Reviewer #1 (Public Review):

This study makes a substantial contribution to our understanding of the molecular evolutionary dynamics of microbial genomes by proposing a model that incorporates relatively frequent adaptive reversion mutations. In many ways, this makes sense from my own experience with evolutionary genomic data of microbes, where reversions are surprisingly familiar as evidence of the immense power of selection in large populations.

One criticism is the reliance on one major data set of B. fragilis to test fits of these models, but this is relatively minor in my opinion and can be caveated by discussion of other relevant datasets for parallel investigation.

Another point is that this problem isn't as new as the manuscript indicates, see for example https://journals.asm.org/doi/10.1128/aem.02002-20.

Nonetheless, the paper succeeds by both developing theory and offering concrete parameters to illustrate the magnitudes of the problems that distinguish competing ideas, for example, the risk of mutational load posed in the absence of frequent back mutation.

Reviewer #1 (Public Review):

(1) Napthylamine (1NA), an industrial reagent used in the manufacturing of dyes and pesticides is harmful to humans and the environment. In the current manuscript, the authors report the successful isolation of a Pseudomonas strain from a former naphthylamine manufacturing site that is capable of degrading 1NA. Using genetic and enzymatic analysis they identified the initial stages of 1NA degradation and the enzymes responsible for downstream processing of 1,2-dihydroxynapthalene and Salicylate. The authors determined the molecular structure of NpaA1, the first enzyme in the pathway responsible for glutamylation of 1NA. NpaA1 has a border substrate specificity compared to previously characterized enzymes involved in aromatic amine degradation. They carried out structural comparison of NpaA1 with glutamine synthase structures, alfa-fold models of similar enzymes and put forth hypothesis to explain the broad substrate specificity of NpaA1.

The manuscript is well written and easy to understand. The authors carried out careful genetic analysis to identify the genes/enzymes responsible for degradation of 1NA to catechol. They characterized the first enzyme in the pathway, NpaA1 which is responsible glutamylation of 1NA. and determined the molecular structure of apo-NpaA1, NpaA1 - AMPPNP complex and Npa1 - ADP - Met-Sox-P complex using X-ray crystallography.<br /> The proposed mechanism of broad substrate specificity of NpaA1, however, is based on comparison of 1NA docked NpaA1 structure with St-GS (Glutamate synthase) and Alphafold2 predicted model of AtdA1 from an aniline degrading strain of Acinetobacter sp. Lack of molecular structure or mutational studies to back the proposed mechanism makes it difficult to agree with the proposed mechanism.

Reviewer #1 (Public Review):<br /> <br /> The impact of this paper is that it shows conclusively the bone defects caused by ninein depletion, albeit transient defects, which has been indirectly deduced in past studies. The paper is largely descriptive including the cytoskeletal analysis of osteoclasts thus it remains unclear how ninein reduction causes bone defects and why this defect is transient. The Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects in ninein-null mice.

Other points:<br /> Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast and osteocyte presence and activity in ninein del/del mice to strengthen the study.

In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.

The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?

Some text needs clarification. For instance, "On days 3 and 4, we found only about half as many large syncytia in cultures from ninein-deleted mice, compared to controls, but on day 5 large syncytia lacking ninein exceeded 90% of control levels. Altogether, this suggests that fusion deficiencies are a transient phenomenon in in vitro-induced adult osteoclasts. On later days of culture, fusion efficiency started to diminish." What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.

Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.

Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.

Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.

I.e., 3f+1 is not suitable for open peer-to-peer systems.

Reviewer #1 (Public Review):

This study offers good evidence pointing to a genetic basis for Arabidopsis thaliana's response to elevated CO2 (eCO2) levels and its subsequent impact on the leaf ionome. The natural variation analyses in the study support the hypothesis that genetic factors, rather than local adaptation, guide the influence of eCO2 on the ionome of rosette leaves in Arabidopsis.

Comments on current version:

I appreciate the revisions and the effort the authors have made.

Most of the abstract now accurately reflects the results and methods. It would be nice to have a few more technical details in the abstract, such as:<br /> * What was the CO2 level?<br /> * Which gene was identified?

I still have a problem with this sentence:

"The elevation of atmospheric CO2 leads to a decline in plant mineral content, which might pose a significant threat to food security in the coming decades."

The authors provide a wide range of published studies that support this statement. I fully agree that this is what the literature suggests. However, I think the literature has asked the wrong question.

In general, these studies addressed the question: Given no time for adaptation, do plants grown under high CO2 have a different mineral composition? The answer is yes.

But a more important question is: Can plants and food crops adapt in time? I believe the strength of this study is that it tests this, and it suggests that the answer is yes. I also think there is a lot of unpublished results and greenhouse breeding success that supports the contention that most plants can adapt to the CO2.

"The artificial elevation of atmospheric CO2 leads to a physiological response and decline in plant mineral content, which might pose a significant threat to food security in the coming decades if plants cannot adapt."

It needs to be made clear throughout the paper when high CO2 levels lead to low mineral composition. These are all artificial manipulations without allowing the plants to adapt to the new environment.

"The elevation of atmospheric CO2 concentration leads to a decline in the mineral composition of C3 plants (Gojon et al., 2023)." - this is well supported in artificial environments.

Do wild plants have fewer minerals in their leaves today compared to plants in 1950? This would be great evidence and framing for this experiment.

Crop plants having lower nitrogen and different mineral compositions over time is substantially a product of breeders initially increasing inputs and then, over the last decade, selecting for higher input efficiency.

At the end of the introduction or the beginning of the results, please define why the CO2 level was chosen and its context as being at the high end of current predictions.

"According to the literature, this results in a 20-25% reduction in vitamin C or lycopene and requires a significantly higher nitrogen and water intake to reach expected sugar levels (Doddrell H (2023), Horticulture Research). In addition, the negative effect of elevated CO2 on tomato nutrient content seems to have significant repercussions on nutrition-health properties (Boufeldja (2023), Molecules)."

Thank you for sharing these reviews. These suggest to me that breeders favored the 80% yield bump over other traits. Either there was no breeding, or the breeding focused on other traits. It is important to mention that breeders should include mineral nutrition in their selection index while they maximize yield. Simpler breeding strategies can sometimes heavily favor one trait over others, but cattle breeders today regularly use selection indices that incorporate weights for two dozen traits.

This study provides nice evidence that an annual weed species is likely to be able to adapt easily to high eCO2. Whether perennial species will be able to adapt in time is clearly a topic that needs to be investigated.

Reviewer #1 (Public Review):

Summary:

Zai et al test if songbirds can recover the capacity to sing auditory targets without singing experience or sensory feedback. Past work showed that after the pitch of targeted song syllables are driven outside of birds' preferred target range with external reinforcement, birds revert to baseline (i.e. restore their song to their target). Here the authors tested the extent to which this restoration occurs in muted or deafened birds. If these birds can restore, this would suggest an internal model that allows for sensory-to-motor mapping. If they cannot, this would suggest that learning relies entirely on feedback dependent mechanisms, e.g. reinforcement learning (RL). The authors find that deafened birds exhibit moderate but significant restoration, consistent with the existence of a previously under-appreciated internal model in songbirds.

Strengths:

The experimental approach of studying vocal plasticity in deafened or muted birds is innovative, technically difficult and perfectly suited for the question of feedback-independent learning. The finding in Figure 4 that deafened birds exhibit subtle but significant plasticity toward restoration of their pre-deafening target is surprising and important for the songbird and vocal learning fields, in general.

In this revision, the authors suitably addressed confusion about some statistical methods related to Fig. 4, where the main finding of vocal plasticity in deafened birds was presented.

There remain minor issues in the presentation early in the results section and in Fig. 4 that should be straightforward to clarify in the revision.

Reviewer #1 (Public Review):

Summary:

In the work: "Endosomal sorting protein SNX4 limits synaptic vesicle docking and release" Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly developed in vitro KO model, with live imaging experiments, electrophysiological recordings, and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with a higher number of docked vesicles and shorter AZ. The evaluation of the presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that resulted in chronical deletion of SNX4 in neurons. A convincing sequence of experimental techniques is applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at the presynaptic site is relevant and of high interest in the field of synaptic physiology and in the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow us to address the physiological roles of SNX4 at the synaptic site and speculate on the underlying molecular mechanisms. In addition, due to the described role of SNX4 in autophagy and the high interest in the regulation of synaptic autophagy in the field of synaptic physiology, an initial evaluation of the autophagy phenotype in the neuronal SNX4KO model is important, and not to be only restricted to the discussion section.

Reviewer #1 (Public Review):

Summary:

This work identified new NMD inhibitors and tested them for cancer treatment, based on the hypothesis that inhibiting NMD could lead to the production of cancer neoantigens from the stabilized mutant mRNAs, thereby enhancing the immune system's ability to recognize and kill cancer cells. Key points of the study include:

• Development of an RNA-seq based method for NMD analysis using mixed isogenic cells that express WT or mutant transcripts of STAG2 and TP53 with engineered truncation mutations.

• Application of this method for a drug screen and identified several potential NMD inhibitors.

• Demonstration that one of the identified compounds, LY3023414, inhibits NMD by targeting the SMG1 protein kinase in the NMD pathway in cultured cells and mouse xenografts.

• Due to the in vivo toxicity observed for LY3023414, the authors developed 11 new SMG1 inhibitors (KVS0001-KVS0011) based on the structures of the known SMG1 inhibitor SMG1i-11 and the SMG1 protein itself.

• Among these, KVS0001 stood out for its high potency, excellent bioavailability, and low toxicity in mice. Treatment with KVS0001 caused NMD inhibition and increased presentation of neoantigens on MHC-I molecules, resulting in the clearance of cancer cells in vitro by co-cultured T cells and cancer xenografts in mice by the immune system.

These findings support the strategy of targeting the NMD pathway for cancer treatment and provide new research tools and potential lead compounds for further exploration.

Strengths:

The RNA-seq-based NMD analysis, using isogenic cell lines with specific NMD-inducing mutations, represents a novel approach for the high-throughput identification of potential NMD modulators or genetic regulators. The effectiveness of this method is exemplified by the identification of a new activity of AKT1/mTOR inhibitor LY3023414 in inhibiting NMD.

The properties of KVS0001 described in the manuscript as a novel SMG1 inhibitor suggest its potential as a lead compound for further testing the NMD-targeting strategies in cancer treatment. Additionally, this compound may serve as a useful research tool.

The results of the in vitro cell killing assay and in vivo xenograft experiments in both immuno-proficient and immune-deficient mice indicate that inhibiting NMD could be a viable therapeutic strategy for certain cancers.

Weaknesses:

The authors did not address the potential effects of NMD/SMG1 inhibitors on RNA splicing. Given that the transcripts of many RNA-binding proteins are natural targets of NMD, inhibiting NMD could significantly alter splicing patterns. This, in turn, might influence the outcomes of the RNA-seq-based method for NMD analysis and result interpretation.

While the RNA-seq-based approach offers several advantages for analyzing NMD, the effects of NMD/SMG1 inhibitors observed through this method should be confirmed using established NMD reporters. This step is crucial to rule out the possibility that mutations in STAG2 or TP53 affect NMD in cells, as well as to address potential clonal variations between different engineered cell lines.

The results from the SMG1/UPF1 knockdown and SMG1i-11 experiments presented in Figure 3 correlate with the effects seen for LY3023414, but they do not conclusively establish SMG1 as the direct target of LY3023414 in NMD inhibition. An epistatic analysis with LY3023414 and SMG1-knockdown is needed.

Reviewer #2 (Public Review):

Summary:<br /> The authors used four datasets spanning 30 countries to examine funding success and research quality score for various disciplines. They examined whether funding or research quality score were influenced by majority gender of the discipline and whether these affected men, women, or both within each discipline. They found that disciplines dominated by women have lower funding success and research quality score than disciplines dominated by men. These findings, are surprising because even the men in women-dominated fields experienced lower funding success and research quality score.

Strengths:<br /> - The authors utilized a comprehensive dataset covering 30 countries to explore the influence of the majority gender in academic disciplines on funding success and research quality scores.<br /> - Findings suggest a systemic issue where disciplines with a higher proportion of women have lower evaluations and funding success for all researchers, regardless of gender.<br /> - The manuscript is notable for its large sample size and the diverse international scope, enhancing the generalizability of the results.<br /> - The work accounts for various factors including age, number of research outputs, and bibliometric measures, strengthening the validity of the findings.<br /> - The manuscript raises important questions about unconscious bias in research evaluation and funding decisions, as evidenced by lower scores in women-dominated fields even for researchers that are men.<br /> - The study provides a nuanced view of gender bias, showing that it is not limited to individuals but extends to entire disciplines, impacting the perception and funding and quality or worth of research.<br /> - This work underscores the need to explore motivations behind gender distribution across fields, hinting at deep-rooted societal and institutional barriers.<br /> - The authors have opened a discussion on potential solutions to counter bias, like adjusting funding paylines or anonymizing applications, or other practical solutions.<br /> - While pointing out limitations such as the absence of data from major research-producing countries, the manuscript paves the way for future studies to examine whether its findings are universally applicable.

Weaknesses:<br /> - The study does not provide data on the gender of grant reviewers or stakeholders, which could be critical for understanding potential unconscious bias in funding decisions. These data are likely not available; however, this could be discussed. Are grant reviewers in fields dominated by women more likely to be women?<br /> - There could be more exploration into whether the research quality score is influenced by inherent biases towards disciplines themselves, rather than only being gender bias.<br /> - The manuscript should discuss how non-binary gender identities were addressed in the research. There is an opportunity to understand the impact on this group.<br /> - A significant limitation is absence of data from other major research-producing countries like China and the United States, raising questions about the generalizability of the findings. How comparable are the findings observed to these other countries?<br /> - The motivations and barriers that drive gender distribution in various fields could be expanded on. Are fields striving to reach gender parity through hiring or other mechanisms?<br /> - The authors could consider if the size of funding awards correlates with research scores, potentially overlooking a significant factor in the evaluation of research quality. Presumably there is less data on smaller 'pilot' funds and startup funds for disciplines where these are more common. Would funding success follow the same trend for these types of funds?<br /> - The language used in the manuscript at times may perpetuate bias, particularly when discussing "lower quality disciplines," which could influence the reader's perception of certain fields.<br /> - The manuscript does not clarify how many gender identities were represented in the datasets or how gender identity was determined, potentially conflating gender identity with biological sex.

Reviewer #1 (Public Review):

Summary:

The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

Strengths:

The data and analysis is highly compelling regarding the existence of cycle skipping.

Comments on the revised version:

All previous recommendations were addressed in this revision.

Reviewer #1 (Public Review):

Summary:

The pituitary gonadotropins, FSH and LH, are critical regulators of reproduction. In mammals, synthesis and secretion of FSH and LH by gonadotrope cells are controlled by the hypothalamic peptide, GnRH. As FSH and LH are made in the same cells in mammals, variation in the nature of GnRH secretion is thought to contribute to the differential regulation of the two hormones. In contrast, in fish, FSH and LH are produced in distinct gonadotrope populations and may be less (or differently) dependent on GnRH than in mammals. In the present manuscript, the authors endeavored to determine whether FSH may be independently controlled by a distinct peptide, cholecystokinin (CCK), in zebrafish.

Strengths:

The authors demonstrated that the CCK receptor is enriched in FSH-producing relative to LH-producing gonadotropes, and that genetic deletion of the receptor leads to dramatic decreases in gonadotropin production and gonadal development in zebrafish. Also, using innovative in vivo and ex vivo calcium imaging approaches, they show that LH- and FSH-producing gonadotropes preferentially respond to GnRH and CCK, respectively. Exogenous CCK also preferentially stimulated FSH secretion ex vivo and in vivo.

Weaknesses:

The concept that there may be a distinct FSH-releasing hormone (FSHRH) has been debated for decades. As the authors suggest that CCK is the long-sought FSHRH (at least in fish), they must provide data that convincingly leads to such a conclusion. In my estimation, they have not yet met this burden. In particular, they show that CCK is sufficient to activate FSH-producing cells, but have not yet demonstrated its necessity. Their one attempt to do so was using fish in which they inactivated the CCK receptor using CRISPR-Cas9. While this manipulation led to a reduction in FSH, LH was affected to a similar extent. As a result, they have not shown that CCK is a selective regulator of FSH. Moreover, they do not yet demonstrate that the effects observed reflect the loss of the receptor's function in gonadotropes, as opposed to other cell types. It also is not clear whether the phenotypes of the fish reflect perturbations in pituitary development vs. a loss of CCK receptor function in the pituitary later in life. Ideally, the authors would attempt to block CCK signaling in adult fish that develop normally. For example, if CCK receptor antagonists are available, they could be used to treat fish and see whether and how this affects FSH vs. LH secretion.

In the Discussion, the authors suggest that CCK, as a satiety factor, may provide a link between metabolism and reproduction. This is an interesting idea, but it is not supported by the data presented. That is, none of the results shown link metabolic state to CCK regulation of FSH and fertility. Absent such data, the lengthy discussion of the link is speculative and not fully merited.

Also in the Discussion, the authors argue that "CCK directly controls FSH cells by innervating the pituitary gland and binding to specific receptors that are particularly abundant in FSH gonadotrophs." However, their imaging does not demonstrate innervation of FSH cells by CCK terminals (e.g., at the EM level). Moreover, they have not demonstrated the binding of CCK to these cells. Indeed, no CCK receptor protein data are shown. The calcium responses of FSH cells to exogenous CCK certainly suggest the presence of functional CCK receptors therein; but, the nature of the preparations (with all pituitary cell types present) does not demonstrate that CCK is acting directly in these cells. Indeed, the asynchrony in responses of individual FSH cells to CCK (Figure 4) suggests that not all cells may be activated in the same way. Contrast the response of LH cells to GnRH, where the onset of calcium signaling is similar across cells (Figure 3). Finally, as the authors note in the Discussion, the data presented do not enable them to conclude that the endogenous CCK regulating FSH (assuming it does) is from the brain as opposed to other sources (e.g., the gut).

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, ITP has functions on feeding, drinking, metabolism, excretion, and circadian rhythm. In the current study, the authors characterized and compared the expression of all three ITP isoforms (ITPa and ITPL1&2) in the CNS and peripheral tissues of Drosophila. An important finding is that they functionally characterized and identified Gyc76C as an ITPa receptor in Drosophila using both in vitro and in vivo approaches. In vitro, the authors nicely confirmed that the inhibitory function of recombinant Drosophila ITPa on MT secretion is Gyc76C-dependent (knockdown Gyc76C specifically in two types of cells abolished the anti-diuretic action of Drosophila ITPa on renal tubules). They also used a combination of multiple approaches to investigate the roles of ITPa and Gyc76C on osmotic and metabolic homeostasis modulation in vivo. They revealed that ITPa signaling to renal tubules and fat body modulates osmotic and metabolic homeostasis via Gyc76C.

Furthermore, they tried to identify the upstream and downstream of ITP neurons in the nervous system by using connectomics and single-cell transcriptomic analysis. I found this interesting manuscript to be well-written and described. The findings in this study are valuable to help understand how ITP signals work on systemic homeostasis regulation. Both anatomical and single-cell transcriptome analysis here should be useful to many in the field.

Strengths:

- The question (what receptors of ITPa in Drosophila) that this study tries to address is important. The authors ruled out the Bombyx ITPa receptor orthologs as potential candidates. They identified a novel ITP receptor by using phylogenetic, anatomical analysis, and both in vitro and in vivo approaches.

- The authors exhibited detailed anatomical data of both ITP isoforms and Gyc76C (in the main and supplementary figures), which helped audiences understand the expression of the neurons studied in the manuscript.

- They also performed connectomes and single-cell transcriptomics analysis to study the synaptic and peptidergic connectivity of ITP-expressing neurons. This provided more information for better understanding and further study on systemic homeostasis modulation.

Weaknesses:

In the discussion section, the authors raised the limitations of the current study, which I mostly agree with, such as the lack of verification of direct binding between ITPa and Gyc76C, even though they provided different data to support that ITPa-Gyc76C signaling pathway regulates systemic homeostasis in adult flies.

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates.

The authors have adequately addressed my previous concerns.

Reviewer #1 (Public Review):

Summary:

Thakare et al propose a gravimetric method to evaluate feeding from solid food in Drosophila adults that can be used to evaluate the nutritional impact of high-fat food.

Strengths:

This method is new and fills a gap in the methods used in Drosophila research.

Weaknesses:

The data presented address a number of questions that are mainly interesting for people needing to reproduce such experiments. The work could be improved by being presented within a broader scope.

Reviewer #1 (Public Review):

Summary:

In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high-fat diet are due in part to adipokinetic hormone (Akh) signaling activation. High-fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on a high-fat diet. Elimination of one of two AkhR-expressing cardiac neurons results in arrhythmia similar to a high-fat diet.

Strengths:

The authors propose a novel mechanism for high-fat diet-induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.

Weaknesses:

Major comments:

(1) The authors state, "Arrhythmic pathology is rooted in the cardiac conduction system." This assertion is incorrect as a blanket statement on arrhythmias. There are certain arrhythmias that have been attributable to the conduction system, such as bradycardic rhythms, heart block, sinus node reentry, inappropriate sinus tachycardia, AV nodal reentrant tachycardia, bundle branch reentry, fascicular ventricular tachycardia, or idiopathic ventricular fibrillation to name a few. However the etiological mechanism of many atrial and ventricular arrhythmias, such as atrial fibrillation or substrate-based ventricular tachycardia, are not rooted in the conduction system. The introduction should be revised to reflect a clear focus on atrial fibrillation (AF). In addition, AF susceptibility is known to be modulated by autonomic tone, which is topically relevant to this manuscript.

(2) The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high-fat diet compared to normal-fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?

(3) The authors state, "to test whether the HFD-induced increase in Akh in the APC affects APC neuron activity, we used CaLexA (https://doi.org/10.3109/01677063.2011.642910)." According to the reference, CaLexA is a tool to map active neurons and would not indicate, as the authors state, whether Akh affects APC neuron activity specifically. It is equally possible that APC neurons may be activated by HFD and produce more Akh. Please clarify this language.

