information we can use in analyzing simple substitution is the letter frequencies of the English language or any language If you looked at a histogram of English letter frequencies, you'd see that e is the most frequent letter just above 12 percent, T is the second most frequent at somewhere around nine percent, a is very frequent at more than eight percent and so forth, so you would see this familiar pattern in any text that you encrypted. For example, if you encrypted it using the simple substitution Cipher, you might get this pattern, but you'd still see a peak where the letter that was substituted for e occurs. And maybe this is the peak for T, and maybe this is the peak for a. By analyzing those peaks and valleys, you can determine how the message was encrypted, can figure out the key. But, wait a minute… frequency analysis works!<br /> Slide 21, 22 chart

E.G. If you sorted by frequencies. Now, you can see this very clearly if you sort the two histograms, they're practically identical, and that's what lets the analyst with some effort, of course, figure out the key and break the message so you can break simple substitution cipher using frequency analysis. That makes Eve happy, Alice and Bob are not very happy about that. Eve wins … you don’t need brute force. Frequency analysis will break simple substitution. slide 23, 24 chart

The last question how many keys would you have to try in order to perform a Brute Force attack on simple substitution? Let's look at that question again. The answer is an enormous number 26 factorial Keys. That's the number of ways you could shuffle the plain text alphabet of 26 letters. So that's way too hard. And that fact makes Alice and Bob happy and Eve not very happy.

Simple Substitution Cipher

Key: Randomly mix up the plain alphabet to get a cipher alphabet.

So the key basically tells what to do with getting a cipher alphabet to be able to encrypt either the key consisting of a number for shifting each letter or a keyword to put in beginning as a way of scrambling

Algorithm: Substitute plain letters with shuffled letters.

This tells how to encrypt the message using to cipher alphabet

Well, let's look at a slightly more complex Cipher, another example, of what's called a simple substitution Cipher, which means generally that you randomly mix up the alphabet to get a cipher alphabet and then you substitute plain letters with shuffled letters, so we're gonna use a little trick here that we're going to have a keyword that'll help us (one way to) construct our Cipher alphabet, which is our actual key by putting the keyword at the beginning and then putting all the letters that aren't in the word, zebrafish following it in alphabetical order. Now, obviously, there are lots of ways we could do this, but that gives us an easy way to transmit the key between Alice and Bob. Alice just tells Bob zebrafish and he knows how to construct this alphabet. But if they wanted, Alice could simply create a permutation or shuffling of this alphabet, not using the keyword and pass that along to Bob. Once you've created the cipher alphabet, encrypting and decrypting, in simple substitution cipher works the same as it did in Caesar Cypher. For each letter in the plane text message, you replace it with the corresponding letter from the cipher alphabet. Of course, you have many more possible Cipher alphabets because the alphabets aren't merely a shifting of the original plain alphabet. chart

How do you crack a Caesar Cipher? In other words, how does Eve break these messages? Well, the one question you might ask is, well, how many different keys would Eve have to try to do what's called a Brute Force attack? That means try every possible key. As you've probably discovered, the answer is 25 Keys. That's way too easy, so Eve is very happy about that because she can easily break Caesar Cypher. chart

This chart is VERY useful and a daily reminder to ensure I am on the right track with achieving my goals.

Is that a bad thing though? I mean, in the end, you were successful, no matter the reason. The goal was achieved, end of story. So is it ONLY to be motivated by the goals we set and not have other reasons to achieve a goal?

The small tasks add up and contribute to achieving your goal.

This is a good strategy that I will definitely use in the future since I had trouble finding more than one good source. Furthermore, I should broaden my key words in order to get better results as well.

These are not tangible, so not only does the working environment include physical visual things it includes the things we cannot see, but what we feel.

I never thought of this as an issue! Makes sense though.

I agree, I personally find that where I choose, or am able to complete my work is very important in keeping me focused and lesses distractions.

There's always room to readjust a plan. Have a plan A, B, C, D, etc.