(4) Are the AkhR+ neurons parasympathetic or sympathetic? Please provide additional experimentation that characterizes these neurons. The AkhR+ neurons appear to be anti-arrhythmic. Please expand the discussion to include a working hypothesis of the overall findings on Akh, AkhR, and AkhR+ neurons.

(5) The authors state, "Heart function is dependent on glucose as an energy source." However, the heart's main energy source is fatty acids with minimal use of glucose (doi: 10.1016/j.cbpa.2006.09.014). Glucose becomes more utilized by cardiomyocytes under heart failure conditions. Please amend/revise this statement.

Reviewer #1 (Public Review):

Summary:

The manuscript by Jang et al. describes the application of new methods to measure the localization of GTP-binding signaling proteins (G proteins) on different membrane structures in a model mammalian cell line (HEK293). G proteins mediate signaling by receptors found at the cell surface (GPCRs), with evidence from the last 15 years suggesting that GPCRs can induce G-protein mediated signaling from different membrane structures within the cell, with variation in signal localization leading to different cellular outcomes. While it has been clearly shown that different GPCRs efficiently traffic to various intracellular compartments, it is less clear whether G proteins traffic in the same manner, and whether GPCR trafficking facilitates "passenger" G protein trafficking. This question was a blind spot in the burgeoning field of GPCR localized signaling in need of careful study, and the results obtained will serve as an important guidepost for further work in this field. The extent to which G proteins localize to different membranes within the cell is the main experimental question tested in this manuscript. This question is pursued through two distinct methods, both relying on genetic modification of the G-beta subunit with a tag. In one method, G-beta is modified with a small fragment of the fluorescent protein mNG, which combines with the larger mNG fragment to form a fully functional fluorescent protein to facilitate protein trafficking by fluorescent microscopy. This approach was combined with the expression of fluorescent proteins directed to various intracellular compartments (different types of endosomes, lysosome, endoplasmic reticulum, Golgi, mitochondria) to look for colocalization of G-beta with these markers. These experiments showed compelling evidence that G-beta co-localizes with markers at the plasma membrane and the lysosome, with weak or absent co-localization for other markers. A second method for measuring localization relied on fusing G-beta with a small fragment from a miniature luciferase (HiBit) that combines with a larger luciferase fragment (LgBit) to form an active luciferase enzyme. Localization of G-beta (and luciferase signal) was measured using a method known as bystander BRET, which relies on the expression of a fluorescent protein BRET acceptor in different cellular compartments. Results using bystander BRET supported findings from fluorescence microscopy experiments. These methods for tracking G protein localization were also used to probe other questions. The activation of GPCRs from different classes had virtually no impact on the localization of G-beta, suggesting that GPCR activation does not result in the shuttling of G proteins through the endosomal pathway with activated receptors.

Strengths:

The question probed in this study is quite important and, in my opinion, understudied by the pharmacology community. The results presented here are an important call to be cognizant of the localization of GPCR coupling partners in different cellular compartments. Abundant reports of endosomal GPCR signaling need to consider how the impact of lower G protein abundance on endosomal membranes will affect the signaling responses under study.

The work presented is carefully executed, with seemingly high levels of technical rigor. These studies benefit from probing the experimental questions at hand using two different methods of measurement (fluorescent microscopy and bystander BRET). The observation that both methods arrive at the same (or a very similar) answer inspires confidence about the validity of these findings.

Weaknesses:

The rationale for fusing G-beta with either mNG2(11) or SmBit could benefit from some expansion. I understand the speculation that using the smallest tag possible may have the smallest impact on protein performance and localization, but plenty of researchers have fused proteins with whole fluorescent proteins to provide conclusions that have been confirmed by other methods. Many studies even use G proteins fused with fluorescent proteins or luciferases. Is there an important advantage to tagging G-beta with small tags? Is there evidence that G proteins with full-size protein tags behave aberrantly? If the studies presented here would not have been possible without these CRISPR-based tagging approaches, it would be helpful to provide more context to make this clearer. Perhaps one factor would be interference from newly synthesized G proteins-fluorescent protein fusions en route to the plasma membrane (in the ER and Golgi).

As noted by the authors, they do not demonstrate that the tagged G-beta is predominantly found within heterotrimeric G protein complexes. If there is substantial free G-beta, then many of the conclusions need to be reconsidered. Perhaps a comparison of immunoprecipitated tagged G beta vs immunoprecipitated supernatant, with blotting for other G protein subunits would be informative.

Additional context and questions:

(1) There exists some evidence that certain GPCRs can form enduring complexes with G-beta-gamma (Pubmed: 23297229, 27499021). That would seem to offer a mechanism that would enable receptor-mediated transport of G protein subunits. It would be helpful for the authors to place the findings of this manuscript in the context of these previous findings since they seem somewhat contradictory.

(2) There is some evidence that GaS undergoes measurable dissociation from the plasma membrane upon activation (see the mechanism of the assay in Pubmed: 35302493). It seems possible that G-alpha (and in particular GaS) might behave differently than the G-beta subunit studied here. This is not entirely clear from the discussion as it now stands.

(3) The authors say "The presence of mNG-b1 on late endosomes suggested that some G proteins may be degraded by lysosomes". The mechanism of lysosomal degradation by proteins on the outside of the lysosome is not clear. It would be helpful for the authors to clarify.

(4) Although the authors do a good job of assessing G protein dilution in endosomal membranes, it is unclear how this behavior compares to the measurement of other lipid-anchored proteins using the same approach. Is the dilution of G proteins what we would expect for any lipid-anchored protein at the inner leaflet of the plasma membrane?

Reviewer #1 (Public Review):

Summary:

In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupt its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiological relevance.

Strengths:

(1) This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

(2) The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

Reviewer #1 (Public Review):

Wang, He et al have constructed comprehensive single nucleus atlas for the gills of the deep sea Bathymodioline mussels, which possess intracellular symbionts that provide a key source of carbon and allow them to live in these extreme environments. They provide annotations of the different cell states within the gills, shedding light on how multiple cell types cooperate to give rise to the emergent functions of the composite tissues and the gills as a whole. They pay special attention to characterizing the bacteriocyte cell populations and identifying sets of genes that may play a role in their interaction with the symbiotes.

Wang, He et al sample mussels from 3 different environments: animals from their native methane rich environment, animals transplanted to a methane-poor environment to induce starvation and animals that have been starved in the methane-poor environment and then moved back to the methane-rich environment. They demonstrated that starvation had the biggest impact on bacteriocyte transcriptomes. They hypothesize that the up-regulation of genes associated with lysosomal digestion leads to the digestion of the intracellular symbiont during starvation, while the non-starved and reacclimated groups more readily harvest the nutrients from symbiotes without destroying them. Further work exploring the differences in symbiote populations between ecological conditions will further elucidate the dynamic relationship between host and symbiote. This will help disentangle specific changes in transcriptomic state that are due to their changing interactions with the symbiotes from changes associated with other environmental factors.

This paper makes available a high quality dataset that is of interest to many disciplines of biology. The unique qualities of this non-model organism and collection of conditions sampled make it of special interest to those studying deep sea adaptation, the impact of environmental perturbation on Bathymodioline mussels populations, and intracellular symbiotes. The authors also use a diverse array of tools to explore and validate their data.

Reviewer #1 (Public Review):

Summary:

Federer et al. tested AAVs designed to target GABAergic cells and parvalbumin-expressing cells in marmoset V1. Several new results were obtained. First, AAV-h56D targeted GABAergic cells with >90% specificity, and this varied with serotype and layer. Second, AAV-PHP.eB.S5E2 targeted parvalbumin-expressing neurons with up to 98% specificity. Third, the immunohistochemical detection of GABA and PV was attenuated near viral injection sites.

Strengths:

Vormstein-Schneider et al. (2020) tested their AAV-S5E2 vector in marmosets by intravenous injection. The data presented in this manuscript are valuable in part because they show the transduction pattern produced by intraparenchymal injections, which are more conventional and efficient.

Weaknesses:

The conclusions regarding the effects of serotype are based on data from single injection tracks in a single animal. I understand that ethical and financial constraints preclude high throughput testing, but these limitations do not change what can be inferred from the measurements. The text asserts that "...serotype 9 is a better choice when high specificity and coverage across all layers are required". The data presented are consistent with this idea but do not make a strong case for it.

A related criticism extends to the analysis of injection volume on viral specificity. Some replication was performed here, but reliability across injections was not reported. My understanding is that individual ROIs were treated as independent observations. These are not biological replicates (arguably, neither are multiple injection tracks in a single animal, but they are certainly closer). Idiosyncrasies between animals or injections (e.g. if one injection happened to hit one layer more than another) could have substantial impacts on the measurements. It remains unclear which results regarding injection volume or serotype would hold up had a large number of injections been made into a large number of marmosets.

Reviewer #1 (Public Review):

Summary:

In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment. The patch clamp experiments are comprehensive and overall solid but a direct demonstration of the role of these conductances in being necessary for surge generation (or at least having a direct physiological consequence on surge properties) is lacking, substantially reducing the impact of the findings.

Strengths:

(1) Examination of multiple types of calcium and potassium currents, both through electrophysiology and molecular biology.

(2) Focus on arcuate kisspeptin neurons during the surge is relatively conceptually novel as the anteroventral periventricular nucleus (AVPV) kisspeptin neurons have received much more attention as the "surge generator" population.

(3) The modeling studies allow for direct examination of manipulation of single and multiple conductances, whereas the electrophysiology studies necessarily require examination of each current in isolation. The construction of an arcuate kisspeptin neuron model promises to be of value to the reproductive neuroendocrinology field.

Weaknesses:

(1) The novelty of some of the experiments needs to be clarified. This reviewer's understanding is that prior experiments largely used a different OVX+E2 treatment paradigm mimicking periods of low estradiol levels, whereas the present work used a "high E2" treatment model. However, Figures 10C and D are repeated from a previous publication by the same group, according to the figure legend. Findings from "high" vs. "low" E2 treatment regimens should be labeled and clearly separated in the text. It would also help to have direct comparisons between results from low E2 and high E2 treatment conditions.

(2) In multiple places, links are made between the changes in conductances and the transition from peptidergic to glutamatergic neurotransmission. However, this relationship is never directly assessed. The data that come closest are the qPCR results showing reduced Tac2 and increased Vglut2 mRNA, but in the figure legend, it appears that these results are from a prior publication using a different E2 treatment regimen.

(3) Similarly, no recordings of arcuate-AVPV glutamatergic transmission are made so the statements that Kiss1ARH neurons facilitate the GnRH surge via this connection are still only conjecture and not supported by the present experiments.

(4) Figure 1 is not described in the Results section, and is only tenuously connected to the statement in the introduction in which it is cited. The relevance of panels C and D is not clear. In this regard, much is made of the burst firing pattern that arises after E2 treatment in the model, but this burst firing pattern is not demonstrated directly in the slice electrophysiology examples.

(5) In Figure 3, it would be preferable to see the raw values for R1 and R2 in each cell, to confirm that all cells were starting from a similar baseline. In addition, it is unclear why the data for TTA-P2 is not shown, or how many cells were recorded to provide this finding.

(6) In Figure 5, panel C lists 11 cells in the E2 condition but panel E lists data from 37 cells. The reason for this discrepancy is not clear.

(7) In all histogram figures, it would be preferable to have the data for individual cells superimposed on the mean and SEM.

(8) The CRISPR experiments were only performed in OVX mice, substantially limiting interpretation with respect to potential roles for TRPC5 in shaping arcuate kisspeptin neuron function during the preovulatory surge.

(9) Furthermore, there are no demonstrations that the CRISPR manipulations impair or alter the LH surge.

(10) The time of day of slice preparation and recording needs to be specified in the Methods.

Reviewer #1 (Public Review):

The manuscript by Zhao et al describes the identification of RAPSYN, a NEDD8 E3 ligase previously studied for its role in acetylcholine receptor clustering and neuromuscular junction formation, as a factor promoting the stabilisation of the BCR-ABL oncogene in Chronic Myeloid Leukemia (CML) cells. The authors have identified that NEDDylation of BCR-ABL by RAPSYN antagonises its poly-ubiquitin and subsequent proteasome-based degradation. Knocking down RAPSYN with shRNA led to increased poly-ubiquitination and faster turnover of BCR-ABL. Furthermore, they describe that SRC-dependent phosphorylation of RAPSYN facilitates its NEDD8-ligase activity.

The authors' findings are primarily rooted in a series of well-conducted in vitro experiments using two CML cell lines, K562 and MEG-01. They have performed some further validations using primary CML samples, which have strengthened their claims.

The author's initial discoveries have come from interrogating a number of publicly available gene expression datasets, both microarray-based and RNA-seq, which revealed that RAPSYN is increased at the protein level but that RNA levels are not different between healthy and CML samples. This is a very interesting observation which warrants further future investigation.

The conclusions of this revised manuscript are broadly supported by the data and the analyses. It also describes novel findings that can spur future studies, both into the basic cellular biology of CML as well as into potential new therapeutic strategies.

Comments on revised version:

I thank the authors for addressing my concerns in the initial review. The revised manuscript with additional data is much stronger.

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Comments on revised version:

All my concerns have been properly addressed.

Reviewer #1 (Public Review):

Summary:

This paper by Watanabe et al described an expression system that can express the paired heavy and light chains of IgG antibodies from single cell B cells. In addition, they used FACS sorting for specific antigens to screen/select the specific populations for more targeted cloning of mAb genes. By staining with multiple antigens, they were able to zoom in to cross-reactive antibodies.

Strengths:

A highly efficient process that combines selection/screening with dua expression of both antibody chains. It is particularly suitable for the isolation of cross-reactive antibodies against conserved epitopes of different antigens, such as surface proteins of related viruses.

Weaknesses:

(1) The overall writing is very difficult to follow and the authors need to work on significant re-writing.

(2) The paper in its current form really lacks detail and it is NOT possible for readers to repeat or follow their methods. For example: a) It is not clear whether the authors checked the serum to see if the mice were producing antibodies before they sacrificed them to harvest spleen/blood i.e. using ELISA? b) How long after administration of the second dose were the mice sacrificed? c) What cell types are taken for single B cell sorting? Splenocytes or PBMC? These are just some of the questions which need to be addressed.

(3) According to the authors, 77 clones were sorted from the PR8+ and H2+ double positive quadrant. It is surprising that after transfection and re-analysing of bulk antibody presenting EXPI cells on FACS, only 13 clones (or 8 clones? - unclear) seemed to be truly cross-reactive. If that is the case, the approach is not as efficient as the authors claimed.

Reviewer #1 (Public Review):

This is a very nice study of Belidae weevils using anchored phylogenomics that presents a new backbone for the family and explores, despite a limited taxon sampling, several evolutionary aspects of the group. The phylogeny is useful to understand the relationships between major lineages in this group and preliminary estimation of ancestral traits reveals interesting patterns linked to host-plant diet and geographic range evolution. I find that the methodology is appropriate, and all analytical steps are well presented. The paper is well-written and presents interesting aspects of Belidae systematics and evolution. The major weakness of the study is the very limited taxon sampling which has deep implications for the discussion of ancestral estimations.

Reviewer #1 (Public Review):

Summary:

Páramo et al. used 3D geometric morphometric analyses of the articulated femur, tibia, and fibula of 17 macronarian taxa (known to preserve these three skeletal elements) to investigate morphological changes that occurred in the hind limb through the evolutionary history of this sauropod clade. A principal components analysis was completed to understand the distribution of the morphological variation. A supertree was constructed to place evolutionary trends in morphological variation into phylogenetic context, and hind limb centroid size was used to investigate potential relationships between skeletal anatomy and gigantism. The majority of the results did not yield statistically significant differences, but they did identify interesting shape-change trends, especially within subclades of Titanosauria. Many previous studies have attempted to elucidate a link between wide-gauge posture and gigantism, which in this study Páramo et al. investigate among several titanosaurian subclades. They propose that morphologies associated with wide-gauge posture arose in parallel with increasing body size among basal members of Macronaria and that this connection became less significant once wide-gauge posture was acquired within Titanosauria. The authors also suggest that other biomechanical factors influenced the independent evolution of subclades within Titanosauria and that these influences resulted in instances of convergent evolution. Therefore, they infer that, overall, wide-gauge posture was not significantly correlated with gigantism, though some morphological aspects of hind limb skeletal anatomy appear to have been associated with gigantism. Their work also supports previous findings of a decrease in body size within Titanosauriformes (which they found to be not significant with shape variables but significant with Pagel's lambda). Collectively, their results support and build on previous work to elucidate more specifics on the evolution of this enigmatic clade. Further study will show if their hypotheses stand or if the inclusion of additional specimens and taxa yields alternative results.

Strengths:

Páramo et al. were diligent in their efforts to digitize and prepare specimens for this study while also minimizing user bias. Their previous work provided a strong platform for this study, specifically for their robust methodology. Between their supplemental files (which include details about specimen digitization and preparation) and the main body of the manuscript, the authors fully provide their results in detailed tables and figures. Their conclusions on evolutionary trends within Titanosauria are reasonably well supported (see weaknesses below) and they provide important details that enhance our understanding of the evolution of this clade and complement previous findings. Their discussion of links between morphology and various biomechanical adaptations is important, and future studies can use these results to investigate such biomechanical adaptations. The trends they identify within the subclades of Titanosauria are very interesting and highlight the diversity of this clade. It is possible that additional investigations of the evolution of these subclades could unite findings in sauropod myology and biomechanics, each of which has been suggested to vary among titanosaurian taxa without a clear phylogenetic or evolutionary distribution. The authors suggest that certain common morphologies arose via convergent evolution among titanosaurian subclades, such as members of Colossosauria exhibiting morphologies more similar to basal titanosaurians than derived saltasaurines. While this conclusion about convergent evolution is not well explained, only future testing will determine if this hypothesis remains supported. Additionally, the authors discuss the influence of uncertainty on the phylogenetic position of some taxa, and this reminds readers to view their findings as tentative trends that may be illuminated through further quantitative analyses. If one accepts the use of hind limb centroid size as a reliable approximation of body size (see concerns in Weaknesses below) then their data also support a hypothesis of decreasing body size through titanosaurian evolution (with PC 2 further differentiating small titanosaurian taxa from one another), providing an opportunity for future analyses to further investigate these interesting trends.

Weaknesses:

Several sentences throughout the manuscript could benefit from citations. For example, the discussion of using hind limb centroid size as a proxy for body mass has no citations attributed. This should be cited or described as a new method for estimating body mass with data from extant taxa presented in support of this relationship. This particular instance is a very important point to include supporting documentation because the authors' conclusions about evolutionary trends in body size are predicated on this relationship.

An additional area of concern is the lack of any discussion of taphonomic deformation in Section 3.3 Caveats of This Study, the results, or the methods. The authors provide a long and detailed discussion of taphonomic loss and how this study does a good job of addressing it; however, taphonomic deformation to specimens and its potential effects on the ensuing results were not addressed at all. Hedrick and Dodson (2013) highlight that, with fossils, a PCA typically includes the effects of taphonomic deformation in addition to differences in morphology, which results in morphometric graphs representing taphomorphospaces. For example, in this study, the extreme negative positioning of Dreadnoughtus on PC 2 (which the authors highlight as "remarkable") is almost certainly the result of taphonomic deformation to the distal end of the holotype femur, as noted by Ullmann and Lacovara (2016).

The authors investigated 17 taxa and divided them into 9 clades, with only Titanosauria and Lithostrotia including more than two taxa (and four clades are only represented by one taxon). While some of these clades represent the average of multiple individuals, the small number of plotted taxa can only weakly support trends within Titanosauria. If similar general trends could be found when the taxa are parsed into fewer, more inclusive clades, it would support and strengthen their claims. Of course, the authors can only study what is preserved in the fossil record, and titanosaurian remains are often highly fragmentary; these deficiencies should therefore not be held against the authors. They clearly put effort and thought into their choices of taxa to include in this study, but there are limitations arising from this low sample size that inherently limit the confidence that can be placed on their conclusions, and this caveat should be more clearly discussed. Specifically, the authors note that their dataset contains many lithostrotians, but they do not discuss unevenness in body size sampling. As neither their size-category boundaries nor the taxa which fall into each of them are clearly stated, the reader must parse the discussion to glean which taxa are in each size category. It should be noted that the authors include both Jainosaurus and Dreadnoughtus as 'large' taxa even though the latter is estimated to have been roughly five times the body mass of the former, making Dreadnoughtus the only taxon included in this extreme size category. The effects that this may have on body size trends are not discussed. Additionally, few taxa between the body masses of Jainosaurus and Dreadnoughtus have been included even though the hind limbs of several such macronarians have been digitized in prior studies (such as Diamantinasaurus and Giraffititan; Klinkhamer et al. 2018). Also, several members of Colossosauria are more similar in general body size to Dreadnoughtus than Jainosaurus, but unfortunately, they do not preserve a known femur, tibia, and fibula, so the authors could not include them in this study. Exclusion of these taxa may bias inferences about body size evolution, and this is a sampling caveat that could have been discussed more clearly. Future studies including these and other taxa will be important for further evaluating the hypotheses about macronarian evolution advanced by Páramo et al. in this study.

Reviewer #1 (Public Review):

Summary:

In the paper entitled "PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis", the authors sought to determine the role of HSCB, a known regulator of Iron sulfur cluster transfer, in the generation of erythrocytes and megakaryocytes. They utilized a human primary cell model of hematopoietic differentiation to identify a novel mechanism whereby HSCB is necessary for activation of erythroid and megakaryocytic gene expression through regulation of the nuclear localization of FOG-1, a essential transcription co-regulator of the GATA transcription factors. Their work establishes this novel regulatory axis as a mechanism by which cytokine signaling through EPO-R and MPL drives the lineage-specification of hematopoietic progenitors to erythrocytes and megakaryocytes, respectively.

Impact:

The major impact of this work is in a greater understanding of how cytokine signaling through EPO/TPO function to promote lineage specification of hematopoietic stem/progenitor cells. While the major kinase cascades downstream of the EPO/TPO receptors have been elucidated, how those cascades effect gene expression to promote a specific differentiation program is poorly understood. For this work, we now understand that nuclear localization of FOG is a critical regulatory node by which EPO/TPO signaling is required to launch FOG-dependent gene expression. However, these cytokine receptors have many overlapping and redundant targets, so it still remains to be elucidated how signaling through the different receptors promotes divergent gene expression programs. Perhaps similar regulatory mechanisms exist for other lineage-specifying transcription factors.

Strengths:

The authors use two different cellular models of erythroid differentiation (K562 and human primary CD34+ cells) to elucidate the multi-factorial mechanism controlling FOG-1 nuclear localization. The studies are well-controlled and rigorously establish their mechanism through complementary approaches. The differentiation effects are established through cell surface marker expression, protein expression, and gene expression analyses. Novel protein interactions discovered by proteomics analyses were validated through bi-directional co-IP experiments in multiple experimental systems. Protein cellular localization findings are supported by both immunofluorescence and cell fractionation immunoblot analyses. The robustness of their experimental findings gives great confidence for the likelihood that the methods and findings can be reproduced in future work based on their conclusions.

Weaknesses:

The one unexplained step in this intricately described mechanism is how HSCB functions to promote TACC3 degradation. It appears that the proteasome is involved since MG-132 reverses the effect of HSCB deficiency, but no other details are provided. Does HSCB target TACC3 for ubiquitination somehow? Future studies will be required to understand this portion of the mechanism.

One weakness of the study design is that no in vivo experiments are conducted. The authors comment that the HSCB mouse phenotype is too dramatic to permit studies of erythropoiesis in vivo; however, a conditional approach could have been pursued.<br /> It should also be noted that a previous study had already shown that TACC3 regulates the nuclear localization of FOG-1, so this portion of the mechanism is not entirely novel. However, the role of HSCB and the proteasomal degradation of TACC3 is entirely novel to my knowledge.

Reviewer #1 (Public Review):

Summary:

This is an interesting study that performs scRNA-Seq on infected and uninfected wounds. The authors sought to understand how infection with E. faecalis influences the transcriptional profile of healing wounds. The analysis demonstrated that there is a unique transcriptional profile in infected wounds with specific changes in macrophages, keratinocytes, and fibroblasts. They also speculated on potential crosstalk between macrophages and neutrophils and macrophages and endothelial cells using NicheNet analysis and CellChat. Overall the data suggest that infection causes keratinocytes to not fully transition which may impede their function in wound healing and that the infection greatly influenced the transcriptional profile of macrophages and how they interact with other cells.

Strengths:

It is a useful dataset to help to understand the impact of wound infection on transcription of specific cell types. The analysis is very thorough in terms of transcriptional analysis and uses a variety of techniques and metrics.

Weaknesses:

Some drawbacks of the study are the following. First the fact that it only has two mice per group, and only looks at one time point after wounding decreases the impact of the study. Wound healing is a dynamic and variable process so understanding the full course of the wound healing response would be very important to understand the impact of infection on the healing wound. The analysis has been bolstered by applying a cross-entropy test on the integrated dataset and to ensure robustness of the datasets (Fig S1F). Including unwounded skin in the scRNA-Seq would also lend a lot more significance to this study. However, this was technically challenging due to constraints with the number of immune cells in unwounded skin as described in the limitations section. Another drawback of the study is that mouse punch biopsies are very different than human wounds as they heal primarily by contraction instead of re-epithelialization like human wounds. The authors mitigated this somewhat be extracting the incisional parts of the wound. So while the conclusions are generally supported the scope of the work is somewhat limited.

Reviewer #1 (Public Review):

The current manuscript revisits previous reports in the literature. The human Pannexin 1 channel is regulated by phosphorylation at two residues by Src kinase. From this series of experiments, the authors conclude that PANX-1 is not phosphorylated at these residues.

The biggest strength of the manuscript is the comprehensiveness of the approach. The authors recapitulate prior experiments in the literature and also add a series of new, orthogonal experiments that all examine the claim of PANX-1 phosphorylation. The breadth of the reported experiments extends over multiple cell lines and protein constructs, in vitro purified proteins, mass spec, different phosphorylation detection reagents and antibodies, and functional electrophysiology assays that show that the addition of Src does not impact gating. The combined weight of all these data strongly suggests that the field should re-examine the claim that PANX-1 is regulated by phosphorylation at Y199 and Y309.

Another strength is that the authors go beyond simply showing that the antibodies do not recognize phosphorylated PANX-1. They also provide potential mechanisms for how the antibodies may be misleading. Both antibodies recognize phosphorylated Src-1. In the case of anti-PANX1-pY308, the authors provide solid mutagenesis evidence that the antibody also weakly recognizes a non-phosphorylated epitope of PANX1 in the same region as the tyrosine. This helps make a convincing case.

Such experiments, while not glamorous, have great practical importance for developing an accurate understanding of how Pannexin channels are regulated.

Reviewer #2 (Public Review):

In this revised manuscript Aguillon and collaborators convincingly demonstrating that CLK is required for free-running behavioral rhythms under constant conditions in the Cnidarian Nematostella. The results also convincingly show that CLK impacts rhythmic gene expression in this organism. This original work thus demonstrates that CLK was recruited very early during animal evolution in the circadian clock mechanism to optimize behavior and gene expression with the time-of-day.

Reviewer #1 (Public Review):

Summary:

The authors analyzed the bacterial colonization of human sperm using 16S rRNA profiling. Patterns of microbiota colonization were subsequently correlated with clinical data, such as spermiogram analysis, the presence of reactive oxygen species (ROS), and DNA fragmentation. The authors identified three main clusters dominated by Streptococcus, Prevotella, and Lactobacillus & Gardnerella, respectively, which aligns with previous observations. Specific associations were observed for certain bacterial genera, such as Flavobacterium and semen quality. Overall, it is a well-conducted study that further supports the importance of the seminal microbiota.

Strengths:

- The authors performed the analysis on 223 samples, which is the largest dataset in semen microbiota analysis so far.<br /> - Inclusion of negative controls to control contaminations.<br /> - Inclusion of a positive control group consisting of men with proven fertility.

Weaknesses:<br /> - The manuscript needs comprehensive proofreading for language and formatting. In many instances, spaces are missing or not required.<br /> - Could the authors explore correlation network analyses to get additional insights into the structure of different clusters?<br /> - The GitHub link is not correct.<br /> - It is not possible to access the dataset on ENA.<br /> - Add the graphs obtained with decontam analysis as a supplementary figure.<br /> - There is nothing about the RPL group in the results section, while the authors discuss this issue in the introduction. What about the controls with proven fertility?<br /> - While correctly stated in the title, the term microbiota should be used throughout the manuscript instead of "microbiome"

Reviewer #1 (Public Review):

Summary:

This manuscript set out to identify selective inhibitors of the pyridoxal phosphatase (PDXP). Previous studies had demonstrated improvements in cognition upon removal of PDXP, and here the authors reveal that this correlates with an increase in pyridoxal phosphate (PLP; PDXP substrate and an active coenzyme form of vitamin B6) with age. Since several pathologies are associated with decreased vitamin B6, the authors propose that PDXP is an attractive therapeutic target in the prevention/treatment of cognitive decline. Following high throughput and secondary small molecule screens, they identify two selective inhibitors. They follow up on 7, 8 dihydroxyflavone (DHF). Following structure-activity relationship and selectivity studies, the authors then solve a co-crystal structure of 7,8 DHF bound to the active site of PDXP, supporting a competitive mode of PDXP inhibition. Finally, they find that treating hippocampal neurons with 7,8 DHF increases PLP levels in a WT but not PDXP KO context. The authors note that 7,8 DHF has been used in numerous rodent neuropathology models to improve outcomes. 7, 8 DHF activity was previously attributed to activation of the receptor tyrosine kinase TrkB, although this appears to be controversial. The present study raises the possibility that it instead/also acts through modulation of PLP levels via PDXP, and is an important area for future work.

Strengths:

The strengths of the work are in the comprehensive, thorough, and unbiased nature of the analyses revealing the potential for therapeutic intervention in a number of pathologies.

Weaknesses:

Potential weaknesses include the poor solubility of 7,8 DHF that might limit its bioavailability given its relatively low potency (IC50= 0.8 uM), which was not improved by SAR. The solubility issues of 7,8 DHF have been discussed at length in the authors' response to Reviewer #3. In particular, the solubility of 7,8 DHF has been found to be variable due to the concentration and buffer conditions. The 7,8 DHF compound has an extended residence time and the co-crystal structure could aid the design of more potent molecules and would be of interest to those in the pharmaceutical industry. The images related to crystal structure have been improved with additional structural analysis of PDXP in a complex of 7,8-DHF (see revised Figure 3).

Reviewer #1 (Public Review):

Summary:

Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:

In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Comments on revised version:

The authors have satisfactorily addressed my concerns.

Reviewer #2 (Public Review):

Summary:

The authors try to establish that there is an Abeta-dependent loss of nuclear pores early in Alzheimer's disease. To do so the authors compared different NUP proteins and assessed their function by analyzing nuclear leakage and resistance to induction of nuclear damage and the associated necroptosis. The authors use a mouse knockin for hAPP with familial Alzheimer's mutations to model amyloidosis related to Alzheimer's disease. Treatment with an inhibitor of beta-amyloid production partially rescued the loss of nuclear pore proteins in young KI neurons, implicating beta-amyloid in Nuclear Pore dysfunction, a mechanism already described in other neurodegenerative diseases but not in Alzheimer's disease.

Comments on revised version:

Upon careful review, some of the critical concerns raised have yet to be fully addressed (the authors did not adequately address the two points of my public review or 5 of my 7 recommendation points), particularly regarding the effects of maturation stage or age. This has negatively impacted my initial enthusiasm for the paper, as the current approach does not fully capture the role of nuclear pore dysfunction in Alzheimer's disease, which is intimately dependent on aging. Here are specific recommendations for further revision:

(1) The manuscript would benefit from a clearer acknowledgement of the limitations concerning the effects of maturation or age. I recommend removing mentions of the effect of time, for example:

(i) Line 1 "4: "By using brain tissues and primary neurons cultured from App KI and wildtype (WT) mice, we observed a loss of NPCs in neuronal nuclei over time. "

(ii) Line 20 "13: "Similarly, in neuron cocultures, there was an 20 increase in intracellular Aβ levels over WT neurons that parallels the reduction of NUPs as neurons 21 mature from DIV "-28. "

(2) The subheading in the Discussion section, "Age-dependent decline in nuclear function during normal aging and in AD," could be more accurately retitled "Nuclear function decline" in AD" to avoid suggesting age dependence without the requisite data.

(3) Because primary neurons differentiate, mature, and age with time in culture, they are required to control for the developmental stage of your cultures. Please include the control data that would support cultures maturation stage, such as staining for axodendritic markers (e.g., MAP2), glial cell distribution (e.g., GFAP), and the balance of excitatory vs. inhibitory neuronal subpopulations (e.g., Gad65). This data is crucial for substantiating the culture conditions and the resulting interpretations.

Reviewer #1 (Public Review):

The study by Longhurst et al. investigates the mechanisms of chemoresistance and chemosensitivity towards three compounds that inhibit cell cycle progression: camptothecin, colchicine, and palbociclib. Genome-wide genetic screens were conducted using the HAP1 Cas9 cell line, revealing compound-specific and shared pathways of resistance and sensitivity. The researchers then focused on novel mechanisms that confer resistance to palbociclib, identifying PRC2.1. Genetic and pharmacological disruption of PRC2.1 function, but not related PRC2.2, leads to resistance to palbociclib. The researchers then show that disruption of PRC2.1 function (for example, by MTF2 deletion), results in locus-specific changes in H3K27 methylation and increases in D-type cyclin expression. It is suggested that increased expression of D-type cyclins results in palbociclib resistance.

Strengths:

The results of this study are interesting and contribute insights into the molecular mechanisms of CDK4/6 inhibitors. Importantly, while CDK4/6 inhibitors are effective in the clinic, tumour recurrence is very high due to acquired resistance.

Weaknesses:

A key resistance mechanism is Rb loss, so it is important to understand if resistance conferred by PRC2.1 loss is mediated by Rb, and whether restoration of PRC2.1 function in Rb-deplete cells results in renewed palbociclib sensitivity. It is also important to understand the clinical implications of the results presented. The inclusion of these data would significantly improve the paper. However, besides some presentation issues and typos as described below, it is my opinion that the results are robust and of broad interest.

Major questions:

(1) Is the resistance to CDK4/6 inhibition conferred by mutation of MTF2 mediated by Rb?

(2) Are mutations in PRC2.1 found in genetic analyses of tumour samples in patients with acquired resistance?

Reviewer #1 (Public Review):

Summary:

In this manuscript, Tung and colleagues identify Calreticulin as a repressor of ATF6 signaling using a CRISPR screen and characterize the functional interaction between ATF6 and CALR.

Strengths:

The manuscript is well written and interesting with an innovative experimental design that provides some new mechanistic insight into ATF6 regulation as well as crosstalk with the IRE1 pathway. The methods used were fit for purpose and reasonable conclusions were drawn from the data presented. Findings are novel and bring together glycoprotein quality control and activation of one sensor of the UPR. This is a novel perspective on how the integration of ER homeostasis signals could be sensed in the ER.

Weaknesses:

Several points remain to be documented to support the authors' model.

Reviewer #1 (Public Review):

Summary:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The diverse methods to validate PAClight1P78A demonstrate a comprehensive approach to sensor engineering by combining biochemical characterization with in vivo studies in rodent brains and zebrafish. This establishes the sensor's biophysical properties (e.g., sensitivity, specificity, kinetics, and spectral properties) and demonstrates its functionality in physiologically relevant settings. Importantly, the inclusion of control sensors and the testing of potential intracellular downstream effects such as G-protein activation underscore a careful consideration of specificity and biological impact.

Strengths:

The fundamental development of PAClight1P78A addresses a significant gap in sensors for Class-B1 GPCRs. The iterative design process -starting from PAClight0.1 to the final PAClight1P78A variant - demonstrates compelling optimization. The innovative engineering results in a sensor with a high apparent dynamic range and excellent ligand selectivity, representing a significant advancement in the field. The rigorous in vitro characterization, including dynamic range, ligand specificity, and activation kinetics, provides a critical understanding of the sensor's utility. Including in vivo experiments in mice and zebrafish larvae demonstrates the sensor's applicability in complex biological systems.

Weaknesses:

The manuscript shows that the sensor fundamentally works in vivo, albeit in a limited capacity. The titration curves show sensitivity in the nmol range at which endogenous detection might be possible. However, perhaps the sensor is not sensitive enough or there are not any known robust paradigms for PACAP release. A more detailed discussion of the sensors's limitations, particularly regarding in vivo applications and the potential for detecting endogenous PACAP release, would be helpful.

There are several experiments with an n=1 and other low single-digit numbers. I assume that refers to biological replicates such as mice or culture wells, but it is not well defined. n=1 in experimental contexts, particularly in Figure 1, raises significant concerns about the exact dynamic range of the sensor, data reproducibility, and the robustness of conclusions drawn from these experiments. Also, ROI for cell cultures, like in Figure 1, is not well defined. The methods mentioned ROIs were manually selected, which appears very selective, and the values in Figure 1c become unnecessarily questionable. The lack of definition for "ROI" is confusing. Do ROIs refer to cells, specific locations on the cell membrane, or groups of cells? It would be best if the authors could use unbiased methods for image analysis that include the majority of responsive areas or an explanation of why certain ROIs are included or excluded.

Reviewer #1 (Public Review):

Summary:

In this study, James Lee, Lu Bai, and colleagues use a multifaceted approach to investigate the relationship between transcription factor condensate formation, transcription, and 3D gene clustering of the MET regulon in the model organism S. cerevisiae. This study represents a second clear example of inducible transcriptional condensates in budding yeast, as most evidence for transcriptional condensates arises from studies of mammalian systems. In addition, this study links the genomic location of transcriptional condensates to the potency of transcription of a reporter gene regulated by the master transcription factor contained in the condensate. The strength of evidence supporting these two conclusions is strong. Less strong is evidence supporting the claim that Met4-containing condensates mediate the clustering of genes in the MET regulon.

Strengths:

The manuscript is for the most part clearly written, with the overriding model and specific hypothesis being tested clearly explained. Figure legends are particularly well written. An additional strength of the manuscript is that most of the main conclusions are supported by the data. This includes the propensity of Met4 and Met32 to form puncta-like structures under inducing conditions, formation of Met32-containing LLPS-like droplets in vitro (within which Met4 can colocalize), colocalization of Met4-GFP with Met4-target genes under inducing conditions, enhanced transcription of a Met3pr-GFP reporter when targeted within 1.5 - 5 kb of select Met4 target genes, and most impressively, evidence that several MET genes appear to reposition under transcriptionally inducing conditions. The latter is based on a recently reported novel in vivo methylation assay, MTAC, developed by the Bai lab.

Weaknesses:

My principal concern is that the authors fail to show convincing evidence for a key conclusion, highlighted in the title, that nuclear condensates per se drive MET gene clustering. Figure 4E demonstrates that Met4 molecules, not condensates per se, are necessary for fostering distant cis and trans interactions between MET6 and three other Met4 targets under -met inducing conditions. In addition, the paper would be strengthened by discussing a recent study conducted in yeast that comes to many of the same conclusions reported here, including the role of inducible TF condensates in driving 3D genome reorganization (Chowdhary et al, Mol. Cell 2022).

Other concerns:

(1) A central premise of the study is that the inducible formation of condensates underpins the induction of MET gene transcription and MET gene clustering. Yet, Figure 1 suggests (and the authors acknowledge) that puncta-like Met4-containing structures pre-exist in the nuclei of non-induced cells. Thus, the transcription and gene reorganization observed is due to a relatively modest increase in condensate-like structures. Are we dealing with two different types of Met4 condensates? (For example, different combinations of Met4 with its partners; Mediator- or Pol II-lacking vs. Mediator- or Pol II-containing; etc.?) At the very least, a comment to this effect is necessary.

(2) Using an in vitro assay, the authors demonstrate that Met4 colocalizes with Met32 LLPS droplets (Figure 2F). Is the same true in vivo - that is, is Met32 required for Met4 condensation? This could be readily tested using auxin-induced degradation of Met32. Along similar lines, the claim that Met32 is required for MET gene clustering (line 250) requires auxin-induced degradation of this protein.

(3) The authors use a single time point during -met induction (2 h) to evaluate TF clustering, transcription (mRNA abundance), and 3D restructuring. It would be informative to perform a kinetic analysis since such an analysis could reveal whether TF clustering precedes transcriptional induction or MET gene repositioning. Do the latter two phenomena occur concurrently or does one precede the other?

(4) Based on the MTAC assay, MET13 does not appear to engage in trans interactions with other Met4 targets, whereas MET6 does (Figures 4C and 4E). Does this difference stem from the greater occupancy of Met4 at MET6 vs. MET13, greater association of another Met co-factor with the chromatin of MET6 vs. MET13, or something else?

Reviewer #1 (Public Review):

There is an undisputable need for better in vitro models recapitulating steatotic liver diseases. This article is from a group of well-known stem cell experts that use human induced pluripotent stem cells (hiPSCs) to build a multicellular steatosis model in vitro. While the model is strong for testing hepatocytes responses, it falls short on translational aspects as well as on non-parenchymal liver cells.

(1) The authors should use the new nomenclature for the disease, MASLD / MASH, as proposed by the scientific societies (Rinella ME, et al. J Hepatol. 2023; 79(6):1542-1556. PMID: 37364790).

(2) There has been a similar approach by the Takebe group (Ouchi R, et al., Cell Metab. 2019; 30(2):374-384, PMID: 31155493). What is different in this model?

(3) The work is very technical and does neither provide any new mechanistic insights nor does it test any new interventions. I do see the clear technical advance in the long-term culture. However, I do not see that this system would allow modelling true "chronic" changes in MASLD, e.g. steatohepatitis and/or fibrosis.

(4) While I am very convinced about the validity of the "hepatocyte" component in this system, the NPC compartment is insufficient. The 3D model does certainly not contain Kupffer cells (which have very distinct characteristics from "M0" macrophages) and does not contain true HSCs (LX-2 is a very insufficient model). Also, the model lacks flow conditions, which does not allow to factor in pathogenic signals from the circulation / portal vein (e.g. gut-liver axis). This will only allow very limited insights into the crosstalk between hepatocytes and NPCs.

(5) The translational value of this model remains unclear to me. The scRNA-seq data should be meticulously compared to sc/snRNA-seq data from human MASLD livers at different stages to understand, what this system is able to model (maybe very early stages of steatosis?).

(6) The study lacks a "use case" to study interventions, e.g. testing resmetirom or any other of the new MASLD drugs in this system.

Reviewer #1 (Public Review):

Summary:

The evolution of non-shivering thermogenesis is of fundamental importance to understand. Here, in small mammals the contractile apparatus of the muscle are shown to increase energy expenditure upon a drop in ambient temperature. Additionally, in the state of torpor, small hibernators did not show an increase in energy expenditure under the same challenge.

Strengths:

The authors have conducted a very well-planned study that has sampled the muscle of large and small hibernators from two continents. Multiple approaches were then used to identify the state of the contractile apparatus, and its energy expenditure under torpor or otherwise.

Weaknesses:

There was only one site of biopsy from the animals used (leg). As the authors state, it would be interesting to know if non-shivering thermogenesis is something that is regionally different in the animal, given the core body and distal limbs have different temperatures.

Reviewer #1 (Public Review):

The Calcium Homeostasis Modulators (CALHM) are a family of large pore channels, of which the physiological role of CALHM1 and 3 is well understood, in particular their key role in taste sensation via the release of the neurotransmitter ATP. The activation mechanism of CALHM1 involves membrane depolarization and a decrease in extracellular Ca concentration, allowing the passage of large cellular metabolites. However, the activation mechanism and physiological roles of other family members are much less well understood. Many structures of homomeric CALHM proteins have been determined, revealing distinct oligomeric assemblies despite a common transmembrane domain topology. CALHM1 and 3 have been shown functionally to form heteromeric assemblies with properties distinct from those of homomeric CALHM1. However, the structural basis of heteromeric CALHM1 and 3 remains unexplored.