We can relate this to the discussions we had in class about why did inmigrants decide to come to the US. It's interesting how a period with a lot of immigration in the US was because of the government policies and corruption in different countries. But, here in the US, people also face governments with corrupt politicians. But I guess there was a lot less than in other countries.

Match this against Zheng et al. 2014's description of AMP.

Me encanta esta fotografía porque están bailando en lo que parece una cárcel (convict camp). Me pregunto por qué estarían ellos ahí. Uno está bailando, otro toca la guitarra, y el otro aplaudiendo. Los demos no participan, pero hay un hombre hasta el fondo que los observa (creo que es un guardia). No me puedo imaginar cómo se dio ese momento, pero me gustaría creer que ellos tuvieron esperanza o, al menos, un breve encuentro con la felicidad.

This is a good example of how states use law and rhetoric to manage religion, and how religious freedom can be more about appearances than practice

While reading this, I thought about how Western narratives today sometimes oversimplify or misrepresent Chinese government policies, especially around religion and ethnicity. For example, news coverage about Uyghur Muslims in Xinjiang often portrays the situation as entirely one sided, which can lead to political pressure or sanctions, even though the reality is more complicated.

Mainstream privilege refers to the advantages or benefits that people get from belonging to the dominant culture, group, or social norm. It’s the “default” status that makes life easier because society is structured around those mainstream values, language, and practices of the majority.

Here, “apologetic tactic” means that Japanese American Buddhists were using the idea of religious freedom to defend or justify their religion to the American majority.

This shows how the government used schools to control Japanese kids and make them more “American.” It connects law, language, and politics, and helps explain how authorities tried to manage immigrant communities in the early 1900s.

What if I do this and still don't find any results? Do I broaden my ideas furthermore? Or do I find another database or scholarly website to find my desired results? I have these questions because I had trouble finding anything that had to do with my topic on the MSU database. So as a result I used google search, I leaned more towards academic associated websites such as Harvard business review. Is that an appropriate solution?

This is breaking down what the aim of the paper is trying to do.

I think it is important to always question your ideas, ask for help, and to know yourself. I think I should ask more help from my teacher and fellow peers, this will make my writing process much easier.

As I become more familiar with the definitions of provenance, this particular instance compels me as I can see many examples of this in some of the media that I consume -

For instance, there is a zine called Sex Change USA by Daisy Thursday [https://luma.com/o1iss5rd] that is a collection of “stories where transgender people were covered in the National Esquire, Sun, and Weekly World News tabloids from 1993-2002.”

What I took away from Sex Change USA was not only a better understanding of the different contexts the pieces from the zine have lived in and do currently live in but a better understanding of the context of it’s current time and how those pieces interact with and inform each other.

I think the only incentive that social media companies have to protect privacy is simply because users desire a service which does that. I feel like this incentive is also weak, as privacy as a resource has been regenotiated over the course of the past thrity years of the internet. Things which previously would have been unthinkable, like uploading SSIDs onto documents through email, are now common (I've had to do so to sign leases multiple times in college). I think its only if the underlying logic of these companies (that being profit and the monopolization of digital existence) changes that an incentive to protect privacy could strengthen.

In my capstone research paper I'm writing this quarter, I place a lot of importance on the reification of thoughts and desires that the collection of behavioral data brings. I feel like there is a huge discomfort in the user of the internet at the prospect of encountering a digital world shaped by their thoughts in a way that they perceive themselves to have little to no control over. The right to be forgotten here resonates with that anxiety and discomfort, and I feel like the US model should definitely adopt something similar. It assumes that anonymity is an inalienable right we have, which is something i also hold to be true.

You can visit the library to help you with resources for your papers and to get ideas for your thesis

Your audience is who reads your writing. You should write in a way that catches the attention of the people it is supposed to.

It is not enough to do research for a paper. It is also important to show the evidence you collected so your readers can understand why you came to the conclusion that you did.

A strong thesis statement concentrates on a specific topic and appears toward the end of the introduction. This maps out the rest of the essay.

final 2019-era fork. aimed to delay the difficulty bomb once more.