In this paper, Drozdzyk et al. present an important study on the structures of heteromeric channels composed of CALHM2 and CALHM4, extending the structural understanding of the CALHM family beyond homomeric channels. The study relies primarily on cryo-EM. Despite the inherent challenges of structural determination due to the similar structural features of CALHM2 and CALHM4, the authors innovatively use synthetic nanobodies to distinguish between the subunits. Their results show a broad distribution of different heteromeric assemblies, with CALHM4 conformation similar to its homomeric form and CALHM2 conformation influenced by its proximity to CALHM4, and provide detailed insights into the interaction between CALHM2 and CALHM4.

The manuscript is well-structured and presents clear results that support the conclusions drawn. The discovery of heteromeric CALHM channels, although currently limited to an overexpressed system, represents a significant advance in the field of large-pore channels and will certainly encourage further investigation into the physiological relevance and roles of heteromeric CALHM channels.

Comments on the revised version:

I appreciate the authors' efforts to try the alternative data processing strategy. Congratulations to the authors for this interesting and important work!

Reviewer #1 (Public Review):

As outlined in my previous public review, Yeo et al. revised the current neuronal intoxication model, common to all serotypes of botulinum neurotoxins. Using a combination of genetic and imaging approaches, they demonstrate that upon internalization, BoNT/A-containing endosomes undergo retro-axonally trafficking to the neuronal soma. Within the soma, this particular serotype then traffics to the endoplasmic reticulum (ER) via the Golgi apparatus. At the ER, the SEC61 translocon complex facilitates the translocation of BoNT/A's metalloprotease domain (light chain, LC) from the ER lumen into the cytosol, where the thioredoxin reductase/thioredoxin system and HSP complexes release and refold the catalytic LC. Subsequently, the LC diffuses and cleaves SNAP25 first in the soma before reaching neurites and synapses.

Although I still acknowledge the well-executed and thoroughly analyzed genome-wide RNAi screen, I must once again highlight significant pitfalls and weaknesses in the paper due to the lack of essential controls and validations. Consequently, I suggest readers to approach the authors' findings with caution, as they may be limited to the combination of one specific cellular model and genetic engineering tools. During the revision process, authors declined to conduct additional experiments that could have strengthened their main conclusions. These include, but are not limited to:

(1) Investigating weather in the newly generated cell line Red-SNAPR, the GFP fragment produced upon toxin cleavage degrades more rapidly in the soma compared to axon terminals, possibly due to differences in proteasome activity in these two compartments.

(2) Validating toxin cleavage activity in the soma before reaching synapses by conducting an additional and more physiological approach, a time course experiment using native BoNT/A and staining BoNT/A-cleaved SNAP25 with specific antibodies.

(3) Assessing whether the addition of mNG1-11 to the LC affects the translocation process itself and quantifying the mean fluorescence intensity (MFI) per cell, taking into consideration the amount of HA-tagged Cyt-mG1-10, which appears predominantly expressed in the cytosol and less detected in neurites. This raises the question of potential bias toward the cell soma in this assay.

(4) Validating major hits (e.g., VPS34 and Sec61) by performing WB or IF analysis to test the cleavage of endogenous SNAP25.

Additionally, during the revision process, the authors raised concerns about the level of scrutiny applied by this reviewer, particularly in comparison to the seminal study of Lilia K. Koriazova & Mauricio Montal published in Nature Structural Biology (PMID: 12459720). In this 2003 paper, Montal's lab pioneered the use of single-channel recordings and substrate proteolysis analysis to reconstitute the translocation of BoNT/A light chain protease across an artificial lipid bilayer via the channel formed by its heavy chain. The authors highlighted that, when converting the experimental conditions from the aforementioned paper into molarity, it appears that the cis compartment was loaded with 10−8 M BoNT/A, and the reported translocated protease activity (measured by substrate cleavage) is equivalent to 10−17 M. This implies that only about 1 LC molecule in 100 million has crossed the membrane. The calculation performed by authors is indeed accurate. However, readers should be informed about another piece of information present in the same paper that might help them to clarify this important point. Koriazova & Montal, by discussing this experiment, have pointed out that this value (10−17 M) corresponds to ≈3600 LC molecules, a number closed to the maximum number of channels that can be formed under the used experimental conditions. Indeed, from the same paper, quotation: 'This number is in close agreement with the maximum number of channels inserted in the bilayer under the assay condition, ≈2000 (Fig. 3a), as estimated from macroscopic membrane conductance ∼1 × 105 pS and γ = 50 pS measured in 0.1 M KCl'. Another aspect that Yeo et al. forgot to mention in their rebuttal letter is that the system used by Koriazova & Montal lacks any chaperones in the trans compartment. Nowadays, we know that upon translocation, the refolding of the L chain is aided by Hsp90 (Azarnia Tehran et al., Cellular microbiology, 2017). Keeping this in mind, is not unrealistic to hypothesize that the number of LC molecules calculated more than 22 years ago by Koriazova & Montal (in an indirect way by checking SNAP25 cleavage using an ELISA-based assay) might be an underestimation. Indeed, the addition of Hsp90 in their system might aid in the refolding of LC molecules that, even if they have successfully be translocated, might not cleave the substrate due to their unfolded state.

As active scientist, I understand the challenges of peer review and publication, which can often be slow and frustrating involving seemingly endless rounds of review. Therefore, I am in favor of the new eLife publishing model. Indeed, this paper has already been published as Reviewed Preprints and will soon be declared as the final Version of Record, accompanied by this public review. Having said that, I hope that the readers of this journal and future scientists will prove me wrong. I hope they will engage with this paper, providing comments, validations (which are currently missing), and citations as frequently as they did for the seminal works of Koriazova & Montal.

Reviewer #1 (Public Review):

The paper combines experiments on freely gliding cyanobacteria, buckling experiments using two-dimensional V shaped corners, and micropipette force measurements with theoretical models to study gliding forces in these organisms. The aim is to quantify these forces and use the results to perhaps discriminate between competing mechanisms by which these cells move. A large data set of possible collision events are analyzed, bucking events evaluated, and critical buckling lengths estimated. A line elasticity model is used to analyze the onset of buckling and estimate the effective (viscous type) friction/drag that controls the dynamics of the rotation that ensues post-buckling. This value of the friction/drag is compared to a second estimate obtained by consideration of the active forces and speeds in freely gliding filaments. The authors find that these two independent estimates of friction/drag correlate with each other and are comparable in magnitude. The experiments are conducted carefully, the device fabrication is novel, the data set is interesting, and the analysis is solid. The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion. While consistent with the data, this conclusion is inferred.

Summary:

The paper addresses important questions on the mechanisms driving the gliding motility of filamentous cyanobacteria. The authors aim to understand these by estimating the elastic properties of the filaments, and by comparing the resistance to gliding under a) freely gliding conditions, and b) in post-buckled rotational states. Experiments are used to estimate the propulsion force density on freely gliding filaments (assuming over damped conditions). Experiments are combined with a theoretical model based on Euler beam theory to extract friction (viscous) coefficients for filaments that buckle and begin to rotate about the pinned end. The main results are estimates for the bending stiffness of the bacteria, the propulsive tangential force density, the buckling threshold in terms of the length, and estimates of the resistive friction (viscous drag) providing the dissipation in the system and balancing the active force. It is found that experiments on the two bacterial species yield nearly identical value of 𝑓 (albeit with rather large variations). The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion.

Strengths of the paper:

The strengths of the paper lie in the novel experimental setup and measurements that allow for the estimation of the propulsive force density, critical buckling length, and effective viscous drag forces for movement of the filament along its contour - the axial (parallel) drag coefficient, and the normal (perpendicular) drag coefficient (I assume this is the case, since the post-buckling analysis assumes the bent filament rotates at a constant frequency). These direct measurements are important for serious analysis and discrimination between motility mechanisms.

Weaknesses:

There are aspects of the analysis and discussion that may be improved. I suggest that the authors take the following comments into consideration while revising their manuscript.

The conclusion that adhesion via focal adhesions is the cause for propulsion rather than slime protrusion, is consistent with the experimental results that the frictional drag correlates with propulsion force. At the same time, it is hard to rule out other factors that may result in this (friction) viscous drag - (active) force relationship while still being consistent with slime production. More detailed analysis aiming to discriminate between adhesion vs slime protrusion may be outside the scope of the study, but the authors may still want to elaborate on their inference. It would help if there was a detailed discussion on the differences in terms of the active force term for the focal adhesion-based motility vs the slime motility.

Can the authors comment on possible mechanisms (perhaps from the literature) that indicate how isotropic friction may be generated in settings where focal adhesions drive motility. A key aspect here would probably be estimating the extent of this adhesion patch and comparing it to a characteristic contact area. Can lubrication theory be used to estimate characteristic areas of contact (knowing the radius of the filament, and assuming a height above substrate)? If the focal adhesions typically cover areas smaller than this lubrication area, it may suggest the possibility that bacteria essentially present a flat surface insofar as adhesion is concerned, leading to transversely isotropic response in terms of the drag. Of course, we will still require the effective propulsive force to act along the tangent.

I am not sure why the authors mention that the power of the gliding apparatus is not rate limiting. The only way to verify this would be to put these in highly viscous fluids where the drag of the external fluid comes into the picture as well (if focal adhesions are on the substrate facing side, and the upper side is subject to ambient fluid drag). Also, the friction referred to here has the form of a viscous drag (no memory effect, and thus not viscoelastic or gel-like), and it is not clear if forces generated by adhesion involve other forms of drag such as chemical friction via temporary bonds forming and breaking. In quasi-static settings and under certain conditions such as separation of chemical and elastic time scales, bond friction may yield overall force proportional to local sliding velocities.

For readers from a non-fluids background, some additional discussion of the drag forces, and the forms of friction would help. For a freely gliding filament if 𝑓 is the force density (per unit length), then steady gliding with a viscous frictional drag would suggest (as mentioned in the paper) 𝑓 ∼ 𝑣! 𝐿 𝜂∥. The critical buckling length is then dependent on 𝑓 and on 𝐵 the bending modulus. Here the effective drag is defined per length. I can see from this that if the active force is fixed, and the viscous component resulting from the frictional mechanism is fixed, the critical buckling length will not depend on the velocity (unless I am missing something in their argument), since the velocity is not a primitive variable, and is itself an emergent quantity.

Reviewer #1 (Public Review):

Summary:

Tsai and Seymen et al. investigate associations between RTE expression and methylation and age and inflammation, using multiple public datasets. The concept of the study is in principle interesting, as a systematic analysis of RTE expression during human aging is lacking. Unfortunately, the reliance on expression microarray data, used to perform the core analysis of the paper places much of the study on shaky ground. The findings of the study would not be sufficiently supported until the authors validate them with more suitable methods.

Strengths:

This is a very important biological problem.

Weaknesses:

RNA microarray probes are obviously biased to genes, and thus quantifying transposon analysis based on them seems dubious. Based on how arrays are designed there should at least be partial (perhaps outdated evidence) that the probe sites overlap a protein-coding or non-coding RNA. The authors state they only used intergenic probes, but based on supplementary files, almost half of RTE probes are not intergenic but intronic (n=106 out of 264). This is further complicated by the fact that not all this small subset of probes is available in all analyzed datasets. For example, 232 probes were used for the MESA dataset but only 80 for the GTP dataset. Thus, RTE expression is quantified with a set of probes which is extremely likely to be highly affected by non-RTE transcripts and that is also different across the studied datasets. Differences in the subsets of probes could very well explain the large differences between datasets in multiple of the analyses performed by the authors, such as in Figure 2a, or 3a. It is nonetheless possible that the quantification of RTE expression performed by the authors is truly interpretable as RTE expression, but this must be validated with more data from RNA-seq. Above all, microarray data should not be the main type of data used in the type of analysis performed by the authors.

Reviewer #2 (Public Review):

Congenital cystic airway abnormalities (CPAM) are a common poorly understood disorder in airway lung development that can be fatal if not effectively treated at birth. This study by Luo and colleagues provides compelling new evidence that bone morphogenetic protein signaling in distal mesenchymal cells is required for normal mouse lung development. Genetic loss of BMP receptor in mice and in fetal mesenchymal cells causes type 2 or alveolar-like CPAM pathology. Furthermore, this is associated with changes in expression of Sox2-Sox9 suggesting defects in the proximal to distal cellularity of the lung. Interestingly, cysts are formed even when SMAD1 and 5, two major downstream effects of BMP signaling are deleted suggesting a role for non-canonical BMP signalling. Furthermore, they were independent of ablating BMP signaling in non-vascular mesenchymal cells. The findings are compelling and provide strong evidence that cystic lung development is caused by loss of non-canonical BMP signaling in mesenchymal cells. The main weakness of the paper is that it does not identify the downstream non-canonical effector of mesenchymal BMP signaling. The authors provide a plausible suggestion that it may be p38 MAPK that deserves further investigation. Despite this minor weakness, the overall findings are novel and considered important because they provide a foundation for new studies, including experiments that may produce drugs designed to prevent or treat newborn infants with CPAM.

Reviewer #1 (Public Review):

Summary:

This manuscript explores the impact of serotonin on olfactory coding in the antennal lobe of locusts and odor-evoked behavior. The authors use serotonin injections paired with an odor-evoked palp-opening response assay and bath application of serotonin with intracellular recordings of odor-evoked responses from projection neurons (PNs).

Strengths:

The authors make several interesting observations, including that serotonin enhances behavioral responses to appetitive odors in starved and fed animals, induces spontaneous bursting in PNs, directly impacts PN excitability, and uniformly enhances PN responses to odors.

Weakness:

The one remaining issue to be resolved is the theoretical discrepancy between the physiology and the behavior. The authors provide a computational model that could explain this discrepancy and provide the caveat that while the physiological data was collected from the antennal lobe, but there could be other olfactory processing stages involved. Indeed other processing stages could be the sites for the computational functions proposed by the model. There is an additional caveat which is that the physiological data were collected 5-10 minutes after serotonin application whereas the behavioral data were collected 3 hours after serotonin application. It is difficult to link physiological processes induced 5 minutes into serotonin application to behavioral consequences 3 hours subsequent to serotonin application. The discrepancy between physiology and behavior could easily reflect the timing of action of serotonin (i.e. differences between immediate and longer-term impact).

Overall, the study demonstrates the impact of serotonin on odor-evoked responses of PNs and odor guided behavior in locust. Serotonin appears to have non-linear effects including changing the firing patterns of PNs from monotonic to bursting and altering behavioral responses in an odor-specific manner, rather than uniformly across all stimuli presented.

Reviewer #1 (Public Review):

The manuscript introduces a bioinformatic pipeline designed to enhance the structure prediction of pyoverdines, revealing an extensive and previously overlooked diversity in siderophores and receptors. Utilizing a combination of feature sequence and phylogenetic approaches, the method aims to address the challenging task of predicting structures based on dispersed gene clusters, particularly relevant for pyoverdines.

Predicting structures based on gene clusters is still challenging, especially pyoverdines as the gene clusters are often spread to different locations in the genome. An improved method would indeed be highly useful, and the diversity of pyoverdine gene clusters and receptors identified is impressive.

However, so far the method basically aligns the structural genes and domains involved in pyoverdine biosynthesis and then predicts A domain specificity to predict the encoded compounds. Both methods are not particularly new as they are included in other tools such as PRISM (10.1093/nar/gkx320 ) or Sandpuma (https://doi.org/10.1093/bioinformatics/btx400) among others. The study claims superiority in A domain prediction compared to existing tools, yet the support is currently limited, relying on a comparison solely with AntiSMASH. A more extensive and systematic comparison with other tools is needed.

Additionally, in contradiction to the authors' claims, the method's applicability seems constrained to well-known and widely distributed gene clusters. The absence of predictions for new amino acids raises concerns about its generalizability to NRPS beyond the studied cases.

The manuscript lacks clarity on how the alignment of structural genes operates when dealing with multiple NRPS gene clusters on different genome contigs. How would the alignment of each BGC work?

Another critical concern is that a main challenge in NRPS structure prediction is not the backbone prediction but rather the prediction of tailoring reactions, which is not addressed in the manuscript at all, and this limitation extensively restricts the applicability of the method.

The manuscript presents a potentially highly useful bioinformatic pipeline for pyoverdine structure prediction, showcasing a commendable exploration of siderophore diversity. However, some of the claims made remain unsubstantiated. Overall, while the study holds promise, further validation and refinement are required to fulfill its potential impact on the field of bioinformatic structure prediction.

Reviewer #1 (Public Review):

The authors provided a detailed analysis of the real-time structural changes in actin filaments resulting from cofilin binding, using High-Speed Atomic Force Microscopy (HSAFM). The cofilin family controls the lifespan of actin filaments in the cells by severing the filament and promoting depolymerization. Understanding the effects of cofilin on actin filament structure is critical. It is widely acknowledged that cofilin binding significantly shortens the pitch of the actin helix. The authors previously reported (1) that this shortening extends to the unbound region of the actin filament on the pointed end side of the cluster. In this study, the authors presented substantially improved AFM images and provide detailed accounts of the dynamics observed. It was found that a minimal cofilin-binding cluster, consisting of 2-4 molecules, could induce changes in the helical parameters over one or more actin crossover repeats. Adjacent to the cofilin-binding clusters, the actin crossovers were observed to shortened within seconds, and this shortening was limited to one side of the cluster. Additionally, the phosphate binding to the actin filament was observed to stabilize the helical twist, suggesting a mechanism in which cofilin preferentially binds to ADP-bound actin filaments. These findings significantly advance our understanding of actin filament dynamics which is essential for a wide of cellular processes.<br /> However, I propose that the sections about MAD and certain parts of the discussions need substantial revisions.

MAD analysis<br /> The authors have presented findings that the mean axial distance (MAD) within actin filaments exhibits a significant dependency on the helical twist, a conclusion not previously derived despite extensive analyses through electron microscopy (EM) and molecular dynamics (MD) simulations. Notably, the MAD values span from 4.5 nm (8.5 pairs per half helical pitch, HHP) to 6.5 nm (4.5 pairs/HHP) as depicted in Figure 3C. The inner domain (ID) of actin remains very similar across C, G, and F forms(2, 3), maintaining similar ID-ID interactions in both cofilactin and bare actin filaments, keeping the identical axial distance between subunits in the both states. This suggests that the ID is unlikely to undergo significant structural changes, even with fluctuations in the filament's twist, keeping the ID-ID interactions and the axial distances. The broad range of MAD values reported poses a challenge for explanation. A careful reassessment of the MAD analysis is recommended to ensure accuracy.<br /> In determining axial distances, the authors extracted measurements from filament line profiles. It is advised to account for potential anomalies such as missing peaks or pseudo peaks, which could arise from noise interference. An example includes the observation of three peaks in HHP6 of Figure Supplement 5C, corresponding to 4.5 pairs. Peak intervals measured from the graph were 5, 11.8, 8.7, and 5.7 nm. The second region (11.8 nm) appears excessively long. If one peak is hidden in the second region, the MAD becomes 5.5 nm.

Compiling histograms of axial distances (ADs) rather than focusing solely on MAD may provide deeper insights. If the AD is too long or too short, the authors should suspect the presence of missing peaks or pseudo-peaks due to noise. If 4.4 or 5.5 pairs/HHP regions tend to contain missing peaks and 7.5-8.5 pairs/HHP regions tend to contain pseudo peaks, this may explain the MAD dependency on the helical twist.

Additionally, Figure 3E indicates a first decay constant of 0.14 seconds, substantially shorter than the frame rate (0.5 sec/frame). This suggests significant variations in line profiles between frames, attributable either to overly rapid dynamics or a low signal-to-noise ratio. Implementing running frame averages (of 2-3 frames) is recommended to distinguish between these scenarios. If the dynamics are indeed fast, the averaged frame's line profile may degrade, complicating peak identification. Conversely, if poor signal-to-noise ratio is the cause, averaging frames could facilitate peak detection. In the latter case, the authors can find the optimal number of frame averages and obtain better line profiles with fewer missing and pseudo-peaks.

Discussions<br /> The authors suggest a strong link between the C-form of actin and the formation of a short pitch helix. However, Oda et al. (3) have demonstrated that the C-form is highly unstable in the absence of cofilin binding, casting doubt on the possibility of the C-form propagating without cofilin binding. Moreover, in one strand of the cofilactin, interactions between actin subunits are limited to those between the inner domains (ID-ID interactions), which are quite similar to the interactions observed in bare actin filaments. This similarity implies that ID-ID interactions alone are insufficient to determine the helical parameters, suggesting that the presence of cofilin is essential for the formation of the short pitch helix in the cofilactin filament. Thus, crossover repeats are not necessarily shortened even if the actin form is C-form.

Narita (4) proposes that the facilitation of cofilin binding may occur through a shortening in the helix pitch, independent of a change to the C-form of actin. Furthermore, the dissociation of the D-loop from an adjacent actin subunit leads directly to the transition of actin to the G-form, which is considered the most stable configuration for the actin molecule (3).

The mechanism by which the shortened pitch propagates remains a critical and unresolved issue. It appears that this propagation is not a result of the C-form's propagation but likely involves an unidentified mechanism. Identifying and understanding this mechanism represents an essential direction for future research.

(1) K. X. Ngo et al., a, Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy. eLife 4, (2015).<br /> (2) K. Tanaka et al., Structural basis for cofilin binding and actin filament disassembly. Nature communications 9, 1860 (2018).<br /> (3) T. Oda et al., Structural Polymorphism of Actin. Journal of molecular biology 431, 3217-3228 (2019).<br /> (4) A. Narita, ADF/cofilin regulation from a structural viewpoint. Journal of muscle research and cell motility 41, 141-151 (2020).