Key Changes 1. Block Reward Reduction, from 5eth to 3 eth. intro of the EIP-649, which brought about difficulty bomb. 2. new opcodes for smart contract, suuport for static calls(via eip -214) eip -140, 196 and 197. 3. light transaction receipt. EIP -658.

EIP-196: EC point addition precompile

EIP-197: EC point scalar multiplication precompile

Curve: alt_bn128

Goal: make heavy cryptography feasible in the EVM

allows for REVERT opcode, this code allows one to retert a failed opration, without comsuming the whole gaas, INVALID as the opcode before it, that actually consumes all gas. revert made possible for debug, and gas retrun on remain.

EIP - 214, brought about static calls, static call allows call the blockchain without a change of state aka ( read only mode)

What resonated most with me in this passage was Cook’s emphasis on collective civic accountability and responsibility in archival practice. Traditionally, archival spaces have frequently functioned as exclusive environments, where access was limited and interaction with records was restricted to a specific, skilled group of professionals. I do not agree that this model serves the greater good. If materials are preserved but remain inaccessible to the communities they serve, the archival mission is then a failure. Cook’s argument reinforces the idea that archives should reflect the perspectives, experiences, and sentiments of the communities they serve. I was affected by the example of Canadian archival initiatives. The Canadian archival system demonstrates how a coordinated and collaborative system can bring together institutions, professionals, and communities in order to support more inclusive access and stewardship. In contrast, the current standings of archival work in the United States appear to be moving in the opposite direction, especially regarding access and preservation. This passage made me reflect on how openness and collaboration have the potential to revitalize archives and align them more closely with the needs of society.

This comment points to how modern social media platforms operate within an attention economy, where user time and engagement are the primary resources being monetized. By analyzing user behavior data and what people click, watch, like, or share: platforms can tailor content and notifications to keep users returning more often and staying longer.

This observation highlights a real issue in data analysis known as data dredging or p-hacking: when people search through large amounts of data without a clear hypothesis, they can often find patterns that look meaningful but are actually just coincidences.

This observation highlights a real issue in data analysis known as data dredging or p-hacking: when people search through large amounts of data without a clear hypothesis, they can often find patterns that look meaningful but are actually just coincidences.

This helped me understand the core principles from which ethnic studies truly derives from.

This solution seems to revolve around the following

• Loot being constant but not generous. This menas the player will always be picoing things up but everything matters.
• Loot being losable. So that everything they have muat be guarded but of course that playing the game is inevitably risking losing it.
• Interdependent. This means that there's no 'win button'. Very little will actually win you the game and as such you're looking for something thatauits your needs. And when you don't find it you're doing your best to use what you have.
• Indirectly supportive og your goal. The racing game here is an excellent example because going faster won't help you win if your turning circle is rubbish. So the core challenge or test must require an interaction of different abilities to overcome.

is this true

has it always been true

must everything wend its way through every shit-filled crevasse

How did the “Lost Cause” help white southerners justify white supremacy and reshape the South’s image to the rest of the nation?

Why do you think people in 1878 were willing to believe that technology could completely end hunger and poverty?

the pockets

on the legs

This is actually a good example of dispatchable demand response - It's like an ipdated version of the practice of making nice at night.

Wow, half of Google's data centers are now using liquid cooling.

The repetitiveness of true-crime stories has resulted in people going back to find more podcasts to quench their thirst for new material.

People enjoy the shock factor that comes in real true crime stories.

Ethos?

Supports my podcast by displaying a similar one that was actually able to help release a man who was wrongfully convicted. This shows that not only are the podcasts trustworthy, but also helps in the outside world.

easy access grants more attraction to podcasts of people looking for news sources or entertainment. Are there podcasts that aren't free?

Important for later incase you need some extra help in this course

It's fascinating how even a simple cell maintains so much control over its metabolic processes. Even though these regulatory mechanisms seem complex, they're actually the most energy-efficient because they prevent the cell from wasting energy.

With how revealing metadata can be, it makes me wonder why platforms even include the metadata in posts. And should media outlets should be more responsible for removing metadata before publishing content that could put people at risk like this example?