Reviewer #1 (Public Review):

Summary:

The study provides valuable insights into the role of PfMORC in Plasmodium's epigenetic regulation, backed by a comprehensive methodological approach. The overarching goal was to understand the role of PfMORC in epigenetic regulation during asexual blood stage development, particularly its interactions with ApiAP2 TFs and its potential involvement in the regulation of genes vital for Plasmodium virulence. To achieve this, they conducted various analyses. These include a proteomic analysis to identify nuclear proteins interacting with PfMORC, a study to determine the genome-wide localization of PfMORC at multiple developmental stages, and a transcriptomic analysis in PfMORCHA-glmS knockdown parasites. Taken together, this study suggests that PfMORC is involved in chromatin assemblies that contribute to the epigenetic modulation of transcription during the asexual blood stage development.

Strengths:

The study employed a multi-faceted approach, combining proteomic, genomic, and transcriptomic analyses, providing a holistic view of PfMORC's role. The proteomic analysis successfully identified several nuclear proteins that may interact with PfMORC. The genome-wide localization offered valuable insights into PfMORC's function, especially its predominant recruitment to subtelomeric regions. The results align with previous findings on PfMORC's interaction with ApiAP2 TFs. Notably, the authors meticulously contextualized their findings with prior research adding credibility to their work.

Weaknesses:

While the study identifies potential interacting partners and loci of binding, direct functional outcomes of these interactions remain an inference. The use of the glmS ribozyme system to achieve a 50% reduction in PfMORC transcript levels makes it difficult to understand the role of PfMORC solely in terms of chromatin architecture without considering its impact on gene expression. Although assessing the overall impact of acute MORC depletion was beyond the scope of the study, it would have been informative.

Reviewer #1 (Public Review):

Summary

The authors use an elegant but somewhat artificial heterodimerisation approach to activate the isolated cytoplasmic domains of different receptor kinases (RKs) including the receptor kinase BRI1 and EFR. The developmental RK BRI1 is known to be activated by the co-receptor BAK1. Active BRI1 is then able to phosphorylate downstream substrates. The immune receptor EFR is also an active protein kinase also activated by the co-receptor BAK1. EFR however appears to have little or no kinase activity but seems to use an allosteric mechanism to in turn enable BAK1 to phosphorylate the substrate kinase BIK1. EFR tyrosine phosphorylation by BAK1 appears to trigger a conformational change in EFR, activating the receptor. Likewise, kinase activating mutations can cause similar conformational transitions in EFR and also in BAK1 in vitro and in planta.

Strengths:

I particularly liked The HDX experiments coupled with mutational analysis (Fig. 2) and the design and testing of the kinase activating mutations (Fig. 3), as they provide novel mechanistic insights into the activation mechanisms of EFR and of BAK1. These findings are nicely extended by the large-scale identification of EFR-related RKs from different species with potentially similar activation mechanisms (Fig. 5).

Weaknesses:

In my opinion, there are currently two major issues with the present manuscript. (1) The authors have previously reported that the EFR kinase activity is dispensible for immune signaling (https://pubmed.ncbi.nlm.nih.gov/34531323/) but the wild-type EFR receptor still leads to a much better phosphorylation of the BIK1 substrate when compared to the kinase inactive D849N mutant protein (Fig. 1). (2) How the active-like conformation of EFR is in turn activating BAK1 is poorly characterized, but appears to be the main step in the activation of the receptor complex. Extending the HDX analyses to resting and Rap-activated receptor complexes could be a first step to address this question, but these HDX studies were not carried out due to technical limitations.

Overall this is an interesting study that aims to advance our understanding of the activation mechanisms of different plant receptor kinases with important functions in plant immunity.

Reviewer #1 (Public Review):

(a) Summary: The present study addresses how the local abundance of metabolites impacts the biology of the tumor microenvironment. The authors enroll patients harboring kidney tumors and use freshly resected tumor material for metabolic studies. Specifically, the authors separate the adjacent normal kidney tissue from the tumor material and then harvest the interstitial fluid from the normal kidney (KIF) or the tumor (TIF) for quantitative metabolomics. The plasma samples from the patient are used for comparison. Additionally, the authors also compare metabolite levels in the plasma of patients with kidney versus lung cancer (or healthy donors) to address how specific tumor types might contribute to circulating levels of metabolites. Altogether, the authors find that the metabolite levels in the KIF and TIF, although vastly different than plasma, are largely overlapping. These findings indicate that tissue of origin appears to have a stronger role in determining the local metabolic environment of tumors than the genetics or biochemistry of the tumor itself.

(b) Strengths: The biggest strength of the current study is the use of human patient-derived samples. The cohort size (~50 patients) is relatively large, which adds to the rigor of the work. The work also relies on a small pool of metabolites that can be quantitatively measured using methods developed by the authors. Focusing on a smaller metabolic pool also likely increases the signal-to-noise ratio and enables the more rigorous determination of any underlying differences. The manuscript is well-written and highlights both the significance of the findings and also acknowledges many of the caveats. The recognition of the metabolic contributions of surrounding normal tissue as the primary driver of local nutrient abundance is a novel finding in the work, which can be leveraged in future studies.

(c) Weaknesses: The work has certain caveats, some of which have been already recognized by the authors. These include the use of steady-state metabolites and the possibility of cross-contamination of some TIF into the adjacent KIF. This study is also unable to distinguish the mechanisms driving the metabolic changes in KIF/TIF relative to circulating levels in plasma.

The relative similarity of KIF and TIF is quite surprising. However, this interpretation is presently based on sampling of only ~100 polar metabolites and ~200 lipid molecules. It is, perhaps, possible that future technological developments that enable more comprehensive quantitative metabolic profiling might distinguish between KIF and TIF composition.

In vitro tissue culture is recognized to suffer from 'non-physiological' nutrient dependencies, which are impacted by the composition of culture media. Thus, in vivo studies remain our current gold-standard in mechanistic studies of tumor metabolism. It is presently unclear whether the findings of this work will be recapitulated in any of the kidney cancer in vivo models and thus be functionally testable.

The authors have acknowledged these caveats and where possible provided textual clarifications and updated figures in their revised manuscript. Future work will be required to model these changes in animal models.

Reviewer #1 (Public Review):

The study is designed to assess the role of Syngap1 in regulating the physiology of the MGE-derived PV+ and SST+ interneurons. Syngap1 is associated with some mental health disorders, and PV+ and SST+ cells are the focus of many previous and likely future reports from studies of interneuron biology, highlighting the translational and basic neuroscience relevance of the authors' work.

Strengths of the study are using well-established electrophysiology methods and the highly controlled conditions of ex vivo brain slice experiments combined with a novel intersectional mouse line, to assess the role of Syngap1 in regulating PV+ and SST+ cell properties. The findings revealed that in the mature auditory cortex, Syngap1 haploinsufficiency decreases both the intrinsic excitability and the excitatory synaptic drive onto PV+ neurons from Layer 4. In contrast, SST+ interneurons were mostly unaffected by Syngap1 haploinsufficiency. Pharmacologically manipulating the activity of voltage-gated potassium channels of the Kv1 family suggested that these channels contributed to the decreased PV+ neuron excitability by Syngap insufficiency. These results therefore suggest that normal Syngap1 expression levels are necessary to produce normal PV+ cell intrinsic properties and excitatory synaptic drive, albeit, perhaps surprisingly, inhibitory synaptic transmission was not affected by Syngap1 haploinsufficiency.

Since the electrophysiology experiments were performed in the adult auditory cortex, while Syngap1 expression was potentially affected since embryonic stages in the MGE, future studies should address two important points that were not tackled in the present study. First, what is the developmental time window in which Syngap1 insufficiency disrupted PV+ neuron properties? Albeit the embryonic Syngap1 deletion most likely affected PV+ neuron maturation, the properties of Syngap-insufficient PV+ neurons do not resemble those of immature PV+ neurons. Second, whereas the observation that Syngap1 haploinsufficiency affected PV+ neurons in auditory cortex layer 4 suggests auditory processing alterations, MGE-derived PV+ neurons populate every cortical area. Therefore, without information on whether Syngap1 expression levels are cortical area-specific, the data in this study would predict that by regulating PV+ neuron electrophysiology, Syngap1 normally controls circuit function in a wide range of cortical areas, and therefore a range of sensory, motor and cognitive functions. These are relatively minor weaknesses regarding interpretation of the data in the present study that the authors could discuss.

Reviewer #1 (Public Review):

In this manuscript, the authors described a computational method catELMo for embedding TCR CDR3 sequences into numeric vectors using a deep-learning-based approach, ELMo. The authors applied catELMo to two applications: supervised TCR-epitope binding affinity prediction and unsupervised epitope-specific TCR clustering. In both applications, the authors showed that catELMo generated significantly better binding prediction and clustering performance than other established TCR embedding methods.

The authors have addressed all of my concerns except for one as following:

(5) GIANA's result is like

– ## TIME:2020-12-14 14:45:14|cmd: GIANA4.py|COVID_test/rawData/hc10s10.txt|IsometricDistance_Thr=7.0|thr_v=3.7|thr_s=3.3|exact=True|Vgene=True|ST=3

– ## Column Info: CDR3 aa sequence, cluster id, other information in the input file<br /> CAISDGTAASSTDTQYF 1 TRBV10-3*01 6.00384245917387e-05 0.930103216755186 COVID19:BS-EQ-0002-T1-replacement_TCRB.tsv<br /> CAISDGTAASSTDTQYF 1 TRBV10-3*01 4.34559031223066e-05 0.918135389545364 COVID19:BS-EQ-0002-T2-replacement_TCRB.tsv<br /> CANATLLQVLSTDTQYF 2 TRBV21-1*01 3.00192122958694e-05 0.878695260046097 COVID19:BS-EQ-0002-T1-replacement_TCRB.tsv<br /> CANATLLQVLSTDTQYF 2 TRBV21-1*01 1.44853010407689e-05 0.768125375525736 COVID19:BS-EQ-0002-T2-replacement_TCRB.ts<br /> ...

as in its example file at: https://raw.githubusercontent.com/s175573/GIANA/master/data/hc10s10--RotationEncodingBL62.txt

The results directly give the clustering results in the second column, and there is no direct distance metric for hierarchical clustering. Therefore, it is still not clear how the authors conducted the hierarchical clustering on GIANA's results. Did the hierarchical clustering apply to each of the original clusters on the CDR3 distances within the same original cluster?

Reviewer #1 (Public Review):

To understand the spinal locomotor circuits, we need to reveal how various types of spinal interneurons work in the circuits. So far, the general roles of the cardinal groups of spinal interneurons (dI6, V0, V1, V2a, V2b, and V3) involved in locomotion have been roughly established but not fully understood. Each group is believed to contain some clades with more detailed functional differences. However, each character and function of these clades has not yet been elucidated.

In this study, Worthy et al. investigated clades of V1 neurons that are one of the main groups of inhibitory neurons in the spinal cord. Previous reports proposed four clades (Renshaw cells, FoxP2, sp8, and pou6f2) in V1 neurons defined by the expression of transcription factors. For V1 neurons in each of the four clades, the authors investigated the birth time and showed the postnatal location in the spinal cord according to the birth time. They found FoxP2-V1 located near LMC motor neurons that project to the limb. Using genetically labeled Foxp2-V1 mice, they showed that most of the synapses of V1 neurons on the cell bodies of motor neurons were from Foxp2-V1 and Renshaw cells. Furthermore, a higher proportion of Foxp2-V1 synapses is observed on LMC motor neurons than on axial motor neurons. They proposed that Foxp2-V1, which represents 60% of V1, can be further classified according to the expression of transcription factors Otp and Foxp4.

These results will be helpful for future analyses of the development and function of V1 neurons. In particular, the discovery of strong synaptic connections between Foxp2-V1 and LMC motor neurons will be beneficial in analyzing the role of V1 neurons in motor circuits that generate movement of the limbs.

The conclusions of this paper are well supported by the data obtained using widely used methods. However, for some analyses, the specificity of labeling V1 clades should be clearly described.

(1) In Figure 1, the MafB antibody (Sigma) was used to identify Renshaw cells at P5. However, according to the supplementary Figure 3D, the specificity of the MafB antibody (Sigma) is relatively low. The image of MafB-GFP, V1-INs, and MafB-IR at P5 should be added to the supplementary figure. The specificity of MaFB-IR-Sigma in V1 neurons at P5 should be shown. This image also might support the description of the genetically labeled MafB-V1 distribution at P5 (page 8, lines 28-32).

(2) The proportion of genetically labeled FoxP2-V1 in all V1 is more than 60%, although immunolabeled FoxP2-V1 is approximately 30% at P5. Genetically labeled Otp-V1 included other non-FoxP2 V1 clades (Fig. 8L-M). I wonder whether genetically labeled FoxP2-V1 might include the other three clades. The authors should show whether genetically labeled FoxP2-V1 expresses other clade markers, such as pou6f2, sp8, and calbindin, at P5.

Reviewer #1 (Public Review):

Summary:

Bennion and colleagues present a careful examination of how an earlier set of memories can either interfere with or facilitate memories formed later. This impressive work is a companion piece to an earlier paper by Antony and colleagues (2022) in which a similar experimental design was used to examine how a later set of memories can either interfere with or facilitate memories formed earlier. This study makes contact with an experimental literature spanning 100 years, which is concerned with the nature of forgetting, and the ways in which memories for particular experiences can interact with other memories. These ideas are fundamental to modern theories of human memory, for example, paired-associate studies like this one are central to the theoretical idea that interference between memories is a much bigger contributor to forgetting than any sort of passive decay.

Strengths:

At the heart of the current investigation is a proposal made by Osgood in the 1940s regarding how paired associates are learned and remembered. In these experiments, one learns a pair of items, A-B (cue-target), and then later learns another pair that is related in some way, either A'-B (changing the cue, delta-cue), or A-B' (changing the target, delta-target), or A'-B' (changing both, delta-both), where the prime indicates that item has been modified, and may be semantically related to the original item. The authors refer to the critical to-be-remembered pairs as base pairs. Osgood proposed that when the changed item is very different from the original item there will be interference, and when the changed item is similar to the original item there will be facilitation. Osgood proposed a graphical depiction of his theory in which performance was summarized as a surface, with one axis indicating changes to the cue item of a pair and the other indicating changes to the target item, and the surface itself necessary to visualize the consequences of changing both.

In the decades since Osgood's proposal, there have been many studies examining slivers of the proposal, e.g., just changing targets in one experiment, just changing cues in another experiment. Because any pair of experiments uses different methods, this has made it difficult to draw clear conclusions about the effects of particular manipulations.

The current paper is a potential landmark, in that the authors manipulate multiple fundamental experimental characteristics using the same general experimental design. Importantly, they manipulate the semantic relatedness of the changed item to the original item, the delay between the study experience and the test, and which aspect of the pair is changed. Furthermore, they include both a positive control condition (where the exact same pair is studied twice), and a negative control condition (where a pair is only studied once, in the same phase as the critical base pairs). This allows them to determine when the prior learning exhibits an interfering effect relative to the negative control condition and also allows them to determine how close any facilitative effects come to matching the positive control.

The results are interpreted in terms of a set of existing theories, most prominently the memory-for-change framework, which proposes a mechanism (recursive reminding) potentially responsible for the facilitative effects examined here. One of the central results is the finding that a stronger semantic relationship between a base pair and an earlier pair has a facilitative effect on both the rate of learning of the base pair and the durability of the memory for the base pair. This is consistent with the memory-for-change framework, which proposes that this semantic relationship prompts retrieval of the earlier pair, and the two pairs are integrated into a common memory structure that contains information about which pair was studied in which phase of the experiment. When semantic relatedness is lower, they more often show interference effects, with the idea being that competition between the stored memories makes it more difficult to remember the base pair.

This work represents a major methodological and empirical advance for our understanding of paired-associates learning, and it sets a laudably high bar for future work seeking to extend this knowledge further. By manipulating so many factors within one set of experiments, it fills a gap in the prior literature regarding the cognitive validity of an 80-year-old proposal by Osgood. The reader can see where the observed results match Osgood's theory and where they are inconclusive. This gives us insight, for example, into the necessity of including a long delay in one's experiment, to observe potential facilitative effects. This point is theoretically interesting, but it is also a boon for future methodological development, in that it establishes the experimental conditions necessary for examining one or another of these facilitation or interference effects more closely.

Weaknesses:

One minor weakness of the work is that the overarching theoretical framing does not necessarily specify the expected result for each and every one of the many effects examined. For example, with a narrower set of semantic associations being considered (all of which are relatively high associations) and a long delay, varying the semantic relatedness of the target item did not reliably affect the memorability of that pair. However, the same analysis showed a significant effect when the wider set of semantic associations was used. The positive result is consistent with the memory-for-change framework, but the null result isn't clearly informative to the theory. I call this a minor weakness because I think the value of this work will grow with time, as memory researchers and theorists use it as a benchmark for new theory development. For example, the data from these experiments will undoubtedly be used to develop and constrain a new generation of computational models of paired-associates learning.

Reviewer #1 (Public Review):

Summary/Strengths:

This manuscript describes a stimulating contribution to the field of human motor control. The complexity of control and learning is studied with a new task offering a myriad of possible coordination patterns. Findings are original and exemplify how baseline relationships determine learning.

Weaknesses:

A new task is presented: it is a thoughtful one, but because it is a new one, the manuscript section is filled with relatively new terms and acronyms that are not necessarily easy to rapidly understand.

First, some more thoughts may be devoted to the take-home message. In the title, I am not sure manipulating a stick with both hands is a key piece of information. Also, the authors appear to insist on the term 'implicit', and I wonder if it is a big deal in this manuscript and if all the necessary evidence appears in this study that control and adaptation are exclusively implicit. As there is no clear comparison between gradual and abrupt sessions, the authors may consider removing at least from the title and abstract the words 'implicit' and 'implicitly'. Most importantly, the authors may consider modifying the last sentence of the abstract to clearly provide the most substantial theoretical advance from this study.

It seems that a substantial finding is the 'constraint' imposed by baseline control laws on sensorimotor adaptation. This seems to echo and extend previous work of Wu, Smith et al. (Nat Neurosci, 2014): their findings, which were not necessarily always replicated, suggested that the more participants were variable in baseline, the better they adapted to a systematic perturbation. The authors may study whether residual errors are smaller or adaptation is faster for individuals with larger motor variability in baseline. Unfortunately, the authors do not present the classic time course of sensorimotor adaptation in any experiment. The adaptation is not described as typically done: the authors should thus show the changes in tip movement direction and stick-tilt angle across trials, and highlight any significant difference between baseline, early adaptation, and late adaptation, for instance. I also wonder why the authors did not include a few no-perturbation trials after the exposure phase to study after-effects in the study design: it looks like a missed opportunity here. Overall, I think that showing the time course of adaptation is necessary for the present study to provide a more comprehensive understanding of that new task, and to re-explore the role of motor variability during baseline for sensorimotor adaptation.

The distance between hands was fixed at 15 cm with the Kinarm instead of a mechanical constraint. I wonder how much this distance varied and more importantly whether from that analysis or a force analysis, the authors could determine whether one hand led the other one in the adaptation.

I understand the distinction between task- and end-effector irrelevant perturbation, and at the same time results show that the nervous system reacts to both types of perturbation, indicating that they both seem relevant or important. In line 32, the errors mentioned at the end of the sentence suggest that adaptation is in fact maladaptive. I think the authors may extend the Discussion on why adaptation was found in the experiments with end-effector irrelevant and especially how an internal (forward) model or a pair of internal (forward) models may be used to predict both the visual and the somatosensory consequences of the motor commands.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Liangliang Fu and colleagues propose that a population of CD81-positive fibroblasts exhibiting senescent features activate neutrophils via the C3/C3aR1 axis and contribute to maintaining the inflammatory response in periodontitis. The authors provide evidence that inhibition of cellular senescence by metformin treatment in murine models ameliorated periodontitis progression. This study provides some valuable insights into the impact of periodontitis-induced gingival damage and the significance of stromal senescence.

Strengths:

(1) The work combines a variety of models of periodontitis, including analyses of human samples, primary gingival fibroblast cell culture isolation and cultures, and mouse models of ligature/induced periodontitis. Then, the results are solid in terms of used models.

(2) Comprehensive exhibition of methodologies incorporating histology procedures, micro-CT imaging, bulkRNAseq and scRNAseq transcriptomic profiles (the latter analyses of published datasets), and a number of computational analyses. The paper is robust at the technical level.

(3) This paper is timely and interesting and it opens potential therapeutic avenues for the treatment of periodontitis. Although the interplay of senescence with periodontitis and the use of metformin has been previously reported (e.g. Kuang et al. Biogerontology 2020), I think the proposed mechanism of neutrophils activation by CD81-positive senescent fibroblasts and the inflammatory response is original. The paper is therefore at the forefront of the field, as senescence and its interplay with the immune system is a hot topic and reflects the current directions ("trending topics") of the field.

Weaknesses:

(1) The assessment of Cellular Senescence is limited and would benefit from additional biomarkers and not just p16 and p21, in particular in vivo.

(2) This paper does not include original scRNAseq datasets in periodontitis, but analyses of already published datasets.

(3) The authors claim that cellular senescence of CD81+ fibroblasts could be attributed to disturbances of lipid metabolism, resulting in differentiation arrest and higher expression of SASP factors in CD81+ fibroblast cells. Although the authors found that a series of pathways related to metabolism (metabolism of linoleic acid, linolenic acid, arachidonic acid, or steroid biosynthesis) are upregulated in CD81+ fibroblasts by transcriptomic analyses the hypothesis remains speculative and requires further validations.

(4) Metformin has been reported to downregulate the SASP and lower senescent cell burden (e.g. for review see Kulkarni, Gubbi, and Barzilai. Cell Metab 2020). Although Metformin's senotherapeutic activities can be mediated by anti-inflammatory effects preventing NFkB translocation to the nucleus (Moiseeva et al. Aging Cell 2013) and has been shown to prevent oxidative stress-induce senescence in human periodontal ligament cells (Kuang et al. Biogerontology 2020) it can also drive multiple and pleiotropic effects unrelated to senescence.

(5) Mechanistically, the proposed activation neutrophils by senescent C81+ fibroblasts via the C3/C3aR1 axis would be further supported by using a senolytic approach (e.g. Bcl2 inhibitor) allowing testing of whether eradication of senescent stromal cells results in reduced levels of CD81 and C3 positivity, and prevention of neutrophils infiltration.

Reviewer #1 (Public Review):

Summary:

De Waele et al. reported a dual-branch neural network model for predicting antibiotic resistance profiles using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry data. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF mass spectrometry data and their associated antibiotic susceptibility profiles. The authors used a dual branch neural network approach to simultaneously represent information about mass spectra and antibiotics for a wide range of species and antibiotic combinations. The authors showed consistent performance of their strategy to predict antibiotic susceptibility for different spectrums and antibiotic representations (i.e., embedders). Remarkably, the authors showed how small datasets collected at one location can improve the performance of a model trained with limited data collected at a second location. Despite these promising results, there are several analyses that the authors could incorporate to offer additional support to some of their claims (see weaknesses). In particular, this work would benefit from a more comprehensive comparison of the author's single recommender model vs an ensemble of specialist models, and the inclusion of 1-2 examples that showcase how their model could be translated into the clinic.

Strengths:

• A single AMR recommender system could potentially facilitate the adoption of MALDI-TOF-based antibiotic susceptibility profiling into clinical practices by reducing the number of models to be considered, and the efforts that may be required to periodically update them.

• Authors tested multiple combinations of embedders for the mass spectra and antibiotics while using different metrics to evaluate the performance of the resulting models. Models trained using different spectrum embedder-antibiotic embedder combinations had remarkably good performance for all tested metrics. The average ROC AUC scores for global and spectrum-level evaluations were above 0.9. Average ROC AUC scores for antibiotic-level evaluations were greater than 0.75.

• Authors showed that data collected in one location can be leveraged to improve the performance of models generated using a smaller number of samples collected at a different location. This result may encourage researchers to optimize data integration to reduce the burden of data generation for institutions interested in testing this method.

Weaknesses:

• Although ROC AUC is a widely used metric. Other metrics such as precision, recall, sensitivity, and specificity are not reported in this work. The last two metrics would help readers understand the model's potential implications in the context of clinical research.

• The authors did not hypothesize or describe in any way what an acceptable performance of their recommender system should be in order to be adopted by clinicians.

• Related to the previous comment, this work would strongly benefit from the inclusion of 1-2 real-life applications of their method that could showcase the benefits of their strategy for designing antibiotic treatment in a clinical setting.

• The authors do not offer information about the model features associated with resistance. This information may offer insights about mechanisms of antimicrobial resistance and how conserved they are across species.

• Comparison of AUC values across models lacks information regarding statistical significance. Without this information it is hard for a reader to figure out which differences are marginal and which ones are meaningful (for example, it is unclear if a difference in average AUC of 0.02 is significant). This applied to Figure 2, Figure 3, and Table 2 (and the associated supplementary figures).

• One key claim of this work was that their single recommender system outperformed specialist (single species-antibiotic) models. However, in its current status, it is not possible to determine that in fact that is the case (see comment above). Moreover, comparisons to species-level models (that combine all data and antibiotic susceptibility profiles for a given species) would help to illustrate the putative advantages of the dual branch neural network model over species-based models. This analysis will also inform the species (and perhaps datasets) for which specialist models would be useful to consider.

• Taking into account that the clustering of spectra embeddings seemed to be species-driven (Figure 4), one may hypothesize that there is limited transfer of information between species, and therefore the neural network model may be working as an ensemble of species models. Thus, this work would deeply benefit from a comparison between the authors' general model and an ensemble model in which the species is first identified and then the relevant species recommender is applied. If authors had identified cases to illustrate how data from one species positively influence the results for another species, they should include some of those examples.

Reviewer #1 (Public Review):

Summary:

This work extends previous agent-based models of murine muscle regeneration by the authors (especially Westman et al., 2021) and by others (especially Khuu et al, 2023) by incorporating additional agent rules (altogether now based on over 100 published studies), threshold parameters and interactions with fields of cytokines and growth factors as well as capillaries (dynamically changing through damage and angiogenesis) and lymphatic vessels. The estimation of 52 unknown parameters against three time courses of tissue-scale observables (muscle cross-sectional area recovery, satellite stem cell count and fibroblast cell count) employs the CaliPro algorithm (Joslyn et al., 2021) and sensitivity analysis. The model is validated against additional time courses of tissue-scale observables and qualitative perturbation data, which match almost all conditions. This model is here used to predict (also non-monotonic) responses of (combinations of) cytokine perturbations but it moreover represents a valuable resource for further analysis of emergent behavior across multiple spatial scales in a physiologically relevant system.

Strengths:

This work (almost didactically) demonstrates how to develop, calibrate, validate and analyze a comprehensive, spatially resolved, dynamical, multicellular model. Testable model predictions of (also non-monotonic) emergent behaviors are derived and discussed. The computational model is based on a widely-used simulation platform and shared openly such that it can be further analyzed and refined by the community. The single-used parameter set is a good starting point for future work that can, as outlined in the discussion section of the paper, analyze model results from the full distribution of matching parameter values and for a spectrum of realistic tissue configurations.

Reviewer #1 (Public Review):

Summary:

In this study, Komarova et al. investigate the clinical prognostic ability of cell-level metabolic heterogeneity quantified via the fluorescence lifetime characteristics of NAD(P)H. Fluorescence lifetime imaging microscopy (FLIM) has been studied as a minimally invasive approach to measure cellular metabolism in live cell cultures, organoids, and animal models. Its clinical translation is spearheaded through macroscopic implementation approaches that are capable of large sampling areas and enable access to otherwise constrained spaces but lack cellular resolution for a one-to-one transition with traditional microscopy approaches, making the interpretation of the results a complicated task. The merit of this study primarily lies in its design by analyzing with the same instrumentation and approach colorectal samples in different research scenarios, namely in vitro cells, in vivo animal xenografts, and tumor tissue from human patients. These conform to a valuable dataset to explore the translational interpretation hurdles with samples of increasing levels of complexity. For human samples, the study specifically investigates the prediction ability of NAD(P)H fluorescence metrics for the binary classification of tumors of low and advanced stage, with and without metastasis, and low and high grade. They find that NAD(P)H fluorescence properties have a strong potential to distinguish between high- and low-grade tumors and a moderate ability to distinguish advanced-stage tumors from low-stage tumors. This study provides valuable results contributing to the deployment of minimally invasive optical imaging techniques to quantify tumor properties and potentially migrate into tools for human tumor characterization and clinical diagnosis.

Strengths:

The investigation of colorectal samples under multiple imaging scenarios with the same instrument and approach conforms to a valuable dataset that can facilitate the interpretation of results across the spectrum of sample complexity.

The manuscript provides a strong discussion reviewing studies that investigated cellular metabolism with FLIM and the metabolic heterogeneity of colorectal cancer in general.

The authors do a thorough acknowledgement of the experimental limitations of investigating human samples ex vivo, and the analytical limitation of manual segmentation, for which they provide a path forward for higher throughput analysis.

Weaknesses:

To substantiate the changes in fluorescence properties at the examined wavelength range (associated with NAD(P)H fluorescence) in relationship to metabolism, the study would strongly benefit from additional quantification of metabolic-associated metrics using currently established standard methods. This is especially interesting when discussing heterogeneity, which is presumably high within and between patients with colorectal cancer, and could help explain the particularities of each sample leading to a more in-depth analysis of the acquired valuable dataset. Additionally, NAD(P)H fluorescence does not provide a complete picture of the cell/tissue metabolic characteristics. Including, or discussing the implications of including fluorescence from flavins would comprise a more compelling dataset. These additional data would also enable the quantification of redox metrics, as briefly mentioned, which could positively contribute to the prognosis potential of metabolic heterogeneity.

In the current form of the manuscript, there is a diluted interpretation and discussion of the results obtained from the random forest and SHAP analysis regarding the ability of the FLIM parameters to predict clinicopathological outcomes. This is, not only the main point the authors are trying to convey given the title and the stated goals, but also a novel result given the scarce availability of these type of data, which could have a remarkable impact on colorectal cancer in situ diagnosis and therapy monitoring. These data merit a more in-depth analysis of the different factors involved. In this context, the authors should clarify how is the "trend of association" quantified (lines 194 and 199).

Reviewer #1 (Public Review):

Summary:

The authors propose an improved neuro-muscle co-culture system to study ALS-related functional differences in human pluripotent stem cell lines.

Strengths:

A simple co-culture system with functional readout.

Weaknesses:

There are concerns about the lack of novelty, rigor, and clarity in the approach. The strength of the study is undermined by its reliance on transcription factors used more than a decade ago, low myocyte activity, and inadequate validation methods, such as the lack of single-cell transcriptome analysis and detailed neuromuscular synapse characterization. The evidence presented requires substantial validation through rigorous experimental approaches and resolution of the identified concerns for the study's findings to be considered significant and reliable.

for - sleep hygiene - 1 - regularity

advice - sleep hygiene - 1 - regularity - try to make weekend sleep times same as weekday - don't deviate if possible

Reviewer #1 (Public Review):

Summary:

The authors collected genomic information from public sources covering 423 eukaryote genomes and around 650 prokaryote genomes. Based on pre-computed CDS annotation, they estimated the frequency of alternative splicing (AS) as a single average measure for each genome and computed correlations with this measure and other genomic properties such as genome size, percentage of coding DNA, gene and intergenic span, etc. They conclude that AS frequency increases with genome complexity in a somewhat directional trend from "lower" organisms to "higher" organisms.

Strengths:

The study covers a wide range of taxonomic groups, both in prokaryotes and eukaryotes.

Weaknesses:

The study is weak both methodologically and conceptually. Current high throughput sequencing technologies, coupled with highly heterogeneous annotation methods, can observe cases of AS with great sensitivity, and one should be extremely cautious of the biases and rates of false positives associated with these methods. These issues are not addressed in the manuscript. Here, AS measures seem to be derived directly from CDS annotations downloaded from public databases, and do not account for differing annotation methods or RNA sequencing depth and tissue sample diversity.

There is no mention of the possibility that AS could be largely caused by random splicing errors, a possibility that could very well fit with the manuscript's data. Instead, the authors adopt early on the view that AS is regulated and functional, generally citing outdated literature.

There is no question that some AS events are functional, as evidenced by strongly supported studies. However, whether all AS events are functional is questionable, and the relative fractions of functional and non-functional AS are unknown. With this in mind, the authors should be more cautious in interpreting their data. The "complexity" of organisms also correlates well (negatively) with effective population size. The power of selection to eliminate (slightly) deleterious mutations or errors decreases with effective population size. The correlation observed by the authors could thus easily be explained by a non-adaptive interpretation based on simple population genetics principles.

The manuscript contains evidence that the authors might benefit from adopting a more modern view of how evolution proceeds. Sentences such as "... suggests that only sophisticated organisms optimize alternative splicing by increasing..." (L113), or "especially in highly evolved groups such as mammals" (L130), or the repeated use of "higher" and "lower" organisms need revising.

Because of the lack of controls mentioned above, and because of the absence of discussion regarding an alternative non-adaptive interpretation, the analyses presented in the manuscript are of very limited use to other researchers in the field. In conclusion, the study does not present solid conclusions.

Reviewer #1 (Public Review):<br /> <br /> Summary:

This paper provides a methodology for normative trajectory modeling, using cross-sectional data to set the "norms," and then applying these norms to longitudinal brain observations. An example of schizophrenia trajectories (two time points) is provided. The method assumes a Bayesian mixed effects model, which included some hyperparameters that need to be tuned. The longitudinal assumption is essentially a random intercept model, assuming that the age-based quantiles do not shift, and if they do that is a sign of disease-like trajectories.

Strengths:

Normative modeling of brain feature trajectories is an important topic. Bayesian models are a promising alternative to modeling these. Leveraging large-scale data to provide norms is also potentially useful.

Weaknesses:

The models described are not fundamentally novel, essentially a random intercept model (with a warping function), and some flexible covariate effects using splines (i.e., additive models). The assumption of constant quantiles is very strong, and limits the utility of the model to very short term data. The schizophrenia example leads to a counter-intuitive normalization of trajectories, which leads to suspicions that this is driven by some artifact of the data modeling/imaging pipelines. The method also assumes that the cross-sectional data is from a "healthy population" without describing what this population is (there is certainly every chance of ascertainment bias in large scale studies as well as small scale studies). This issue is completely elided over in the manuscript.

Reviewer #1 (Public Review):

The authors sought to determine the impact of early antiretroviral treatment on the size, composition, and decay of the HIV latent reservoir. This reservoir represents the source of viral rebound upon treatment interruption and therefore constitutes the greatest challenge to achieving an HIV cure. A particular strength of this study is that it reports on reservoir characteristics in African women, a significantly understudied population, of whom some have initiated treatment within days of acute HIV diagnosis. With the use of highly sensitive and current technologies, including digital droplet PCR and near full-length genome next-generation sequencing, the authors generated a valuable dataset for investigation of proviral dynamics in women initiating early treatment compared to those initiating treatment in chronic infection. The authors confirm previous reports that early antiretroviral treatment restricts reservoir size, but further show that this restriction extends to defective viral genomes, where late treatment initiation was associated with a greater frequency of defective genomes. Furthermore, an additional strength of this study is the longitudinal comparison of viral dynamics post-treatment, wherein early treatment was shown to be associated with a more rapid rate of decay in proviral genomes, regardless of intactness, over a period of one year post-treatment. While it is indicated that intact genomes were not detected after one year following early treatment initiation, caution should be taken with interpretation where sequence numbers are low. Defective genomes are more abundant than intact genomes and are therefore more likely to be sampled. Early treatment was also associated with reduced proviral diversity and fewer instances of polymorphisms associated with cytotoxic T-lymphocyte immune selection. This is expected given that rapid evolution and extensive immune selection are synonymous with HIV infection in the absence of treatment, yet points to an additional benefit of early treatment in the context of immune therapies to restrict the reservoir.

Given that this is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C, the manuscript would benefit from placing these findings in the context of what has been reported in other populations, for example, how decay rates of intact and defective genomes compare with that of other subtypes where known. While not a primary outcome of the study, the comparisons of peak viremia in the hyperacute and chronic-treated groups may be confounded by the fact that peak viremia may have been pre-empted by early treatment i.e., the true peak was not reached in early-treated individuals. Indeed, in the abstract, the authors indicate that treatment was initiated before the peak. The use of the term 'peak' viremia in the hyperacute-treated group could perhaps be replaced with 'highest recorded viral load'. The statistical comparison of this measure in the two groups is perhaps more relevant with regards to viral burden over time or area under the curve viral load as these are previously reported as correlates of reservoir size. The analysis of clonal expansion of proviral genomes may be limited by higher sequence homogeneity in hyperacute infection i.e., cells with different proviral integration sites may have a higher likelihood of containing identical genomes than chronic infection.

Overall, these data demonstrate the distinct benefits of early treatment initiation at reducing the barrier to a functional cure for HIV, not only by restricting viral abundance and diversity but also potentially through the preservation of immune function and limiting immune escape. It therefore provides clues to curative strategies even in settings where early diagnosis and treatment may be unlikely.

Reviewer #2 (Public Review):

Summary

In this work, Bartolome and colleagues develop a new approach to identify proteasome interacting proteins and substrates. The approach is based on fusing proteasome subunits with a biotin ligase that will label proteins that come in close physical distance of the ligase. These biotin-labeled proteins (or their resulting tryptic peptides) can be affinity purified using streptavidin and identified by mass spectrometry.

This elegant solution was able to identify a large proportion of known proteasome interactors, as well as multiple potential new interactors. Combining this approach with a proteasome inhibitor allowed also for the enrichment of substrates, due to increased contact time between substrates and the proteasome. Again, the authors were able to identify novel substrates. Finally, the authors implemented this strategy in vivo, providing the hints for potential tissue-specific proteasome interactors.<br /> This novel strategy provides an additional approach to identify new proteasome substrates, which can be particularly powerful for low abundant proteins, e.g., transcription factors. The possibility to implement it in vivo in specific cell types opens the possibility for identifying proteasome interactors in small cell subpopulations or in subpopulations involved in disease.

Strengths

The authors carefully characterized their genetically engineered proteasome-biotin ligase fusions to ensure that proteasome structure and activity was not altered. This is key to ensure that the proteins identified to interact with the proteasome reflect interactions that occur under physiological conditions.

The authors implemented an algorithm that controls the false positive rate of the identified interactors of the proteasome. This is an important aspect to avoid spending time on the characterization of potential interactors that are just an artifact of the experimental setup.

The addition of a proteasome inhibitor allowed the authors to identify substrates of the proteasome. Although there are other strategies to do this (e.g., affinity purification of Gly-Gly modified peptides, which is a marker for ubiquitination), this additional approach can highlight currently unknown substrates. One example are low abundance proteins, such as transcription factors.

The overall strategy developed by the authors can be implemented in vivo, which opens for the possibility of determining cell type-specific proteasome interactors (and perhaps substrates).

Weaknesses

There is a proportion (approximately 38%) of the PSMA4-biotin ligase fusion that remains unassembled (i.e., not part of the functional proteasome) and that can contribute to a small proportion of false positive interactions.

Reviewer #1 (Public Review):

Summary:

This paper by Gao et al. describes the effect of capsaicin on the NRF2/KEAP1 pathway. The authors carried out a set of in vitro experiments that addressed the mechanisms of the protective effect of capsaicin on ethanol-induced cytotoxicity. They also conducted in vivo studies in rats focusing on ethanol-induced gastric mucosal oxidative damage. The authors conclude that capsaicin activates NRF2, which leads to the induction of cytoprotective genes, preventing oxidative damage. This effect has already been shown, and it is well established that capsaicin activates NRF2, but what can be novel in the paper is the demonstration that capsaicin may directly bind to KEAP1 and that it is a noncovalent modification of the Kelch domain. The authors also designed new albumin-coated capsaicin nanoparticles, which were tested for the therapeutic effect in vivo. Apart from novelty concerns, the manuscript may be potentially interesting, but in my opinion, it is not fully technically sound, which weakens the strength of the evidence.

Major concerns:

For studies investigating capsaicin binding to KEAP1, the authors used capsaicin concentrations that are toxic to cells (Figures S1D and 4F, G). In vivo studies were performed only in 3 rats per group. The T-test was used for the comparison of more than two groups. Given the well-known issues with the specificity of the NRF2 antibody, the authors should provide appropriate controls, especially for IF and IHC staining.

Reviewer #1 (Public Review):

The authors build on their previous study that showed the midgut microbiome does not oscillate in Drosophila. Here, they focus on metabolites and find that these rhythms are in fact microbiome-dependent. Tests of time-restricted feeding, a clock gene mutant, and diet reveal additional regulatory roles for factors that dictate the timing and rhythmicity of metabolites. The study is well-written and straightforward, adding to a growing body of literature that shows the time of food consumption affects microbial metabolism which in turn could affect the host.

Some additional questions and considerations remain:

(1) The main finding that the microbiome promotes metabolite rhythms is very interesting. Which microbiota are likely to be responsible for these effects? The author's previous work in this area may shed light on this question. Are specific microbiota linked to some of the metabolic pathways investigated in Figure 5?

(2) TF increases the number of rhythmic metabolites in both microbiome-containing and abiotic flies in Figure 1. This is somewhat surprising given that flies typically eat during the daytime rather than at night, very similar to TF conditions. I would have assumed that in a clock-functioning animal, the effect of restricting food availability should not make a huge difference in the time of food consumption, and thus downstream impacts on metabolism and microbiome. Can the authors measure food intake directly to compare the ad-lib vs TF flies to see if there are changes in food intake? Would restricting feeding to other times of day shift the timing of metabolites accordingly?

(3) In Figure 2, Per loss of function reveals a change in the phase of rhythmic metabolites. In addition, the effect of the microbiome on these is very different = The per mutants show increased numbers of rhythmic metabolites when the microbiome is absent, unlike the controls. Is it possible that these changes are due to altered daily feeding rhythms in per mutants? Testing the time and amount of food consumed by the per mutant flies would address this question. Would TF in the per mutants rescue their metabolite rhythms and make them resemble clock-functioning controls?

(4) The calorie content of each diet - normal vs high protein vs high-sugar are different. The possibility of a calorie effect rather than a difference in nutrition (protein/carbohydrate) should be discussed. Another issue worth considering is the effect of high protein/sugar on the microbiome itself. While the microbiome doesn't seem to affect rhythms in the high-protein diet, the high-sugar diet seems highly microbiome-dependent in Supplementary Fig 8C vs D. Does the diet impact the microbiome and thus metabolite rhythmicity downstream?

(5) It would be good if a supplementary table was provided outlining the specific metabolites that are shown in the radial plots. It is not clear if the rhythms shown in the figures refer to the same metabolites peaking at the same time, or rather the overall abundance of completely different metabolites. This information would be useful for future research in this area.

Reviewer #1 (Public Review):

Summary:

In this work by Wang et al., the authors use single-molecule super-resolution microscopy together with biochemical assays to quantify the organization of Nipah virus fusion protein F (NiV-F) on cell and viral membranes. They find that these proteins form nanoscale clusters which favors membrane fusion activation, and that the physical parameters of these clusters are unaffected by protein expression level and endosomal cleavage. Furthermore, they find that the cluster organization is affected by mutations in the trimer interface on the NiV-F ectodomain and the putative oligomerization motif on the transmembrane domain, and that the clusters are stabilized by interactions among NiV-F, the AP2-complex, and the clathrin coat assembly. This work improves our understanding of the NiV fusion machinery, which may have implications also for our understanding of the function of other viruses.

Strengths:

The conclusions of this paper are well-supported by the presented data. This study sheds light on the activation mechanisms underlying the NiV fusion machinery.