I also connected this to what I see in popular culture and social media. A lot of online “allyship” looks loud and performative, but not always relational. Sue et al.’s point about microinterventions being about reassurance and not leaving someone alone in their experience feels very different from posting something just to be seen as on the right side. It makes me think about how real resistance happens in everyday moments, not just big statements.

This statement from George shows how badly he wants to obtain freedom, and how he is willing to die if he has to, as he would rather die then spend more of his life enslaved.

•   Bronchitis Treatment: Inhaling moist air from a humidifier or steamy shower can help loosen and thin mucus, making breathing easier.
•   Bronchitis Symptoms: Coughing, sore throat, chest congestion, and coughing up thick mucus.
•   Bronchitis Cause: Usually caused by viruses, not bacteria, so antibiotics are generally ineffective.
•   Bronchitis Definition: Inflammation of the bronchial tubes, causing narrowing and excessive mucus production.
•   Mucus Function and Problem: Mucus traps pathogens but excessive production hinders airflow, leading to coughing.
•   Bronchitis Treatment: Thinning mucus for effective coughing through fluids, humidifiers, steam, and expectorants, while avoiding cough suppressants.
•   Reason for Pulse Oximetry: To check Sacheen’s blood oxygen level and ensure clogged airways weren’t impacting her oxygen intake.
•   Difference Between Acute and Chronic Bronchitis: Acute bronchitis is a short-term condition often caused by a cold or flu, while chronic bronchitis is a long-term condition often caused by smoking and associated with COPD.
•   Smoking Cessation Advice: Dr. Tsosie strongly advised Sacheen to quit smoking to prevent future respiratory infections, COPD, and lung cancer.
•   Respiratory System Function: Critical for gas exchange and protecting the body from harmful substances in the air.
•   Respiratory System Vulnerability: Prone to infections and damage from allergens, mold, air pollution, and cigarette smoke.
•   Respiratory System Overview: Comprises the upper respiratory tract (nasal cavity, pharynx, larynx) for air conduction and the lower respiratory tract (trachea, bronchi, bronchioles, lungs) for conduction and gas exchange.
•   Respiratory System Defense: The mucociliary escalator, consisting of mucus-producing cells and cilia, protects the lungs by trapping and expelling harmful particles and pathogens.
•   Breathing Regulation: The respiratory system, controlled by the brain, regulates breathing rate based on carbon dioxide levels in the blood to maintain homeostasis.
•   Breathing Mechanics: Breathing involves inhaling (active process driven by diaphragm contraction) and exhaling (passive process driven by lung elasticity).
•   Gas Exchange Definition: The biological process of transferring gases across cell membranes for entering or leaving the blood.
•   Gas Exchange Mechanism: Occurs by diffusion across cell membranes, moving down a concentration gradient from high to low concentration.
•   Lung Gas Exchange: Takes place in alveoli, where deoxygenated blood picks up oxygen and releases carbon dioxide.
•   Smoking and COPD: Smoking is the primary cause of COPD, reducing lung elasticity and impairing exhalation.
•   Smoking and Lung Cancer: Smoking is the leading cause of lung cancer, a malignant tumor characterized by uncontrolled cell growth in the lungs.
•   Health Risks of Smoking: Smoking poses numerous health risks, including increased risk of various cancers, cardiovascular disease, and other adverse effects.
•   Respiratory System Function: The respiratory system facilitates gas exchange, bringing oxygen into the body and removing carbon dioxide.
•   Gas Exchange Mechanism: Oxygen and carbon dioxide flow across membranes based on concentration gradients, moving from areas of higher to lower concentration.
•   COPD and Blood pH: COPD can lead to elevated carbon dioxide levels, causing respiratory acidosis and prompting the body to compensate by increasing breathing rate.
•   Bronchitis Treatment: Changes to the environment, such as more frequent cleaning, would not help asthma caused by a gene.
•   Bronchitis Definition: Bronchitis is an inflammation of the bronchi, the large and medium-sized airways in the lungs that carry air from the trachea.
•   Bronchodilator Definition: A medication that opens constricted airways.
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