Weaknesses:

The authors provide limited details of the convolutional neural network they developed in this work. Even though custom-codes are made available, a description of the network and specifications of how it was used in this work would aid the readers in assessing its performance and applicability. The same holds for the custom-written OPTICS algorithm. Furthermore, limited details are provided for the imaging setup, oxygen scavenging buffer, and analysis for the single-molecule data, which limits reproducibility in other laboratories. The claim of 10 nm resolution is not backed up by data and seems low given the imaging conditions and fluorophores used. Fourier Ring Correlation analysis would have validated this claim. If the authors refer to localization precision rather than resolution, then this should be specified and appropriate data provided to support this claim.

Reviewer #1 (Public Review):

Summary:

The study "Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling" by Senem Ntourmas et al. contributes to the understanding of phase separation in Dishevelled (DVL) proteins, specifically focusing on DVL2. It builds upon existing research by investigating the endogenous complexes of DVL2 using ultracentrifugation and contrasting them with DVL1 and DVL3 behavior. The study identifies a DVL2-specific region involved in condensate formation and introduces the "two-step" concept of DVL2 condensate formation, enriching the field's knowledge.

Strengths:

A notable strength of this study is the validation of endogenous DVL2 complexes, providing insights into its behavior compared to DVL1 and DVL3. The functional validation of the DVL C-terminus (here termed conserved domain 2 (CD2) and the identification of DVL2-specific regions (here termed LCR4) involved in condensate formation are significant contributions that complement the current knowledge on the importance of DVL DIX domain, DEP domain and intrinsically disordered regions between DIX and PDZ domains. Additionally, the introduction of the concept where oligomerization (step 1) precedes condensate formation (step 2) is an interesting hypothesis, which can be further experimentally challenged in the future.

Weaknesses:

However, the applicability of the findings to full-length DVL2 protein, hence the physiological relevance, is limited. This is mostly due to the fact that the authors almost completely depend on the set of DVL2 mutants, which lack the (i) DEP domain and (ii) nuclear export signal (NES). These variants fail to establish DEP domain-mediated interactions, including those with FZD receptors. Of note, the DEP domain itself represents a dimerization/tetramerization interface, which could affect the protein condensate formation of these mutants. Possibly even more importantly, the used mutants localize into the nucleus, which has different biochemical & biophysical properties than a cytoplasm, where DVL typically reside, which in turn affects the condensate formation. On top, in the nucleus, most of the DVL binding partners, including relevant kinases, which were reported to affect protein condensate formation, are missing.

Second, the use of an overexpression system, while suitable for comparing DVL2 protein condensate features, falls short in functional assays. The study could benefit from employing established "rescue systems" using DVL1/2/3 knockout cells and re-expression of DVL variants for more robust functional assessments.

Furthermore, the discussion and introduction overlook some essential aspects of DVL biology. One such example is the importance of the open/close conformation of DVL and its effects on DVL phase separation and activity. In the context of this study, it is important to say that this conformational plasticity is mediated by DVL C-terminus (CD2 in this study). The second example is the reported roles of DVL1 and DVL3, which can both mediate the Wnt3a signal. How this can be interpreted when DVL1 and DVL3 lack LCR4 and still form condensates?

In order to increase the physiological relevance of the study, I would recommend analyzing several key mutants in the context of the full-length DVL2 protein using the rescue/complementation system. Further, a more thorough discussion and connections with the existing literature on DVL protein condensates/puncta/LLPS can improve the impact of the study.

Reviewer #1 (Public Review):

Summary:

In this study, a chromosome-level genome of the rose-grain aphid M. dirhodum was assembled with high quality, and A-to-I RNA-editing sites were systematically identified. The authors then demonstrated that: 1) Wing dimorphism induced by crowding in M. dirhodum is regulated by 20E (ecdysone signaling pathway); 2) an A-to-I RNA editing prevents the binding of miR-3036-5p to CYP18A1 (the enzyme required for 20E degradation), thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring.

Strengths:

The authors present both genome and A-to-I RNA editing data. An interesting finding is that a A-to-I RNA editing site in CYP18A1 ruin the miRNA binding site of miR-3036-5p. And loss of miR-3036-5p regulation lead to less 20E and winged offspring.

Weaknesses:

How crowding represses the miR-3036-5p is still unclear.

Reviewer #1 (Public Review):

This manuscript presents an extremely exciting and very timely analysis of the role that the nucleosome acidic patch plays in SWR1-catalyzed histone exchange. Intriguingly, SWR1 loses activity almost completely if any of the acidic patches are absent. To my knowledge, this makes SWR1 the first remodeler with such a unique and pronounced requirement for the acidic patch. The authors demonstrate that SWR1 affinity is dramatically reduced if at least one of the acidic patches is absent, pointing to a key role of the acidic patch in SWR1 binding to the nucleosome. The authors also pinpoint a specific subunit - Swc5 - that can bind nucleosomes and engage the acidic patch and obtain a cryo-EM structure of Swc5 bound to a nucleosome. They also identify a conserved arginine-rich motif in this subunit that is critical for nucleosome binding and histone exchange in vitro and for SWR1 function in vivo. The authors provide evidence that suggests a direct interaction between this motif and the acidic patch.

Strengths:

The manuscript is well-written and the experimental data are of outstanding quality and importance for the field. This manuscript significantly expands our understanding of the fundamentally important and complex process of H2A.Z deposition by SWR1 and would be of great interest for a broad readership.

Reviewer #1 (Public Review):

Summary:

Zhao and colleagues employ Drosophila nephrocytes as a model to investigate the effects of a high-fat diet on these podocyte-like cells. Through a highly focused analysis, they initially confirm previous research in their hands demonstrating impaired nephrocyte function and move on to observe the mislocalization of a slit diaphragm-associated protein (pyd). Employing a reporter construct, they identify the activation of the JAK/STAT signaling pathway in nephrocytes. Subsequently, the authors demonstrate the involvement of this pathway in nephrocyte function from multiple angles, using a gain-of-function construct, silencing of an inhibitor, and ectopic overexpression of a ligand. Silencing the effector Stat92E via RNAi or inhibiting JAK/STAT with Methotrexate effectively restored impaired nephrocyte function induced by a high-fat diet, while showing no impact under normal dietary conditions.

Strengths:

The findings establish a link between JAK/STAT activity and the impact of a high-fat diet on nephrocytes. This nicely underscores the importance of organ crosstalk for nephrocytes and supports a potential role for JAK/STAT in diabetic nephropathy, as previously suggested by other models.

Weaknesses:

The analysis is overly reliant on tracer endocytosis and single lines. Immunofluorescence of slit diaphragm proteins would provide a more specific assessment of the phenotypes.

Reviewer #1 (Public Review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium-activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well-characterized calcium-activated lipid scramblase that plays an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study, the group demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium-activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between the TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

The electrophysiology data does not entirely support their claims.

Reviewer #1 (Public Review):

Using a combination of cutting-edge high-resolution approaches (expansion microscopy, SIM, and CLEM) and biochemical approaches (in vitro translocation of actin filaments, cargo uptake assays, and drug treatment), the authors revisit previous results about TbMyo1 and TbACT in the bloodstream form (BSF) of Trypanosoma brucei. They show that a great part of the myosin motor is cytoplasmic but the fraction associated with organelles is in proximity to the endosomal system. In addition, they show that TbMyo1 can move actin filaments in vitro and visualize for the first time this actomyosin system using specific antibodies, a "classical" antibody for TbMyo1, and a chromobody for actin. Finally, using latrunculin A, which sequesters G-actin and prevents F-actin assembly, the authors show the delocalization and eventually the loss of the filamentous actin signal as well as the concomitant loss of the endosomal system integrity. However, they do not assess the localization of TbMyo1 in the same conditions.

Overall the work is well conducted and convincing. The conclusions are not over-interpreted and are supported by the experimental results.

Reviewer #1 (Public Review):

Summary:

The authors profile gene expression, chromatin accessibility, and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T-cell activation.

This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as exploring the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.

Suggestions for improvement:

Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.

The obvious question that the authors don't venture into is why the results are quite different. In principle, this could be due to the differences between:<br /> (a) the cell stimulation procedure<br /> (b) the GWAS datasets used<br /> (c) the types of assay (Hi-C vs Capture Hi-C)<br /> (d) approaches for defining gene-linked regions (loops vs neighbourhoods)<br /> (e) how the GWAS signals at gene-linked regions are aggregated (e.g., the flavours of COGS in Javierre and Burren vs the authors' approach).

Re (a), I'm not sure the authors make it explicitly clear in the main text that the Capture Hi-C-based studies also use *stimulated* CD4 T cells, particularly in the section "Comparative predictive power...". So the cells used are pretty much the same, and the differences likely arise from points (b) to (e).

It would be useful for the community to understand more clearly what is driving these differences, ideally with some added data. Could the authors, for example, take the PCHi-C data from Javierre/Burren and use their GWAS data and variant-to-gene assignment algorithms?

In addition, given that the authors use Hi-C, a popular method for V2G prioritisation for this type of data is currently ABC (Nasser et al, Nature 2021). Could the authors provide a comparative analysis with respect to the V2G assignments in the paper and, if they see it appropriate, also run ABC-based GWAS integration on their own Hi-C data?

Reviewer #1 (Public Review):

In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of  IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.

Strengths:

This study utilizes multiple biochemical, proteomic, functional and cell biology assays to substantiate their results.  Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex- it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.

Limitations/Weaknesses:

The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:

(1) Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation ( in Fig 3A),  cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies. <br /> (2) The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggests that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well. <br /> (3) The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:<br /> - It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.<br /> -It may be of benefit to repeat the Phos tag experiments with full length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating size of migrated bands.<br /> -Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.<br /> -Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK. <br /> -It is unclear why the lower, phosphorylated bands seem to be increasing ( rather than decreasing) with AA starvation/ Rapa in Fig 5H.

Reviewer #1 (Public Review):

Summary:

The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated. Still, the impact of their results builds on recent structural studies and the stated impact is based on these prior works.

Strengths:

(1) After reading through the paper, the major findings are:<br /> - RING2 and pUbl compete for binding to RING0.<br /> - Parkin can dimerize.<br /> - ACT plays an important role in enzyme kinetics.

(2) The use of molecular scissors in their construct represents a creative approach to examining inter-domain interactions.

(3) From my assessment, the experiments are well-conceived and executed.

Weaknesses:

(1) The manuscript, as written, is NOT for a general audience. Admittedly, I am not an expert on Parkin structure and function, but I had to do a lot of homework to try to understand the underlying rationale and impact. This reflects, I think, that the work generally represents an incremental advance on recent structural findings.

(2) To this point, it is hard to understand the impact of this work without more information highlighting the novelty. There are several structures of Parkin in various auto-inhibited states, and it was hard to delineate how this is different.

(3) As noted, I appreciated the use of protease sites in the fusion protein construct. It is unclear how the loop region might affect the protein structure and function. The authors worked to demonstrate that this did not introduce artifacts, but the biological context is missing.

(4) While it is likely that the binding is competitive between the Ubl and RING2 domains, the data is not quantitative. Is it known whether the folding of the distinct domains is independent? Or are there interactions that alter folding? It seems plausible that conformational rearrangements may invoke an orientation of domains that would be incompatible. The biological context for the importance of this interaction was not clear to me.

(5) What is the rationale for mutating Lys211 to Asn? Were other mutations tried? Glu? Ala? Just missing the rationale. I think this may have been identified previously in the field, but not clear what this mutation represents biologically.

(6) I was confused about how the phospho-proteins were generated. After looking through the methods, there appear to be phosphorylation experiments, but it is unclear what the efficiency was for each protein (i.e. what % gets modified). In the text, the authors refer to phospho-Parkin (T270R, C431A), but not clear how these mutations might influence this process. I gather that these are catalytically inactive, but it is unclear to me how this is catalyzing the ubiquitination in the assay.

(7) The authors note that "ACT can be complemented in trans; however, it is more efficient in cis", but it is unclear whether both would be important or if the favored interaction is dominant in a biological context.

(8) The authors repeatedly note that this study could aid in the development of small-molecule regulators against Parkin to treat PD, but this is a long way off. And it is not clear from their manuscript how this would be achieved. As stated, this is conjecture.

Reviewer #1 (Public Review):

Summary:

In this paper, Bruter and colleagues report the effects of inducible deletion of the genes encoding the two paralogous kinases of the Mediator complex in adult mice. The physiological roles of these two kinases, CDK8 and CDK19, are currently rather poorly understood; although conserved in all eukaryotes, and among the most highly conserved kinases in vertebrates, individual knockouts of genes encoding CDK8 homologues in different species have revealed generally rather mild and specific effects, in contrast to Mediator itself. Here, the authors provide evidence that neither CDK8 nor CDK19 are required for adult homeostasis but they are functionally redundant for maintenance of reproductive tissue morphology and fertility in males.

Strengths:

The morphological data on the atrophy of the male reproductive system and the arrest of spermatocyte meiosis are solid and are reinforced by single-cell transcriptomics data, which is a challenging technique to implement in vivo. The main findings are important and will be of interest to scientists in the fields of transcription and developmental biology.

Weaknesses:

There are several major weaknesses.

The first is that data on the general health of mice with single and double knockouts is not shown, nor is there any data on effects in any other tissues. This gives the impression that the only phenotype is in the male reproductive system, which would be misleading if there were phenotypes in other tissues that are not reported. Furthermore, data for the genitourinary system in single knockouts are very sparse; data are described for fertility in Figure 1H, ploidy, and cell number in Figures 2B and C, plasma testosterone and luteinizing hormone levels in Figures 5C and 5D, and morphology of testis and prostate tissue for single Cdk8 knockout in Supplementary Figure 1C (although in this case the images do not appear very comparable between control and CDK8 KO, thus perhaps wider fields should be shown), but, for example, there is no analysis of different meiotic stages or of gene expression in single knockouts. It is worth mentioning that single knockouts seem to show a corresponding upregulation of the level of the paralogue kinase, indicating that any lack of phenotypes might be due to feedback compensation, which would be an interesting finding if confirmed; this has not been mentioned.

The second major weakness is that the correlation between double knockout and reduced expression of genes involved in steroid hormone biosynthesis is portrayed as a causal mechanism for the phenotypes observed. While this is a possibility, there are no experiments performed to provide evidence that this is the case. Furthermore, there is no evidence showing that CDK8 and/or CDK19 are directly responsible for the transcription of the genes concerned.

Finally, the authors propose that the phenotypes are independent of the kinase activity of CDK8 or CDK19 because treatment of mice for a month with an inhibitor does not recapitulate the effects of the knockout, and nor does expression of two steroidogenic genes change in cultured Leydig cells upon treatment with an inhibitor. However, there are no controls for effective target inhibition shown.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

(1) Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> (2) Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Reviewer #1 (Public Review):

Summary:

The authors present a mean-field model that describes the interplay between (protein) aggregation and phase separation. Different classes of interaction complexity and aggregate dimensionality are considered, both in calculations concerning (equilibrium) phase behavior and kinetics of assembly formation.

Strengths:

The present work is, although purely theoretical, of high interest to understanding biological processes that occur as a result of a coupling between protein aggregation and phase separation. Of course, such processes are abundant, in the living cell as well as in in-vitro experiments. I appreciate the consideration of aggregates with various dimensionality, as well as the categorization into different "interaction classes", together with the mentioning of experimental observations from biology. The model is convincing and underlines the complexity associated with the distribution of proteins across phases and aggregates in the living cell.

Weaknesses:

There are a few minor weaknesses.

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as an increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP levels. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Comments on revised submission:

The authors have addressed my comments adequately, actually except for all but one. I have only one comment to do with the last line of the abstract. First, "genetic manipulation" usually means changing DNA. In Mtb pathogenesis I hope there is no DNA modification or change in the bacterial DNA. Also, the authors did not really inactivate the whole PhoP- rv0805-cAMP pathway. It would be best if the last line is made more fact based: Thus, inactivation of PhoP decreases cAMP level, thereby stress tolerance and intracellular survival of the bacillus.

Reviewer #1 (Public Review):

This work presents CTFFIND5, a new version of the software for determination of the Contrast Transfer Function (CTF) that models the distortions introduced by the microscope in cryoEM images. CTFFIND5 can take acquisition geometry and sample thickness into consideration to improve CTF estimation.

To estimate tilt (tilt angle and tilt axis), the input image is split into tiles and correlation coefficients are computed between their power spectra and a local CTF model that includes the defocus variation according to a tilted plane. As a final step, by applying a rescaling factor to the power spectra of the tiles, an average tilt-corrected power spectrum is obtained and used for diagnostic purposes and to estimate the goodness of fit. This global procedure and the rescaling factor resemble those used in Bsoft, Warp, etc, with determination of the tilt parameters being a feature specific of CTFFIND5 (and formerly CTFTILT). The performance of the algorithm is evaluated with tilted 2D crystals and tilt-series, demonstrating accurate tilt estimation in some cases and some limitations in others. Further analysis of CTF determination with tilt-series, particularly showing whether there is accurate or stable estimation at high tilts, might be helpful to show the robustness of CTFFIND5 in cryoET.

CTFFIND5 represents the first CTF determination tool that considers the thickness-related modulation envelope of the CTF firstly described by McMullan et al. (2015) and experimentally confirmed by Tichelaar et al. (2020). To this end, CTFFIND5 uses a new CTF model that takes the sample thickness into account. CTFFIND5 thus provides more accurate CTF estimation and, furthermore, gives an estimation of the sample thickness, which may be a valuable resource to judge the potential for high resolution. To evaluate the accuracy of thickness estimation in CTFFIND5, the authors use the Lambert-Beer law on energy-filtered data and also tomographic data, thus demonstrating that the estimates are reasonable for images with exposure around 30 e/A2. While consideration of sample thickness in CTF determination sounds ideally suited for cryoET, practical application under the standard acquisition protocols in cryoET (exposure of 3-5 e/A2 per image) is still limited. In this regard, the authors are honest in the conclusions and clearly identify the areas where thickness-aware CTF determination will be valuable at present: e.g. in situ single particle analysis and in vitro single particle cryoEM of purified samples at low voltages.

In conclusion, the manuscript introduces novel methods inside CTFFIND5 that improve CTF estimation, namely acquisition geometry and sample thickness. The evaluation demonstrates the performance of the new tool, with fairly accurate estimates of tilt axis, tilt angle and sample thickness and improved CTF estimation. The manuscript critically defines the current range of application of the new methods in cryoEM.

Reviewer #1 (Public Review):

Summary:

The present work from Velloso and collaborators investigated the transcription profiles of resident and recruited hypothalamic microglia. They found sex-dependent differences between males and females and identified the protective role of chemokine receptor CXCR3 against diet-induced obesity.

Strengths:

(1) Novelty<br /> (2) Relevance, since this work provides evidence about a subset of recruited microglia that has a protective effect against DIO. This provides a new concept in hypothalamic inflammation and obesity.

Weaknesses:

(1) Lack of mechanistic insight into the sex-dependent effects.<br /> (2) Analysis of indirect calorimetry data requires more depth.<br /> (3) A deeper analysis of hypothalamic inflammation and ER stress pathways would strengthen the manuscript.

Reviewer #1 (Public Review):

Summary:

This is a follow-up study to the authors' previous eLife report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:

Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

Joint Public Review:

In their revised manuscript additional experiments have been conducted competently, and the interpretation of experiments regarding exit from the ER are convincing. They collectively indicate that the phase partitioning behaviour of the TMDs do not have a significant effect on exit from the ER; they all exit the ER very slowly unless they carry a short cytoplasmic domain from LAT which is sufficient to accelerate ER exit. This data is consistent with available literature supporting a role for a ER-exit signal. Along with new experiments in their revision, they have also toned down the assertion that their data rule out a phase partitioning mechanism at the ER.

The authors, however, continue to interpret their experiments regarding Golgi exit of the transmembrane peptides (with luminal and cytoplasmic domains) as conclusive evidence of the role of lipid rafts in exit from the Golgi. This is once again based on correlation of the phase partitioning behaviour of these proteins in GPMVs, phase separated at low temperatures. They argue that this represents very strong evidence that trafficking out of the Golgi is driven by phase separation. The reviewers consider that there are a number of potential issues with the final model that need to be addressed.

We reiterate that:

(1) the phase segregation in the GPMV at low temperatures is dictated by thermodynamics given its composition and the measurement temperature. However at physiological temperatures at which membrane trafficking is taking place these GPMVs will not exhibit phase separation. Hence it is difficult to argue that a sorting mechanism based solely on the partitioning of the synthetic TM constructs into liquid ordered domain detected at low temperatures in GPMVs provide an explanation of the explanation of the differential kinetics of traffic of the LAT TMD and the allL-TMD constructs, although there is a strong correlation with its phase partitioning behaviour.

(2) The fluctuations of lipid composition resembling lo-domains if persisting at higher temperatures and its conversion into a sorting domain will require a cellular mechanism, that may or may not retain similar properties of these lipidic environments. Additionally, TMDs from TfR/VsVG and GPI prefer different domains in the GPMV assays (Table S1) yet they traffic to the cell surface equally rapidly.

(3) The authors fail to discuss the point raised about the relatively high colocalization of TfR with the GPI probe (seen in Fig 5E) in the Golgi. This is inconsistent with their explanation of traffic correlating with partitioning into distinct domains in the Golgi, since TfR and GPI probes show an opposite preference for lo versus ld domains in cooled GPMVs. TMD-allL and the LAT-allL are segregated from TfR in the Golgi, and end up in a different final destination (ie lysosomes). This could represent yet another membrane specialisation in the Golgi for lysosomal traffic. The segregation that the authors report in the Golgi is therefore not a convincing argument for phase preferences in GPMVs dictating the trafficking behaviour of these molecules towards the plasma membrane.

(4) Despite the authors' claim in their rebuttal that 'we feel that GPMVs are a useful tool for quantifying protein preference for ordered (raft) membrane domains and that this preference is a useful proxy for the raft-associated behavior ... biological membrane with a relevant and measurable cellular outcome, specifically inter-organelle trafficking rates." -several caveats for these observations need to be addressed before they constitute strong evidence for the raft model of membrane trafficking proposed. Phase partitioning in GPMVs is just another operational definition and while more refined (ie the data is derived from the membrane of interest, ie, the plasma membrane) it is not very different conceptually from quantitative measurements of detergent-insolubility.

(5) Further work is necessary to establish that ordered domains are formed at the Golgi at physiological temperatures, into which these proteins may partition; subsequently, there must be a mechanism that selectively traffics these proteins towards the cell surface.

(6) The authors continue to conflate thermodynamic phase separation mechanisms with the real possibility of the formation of functional sorting domains by cellular mechanisms that likely involve lipidic interactions, adding to the confusion in the literature.

Reviewer #1 (Public Review):

Summary:

This manuscript describes the identification and isolation of several phage from deep sea isolates of Lentisphaerae strains WC36 and zth2. The authors observe induction of several putative chronic phages with the introduction of additional polysaccharides to the media. The authors suggest that two of the recovered phage genomes encode AMGs associated with polysaccharide use. The authors also suggest that adding the purified phage to cultures of Pseudomonas stutzeri 273 increased the growth of this bacteria due to augmented polysaccharide use genes from the phage.

While the findings were of interest and relevance to the field, it is my opinion that several of the analysis fall short of supporting the key assertions presented.

Strengths:

Interesting isolate pf deep sea Lentisphaerae strains which will undoubtedly further our understanding of deep sea microbial life.

Weaknesses:

Many of the findings are consistent with a phage contamination in the polysaccharide stock solution.

The genes presented as AMGs are largely well known and studied phage genes which play a role in infection cycles.

The evidence that the isolated phage can infect Pseudomonas stutzeri 273 is lacking, putting into question the dependent results.

Reviewer #1 (Public Review):

This manuscript examines the individual and dual effects of CHIP and LOY in MI employing a cohort of ~460 individuals. CHIP is assessed by NGS and LOY is assessed by PCR. The threshold for CHIP is set at 2% (an arbitrary cutoff that is often used) and LOY at 9% (according to the Discussion text - this reviewer may have missed the section that describes why this threshold was employed). The investigation assessed whether LOY could modulate inflammation, atherosclerotic burden, or MI risk associated with CHIP. Neither CHIP nor LOY independently affected hsCRP, atherosclerotic burden, or MI incidence, nor did LOY presence diminish these outcomes in CHIP+ male subjects.

This study represents the first dual analysis of CHIP and LOY on CVD outcomes. The results are largely negative, contradictory to other studies (many with much larger sample sizes). I would attribute the limitation of sample size as a major contributor to the negative data. While the negative data are suspect, the "positive" finding that LOY abolishes the prognostic significance of CHIP on MI is of interest (and consistent with what is understood from mechanistic studies).

Overall, I enjoyed reading the paper, and it is of interest to the research community. However, I disagree with some of the authors' interpretations of the data. Generally, many conclusions on CHIP interpretation are based on the comparison of findings from very large datasets that have been evaluated by shallow NGS DNA sequencing. These studies lack sensitivity and accuracy, but this is counterbalanced by their very large sample sizes. Thus, they draw conclusions from the sickest individuals (ICD codes) with the largest clones (explaining the 10% VAF threshold). Here, the study has a well-phenotyped cohort, but as far as this reviewer can tell, the DNA sequencing is "shallow" NGS. Typically, to assess smaller datasets, investigators employ an error-correction method (DNA barcodes, duplex sequencing, etc.) for the sensitivity and accuracy of calling variants. Thus, the current study appears to suffer from this limitation (small sample sizes combined with NGS).

While the "negative" data from this study are inconclusive, the positive data (i.e. CHIP being prognostic for MI in the absence but not presence of MI) is of interest. Thus, the investigators may want to consider a shorter report that largely focuses on this finding.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

Strengths:

The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

Weaknesses:

The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice. Only a few experiments were performed using female mice, therefore, more experiments should be performed to complete the story of the role of BEND2 on female fertility. In addition, the title and abstract of the manuscript do not align with the story, as female fertility is only a small portion of the data compared to the male fertility section.

Reviewer #1 (Public Review):

This study by Porter et al reports on outcomes from a small, open-label, pilot randomized clinical trial comparing dornase-alfa to best available care in patients hospitalized with COVID-19 pneumonia. As the number of randomized participants is small, investigators describe also a contemporary cohort of controls and the study concludes with decrease of inflammation (reflected by CRP levels) after 7 days of treatment but no other statistically significant clinical benefit.

I read with interest this manuscript and I find the idea about treatment of COVID-19 patients with dornase-alfa novel and inspiring. I have some major concerns about the methodology the authors followed in this RCT.

My major concerns are:

(1) The authors have chosen a primary outcome that cannot be at least considered as clinically relevant or interesting. After 3 years of the pandemic with so much research, why investigate if a drug reduces CRP levels as we already have marketed drugs that provide beneficial clinical outcomes such as dexamethasone, anakinra, tocilizumab and baricitinib.

(2) ΙΤΤ analysis is not followed

Reviewer #1 (Public Review):

I have reviewed, with interest, the manuscript "Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast". The described findings are relevant and useful for daily practice in periodontology. The paper is concise, professionally written, and easy to read. In this study, Jiayao et al. revealed the role of miR-335-3p in psychological stress-induced osteoporosis. CUMS mice were constructed to observe the femur phenotype, osteoclasts were identified as the primary research object, and miRNA-seq was used to find the key miRNAs linking the brain and peripheral tissues. This study showed that the expression of miR-335-3p was simultaneously reduced in mice's NAC, serum, and bone under psychological stress. The miR-335-3p/Fos/NFATC1 signaling pathway was validated in osteoclasts to reveal the potential mechanism of enhanced osteoclast activity under psychological stress. From a new perspective of miRNAs, this study indicates a possible cause of disturbed bone metabolism due to psychological stress and may suggest a new approach to treating osteoporosis.

Reviewer #2 (Public Review):

The authors analysed functional MRI recordings of brain activity at rest, using state-of-the-art methods that reveal the diverse ways in which information can be integrated in the brain. In this way, they found brain areas that act as (synergistic) gateways for the 'global workspace', where conscious access to information or cognition would occur, and brain areas that serve as (redundant) broadcasters from the global workspace to the rest of the brain. The results are compelling and are consistent with the already assumed role of several networks and areas within the Global Neuronal Workspace framework. Thus, in a way, this work comes to stress the role of synergy and redundancy as complementary information processing modes, which fulfill different roles in the bigger context of information integration.

In addition, to prove that the identified high-order interactions are relevant to the phenomenon of consciousness, the same analysis was performed in subjects under anesthesia or with disorders of consciousness (DOC), showing that indeed the loss of consciousness is associated with a deficient integration of information within the gateway regions.

Reviewer #1 (Public Review):

Existing literature suggests that brain structures implicated in memory such as the hippocampus, and reward/punishment processing such as the striatal regions are also engaged in learning and value-based decision-making. However, how the contributions of these regions to learning and value-based decision-making change over time, particularly in children where these neural systems show protracted maturation was not studied systematically. This is the question the authors are aiming to address in this work in which children 6-to-7-years-old were recruited for a neuroimaging study that involves taking structural scans from this cohort to investigate how they correlate with changes in the way children approach a reinforcement learning task in which they learn to identify the better shape between 2 options through trial-and-error.

Particular strengths of the paper are longitudinally following up a cohort of small children and engaging them in a value-based decision-making task so that the relationship between neural maturation and improvements in reinforcement learning can be studied reliably. Towards this end, the authors make use of well-established computational modelling approaches to extract key parameters such as learning rates (which designate the speed of learning from expected versus actual outcomes) or choice stochasticity (which designate the inherent variation in people's decisions and the tendency to explore between the options) from children's choices so that their structural neural correlates can be established. As a part of this endeavour, the authors rely on methodological choices which do not warrant much criticism. Their data visualization choices are particularly spot-on and highly informative about the details of the raw data.

Also considering the importance of the hippocampal system in human memory, the key contribution of the paper is that the volumetric increases in hippocampus size between 2 assessment points correlated selectively with the delayed, but not immediate, learning score which refers to the learning condition in which the outcome feedback is given to the children after a 5-seconds delay. Although the authors also demonstrate evidence to suggest that changes in the striatal volume are also implicated in learning performance, this was more general as associations were found for both immediate and delayed feedback conditions. Thus, the paper makes an important contribution to the fields of developmental and decision neuroscience. An important question arising from the authors' findings could be that, whether the hippocampus maintains this selective role in value-based learning during the course of neuronal development, for example, whether a similar association would be found in children 8-to-9 years old. A better understanding of how these developmental trajectories map onto changes in learning and decision-making can inform fields outside neuroscience, for example tailoring educational approaches onto neural development pathways to boost learning efficiency in young children.

Reviewer #1 (Public Review):

Summary:

The authors goal here was to explore how a non hebbian form of plasticity, heterosynaptic LTP, could shape neuronal responses and learning. They used several conceptually and technically innovative approaches to answer this. First, they identified a behavioral paradigm that was a subthreshold training paradigm (stimulation of thalamic inputs with a footshock), which could be 'converted' to a memory via homosynaptic LTP (HFS of thalamic inputs). They then find that stimulation of 'cortical' inputs could also convert the subthreshold stimulation to a lasting memory, and that this was associated with a change in neuronal response, akin to LTP. Finally, they provide some slice work which demonstrated that stimulation of cortical inputs could stabilize LTP at thalamic inputs.

Strengths:

(1) The approach was innovative and asked an important question in the field.

(2) The studies are, for the most part, quite rigorous, using a novel dual opsin approach to probe multiple inputs in vivo.

(3) The authors explore neural responses both in vivo and ex vivo, as well as leveraging a 'simple' behavior output of freezing.

Reviewer #1 (Public Review):

Summary:

Li and colleagues describe an experiment whereby sequences of dots in different locations were presented to participants while electroencephalography (EEG) was recorded. By presenting fixed sequences of dots in different locations repeatedly to participants, the authors assumed that participants had learned the sequences during the experiment. The authors also trained classifiers using event-related potential (ERP) data recorded from separate experimental blocks of dots presented in a random (i.e., unpredictable) order. Using these trained classifiers, the authors then assessed whether patterns of brain activity could be detected that resembled the neural response to a dot location that was expected, but not presented. They did this by presenting an additional set of sequences whereby only one of the dots in the learned sequence appeared, but not the other dots. They report that, in these sequences with omitted stimuli, patterns of EEG data resembled the visual response evoked by a dot location for stimuli that could be expected, but were not presented. Importantly, this only occurred for an omitted dot stimulus that would be expected to appear immediately after the dot that was presented in these partial sequences.

This exciting finding complements previous demonstrations of the ability to decode expected (but not presented) stimuli in Blom et al. (2020) and Robinson et al. (2020) that are cited in this manuscript. It suggests that the visual system is able to generate patterns of activity that resemble expected sensory events, approximately at times at which an observer would expect them.

Strengths:

The experiment was carefully designed and care was taken to rule out some confounding factors. For example, gaze location was tracked over time, and deviations from fixation were marked, in order to minimise the contributions of saccades to above-chance decoding of dot position. The use of a separate block of dots (with unpredictable locations) to train the classifiers was also useful in isolating visual responses evoked by each dot location independently of any expectations that might be formed during the experiment. A large amount of data was also collected from each participant, which is important when using classifiers to decode stimulus features from EEG data. This careful approach is commendable and draws on best practices from existing work.

Weaknesses:

While there was clear evidence of careful experiment design, there are some aspects of the data analysis and results that significantly limit the inferences that can be drawn from the data. Both issues raised here relate to the use of pre-stimulus baselines and associated problems. As these issues are somewhat technical and may not be familiar to many readers, I will try to unpack each line of reasoning below. Here, it should be noted that these problems are complex, and similar issues often go undetected even by highly experienced EEG researchers.

Relevant to both issues, the authors derived segments of EEG data relative to the time at which each dot was presented in the sequences (or would have appeared when the stimuli were omitted in the partial sequences). Segments were derived that spanned -100ms to 300ms relative to the actual or expected onset of the dot stimulus. The 300ms post-stimulus time period corresponds to the duration of each dot in the sequence (100ms) plus the inter-stimulus interval (ISI) that was 200ms in duration before the next dot appeared (or would be expected to appear in the partial sequences). Importantly, a pre-stimulus baseline was applied to each of these segments of data, meaning that the average amplitude at each electrode between -100ms and 0ms relative to (actual or expected) stimulus onset was subtracted from each segment of data (i.e., each epoch in common EEG terminology). While this type of baseline subtraction procedure is commonplace in EEG studies, in this study design it is likely to cause problematic effects that could plausibly lead to the patterns of results reported in this manuscript.

First of all, the authors compare event-related potentials (ERPs) evoked by dots in the full as compared to partial sequences, to test a hypothesis relating to attentional tuning. They reported ERP amplitude differences across these conditions, for epochs corresponding to when a dot was presented to a participant (i.e., excluding epochs time-locked to omitted dots). However, these ERP comparisons are complicated by the fact that, in the full sequences, dot presentations are preceded by the presentation of other dots in the sequence. This means that ERPs evoked by the preceding dots in the full sequences will overlap in time with the ERPs corresponding to the dots presented at the zero point in the derived epochs. Importantly, this overlap would not occur in the partial sequence conditions, where only one dot was presented in the sequence. This essentially makes any ERP comparisons between full and partial sequences very difficult to interpret, because it is unclear if ERP differences are simply a product of overlapping ERPs from previously presented dots in the full sequence conditions. For example, there are statistically significant differences observed even in the pre-stimulus baseline period for this ERP analysis, which likely reflects the contributions ERPs evoked by the preceding dots in the full sequences, which are absent in the partial sequences.

The problems with interpreting this data are also compounded by the use of pre-stimulus baselines as described above. Importantly, the use of pre-stimulus baselines relies on the assumption that the ERPs in the baseline period (here, the pre-stimulus period) do not systematically differ across the conditions that are compared (here, the full vs. partial sequences). This assumption is violated due to the overlapping ERPs issue described just above. Accordingly, the use of the pre-stimulus baseline subtraction can produce spurious effects in the time period after stimulus onset (for examples see Feuerriegel & Bode, 2022, Neuroimage). This also makes it very difficult to meaningfully compare the ERPs following dot stimulus onset in these analyses.

The second issue relates to the use of pre-stimulus baselines and concerns the key finding reported in the paper: that EEG patterns corresponding to expected but omitted events can be decoded in the partial sequences. In the partial sequences, there are two critical epochs that were derived: One time-locked to the presentation of the dot, and another that was time-locked to 300ms after the dot was presented (i.e. when the next dot would be expected to appear). The latter epoch was used to test for representations of expected, but omitted, stimulus locations.

For the epochs in which the dots were presented, above-chance decoding can be observed spanning a training time range from around 100-300ms and a testing time range of a similar duration (see the plot in Figure 4b). This plot indicates that, during the time window of around 200-300ms following dot stimulus onset, the position of the dot can be decoded not only from trained classifiers using the same time windows spanning 200-300ms, but also using classifiers trained using earlier time windows of around 100-200ms.

This is important because the 200-300ms time period after dot onset in the partial sequences is the window used for pre-stimulus baseline subtraction when deriving epochs corresponding to the first successor representation (i.e., the first stimulus that might be expected to follow from the presented dot, but did not actually appear). In other words, the 200-300ms time window from dot onset corresponds to the -100 to 0 ms time window in the first successor epochs. Accordingly, the pattern that is indicative of the preceding, actually presented dot position would be subtracted from the EEG data used to test for the successor representation. Notably, the first successor condition would always be in another visual field quadrant (90-degree rotated or the opposite quadrant) as stated in the methods. In other words, the omitted stimulus would be expected to appear in the opposite vertical and/or horizontal visual hemifield as compared to the previously presented dot in these partial sequences.

This is relevant because ERPs tend to show reversed polarity across hemifields. For example, a stimulus presented in the right hemifield will have reversed polarity patterns at the same electrode as compared to an equivalent stimulus presented in the left hemifield (e.g., Supplementary Figure 3 in the comparable study of Blom et al., 2020). By subtracting the ERP patterns evoked by the presented dot in the partial sequences during the time period of 200-300ms (corresponding to the -100 to 0ms baseline window), this would be expected to bias patterns of EEG data in the first successor epochs to resemble stimulus positions in opposite hemifields. This could plausibly produce above-chance decoding accuracy in the time windows identified in Figure 5a, where the training time windows broadly correspond to the periods of above-chance decoding during 200-300ms from dot stimulus onset in Figure 4b.

In other words, the above-chance decoding of the first successor representation may plausibly be an artefact of the pre-stimulus baseline subtraction procedure used when deriving the epochs. This casts some doubt as to whether genuine successor representations were actually detected in the study. Additional tests for successor representations using ERP baselines prior to the presented dot in the partial sequences may be able to get around this, but such analyses were not presented, and the code and data were not accessible at the time of this review.

Although the study is designed well and a great amount of care was taken during the analysis stage, these issues with ERP overlap and baseline subtraction raise some doubts regarding the interpretability of the findings in relation to the analyses currently presented.

Reviewer #1 (Public Review):

Summary:

This is a strong paper that sets the foundation for future work that will explore the innervation of the giant fiber, allowing experiments that will link molecular/developmental mechanisms to circuit function at a level of resolution that has not previously been possible. In the course of this work the investigators discover an axon-axon competition that reflects the order of innervation of the target. In addition, a host of reagents are developed that will be of wide use in dissecting this system.

Strengths:

(1) The developmental, functional and connectomic characterization of the wiring pattern to be dissected is impressively thorough and quantitative.<br /> (2) The reagents that the authors establish will be foundational to subsequent effort.<br /> (3) The discovery that axon-axon competition is involved in patterning this system, and might combined with innervation order to give a deterministic outcome is an interesting one (and might be useful to address variation in cell number (see below)!

Weaknesses:

(1) In my opinion, the authors miss an opportunity to leverage their connectomics characterization somewhat more. That is, from characterization of the connectomes of two flies, the authors describe substantial variation in the number of pre-synaptic cells providing inputs (for example, in FAFB, there are 55 LC4 cells, while in the hemibrain, there are 71 - almost 30 percent more), yet the number of total synapses provided by each class of cell types is remarkably stereotyped 2442 synapses versus 2290 synapses). And the ratio of LC4 to LPLC2 synapses is even more stereotyped... As this kind of stereotypy would be consistent with the authors competition model, but inconsistent with a model in which each cell makes a similar number of synapses (which would be the model from the periphery of the visual system), the authors should comment a bit more on what they see. Perhaps the wiring model the authors advocate for compensates for what appears to be quite significant variation in the numbers of LC neurons?

(2) I appreciate how the authors pivoted to interpreting their results using Kir2.1 to reflect the effects of cell ablation. However, I worry that since the mechanism behind Kir2.1 mediated ablation is unknown, there could be other effects associated with this perturbation, creating indirect effects that alter LPLC2 cells somehow. I would therefore ask that the authors repeat these experiments with a more standard cell ablation strategy (such as a light gated caspase, or ricin). More crucially, the author's model that arrival order is functionally important would be greatly strengthened if they did the reciprocal ablation of LPLC2 and asked what happens to LC4. One could easily imagine a model in which these two cell types mutually compete for real estate, after an initial bias is set by arrival order.

Reviewer #1 (Public Review):

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crest-derived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

I appreciate the amount of work the authors have put into generating the mouse models to detect these cells, but there isn't any new insight on either the nature of ENCDC development or the role of Ret and Ednrb. Also, there are sophisticated single-cell genomics methods to detect rare cell type/states these days and the authors should either employ some of those themselves in these mouse models or look at extensively publicly available single-cell datasets of the developing wildtype and mutant mouse and human ENS to map out the global transcriptional profile of these cells. A more detailed analysis of these Pax2-positive cells would be really helpful to both the ENS community as well as researchers studying gut motility disorders.

Reviewer #1 (Public Review):

The manuscript entitled "A septo-hypothalamic-medullary circuit directs stress-induced analgesia" by Shah et al., showed that the dLS-to-LHA circuit is sufficient and necessary for stress-induced analgesia (SIA), which is mediated by the rostral ventromedial medulla (RVM) in a opioid-dependent manner. This study is interesting and important and the conclusions are largely supported by the data. I have a few concerns as follows:

(1) The present data show that activation of dLS neurons produces SIA, however, this manipulation is non-specific. It may be better to see the effect of specific manipulation of stress-activated c-Fos positive neurons in the dLS using a combination of the Tet-Off system and chemogenetic/optogenetic tools.

(2) Depending on its duration, and intensity, stress can exert potent and bidirectional modulatory effects on pain, either reducing pain (SIA) or exacerbating it (stress-induced hyperalgesia, SIH). Is the circuit in the manuscript involved in SIH?

(3) It is well-accepted that opioid and cannabinoid receptors participate in the SIA, and the evidence is especially strong for the RVM endocannabinoid system. Given this, why did the authors focus their study on the opioid system?

(4) Does silencing of the dLS neurons affect stress-induced anxiety-like behaviors? Alternatively, what is the relationship between SIA and the level of stress-induced anxiety?

(5) Direct electrophysiological evidence should be provided to confirm the efficacy of the MP-CNO.

(6) Is the LHA a specific downstream target for SIA, and is the LHA involved in stress-induced anxiety-like behaviors?

(7) Do LHA neurons have direct projections to the RVM? If yes, what is its role in the SIA?

Joint Public Review:

Summary:

This manuscript investigates how energetic demands affect the sleep-wake cycle in Drosophila larvae. L2 stage larvae do not show sleep rhythm and long-term memory (LTM), however, L3 larvae do. The authors manipulate food content to provide insufficient nutrition, which leads to more feeding, no LTM, and no sleep even in older larvae. Similarly, activation of NPF neurons suppresses sleep rhythm. Furthermore, they try to induce a sleep-like state using pharmacology or genetic manipulations in L2 larvae, which can mimic some of the L3 behaviours. A key experimental finding is that activation of DN1a neurons activate the downstream DH44 neurons, as assayed by GCaMP calcium imaging. This occurs only in third instar and not in second instar, in keeping with the development of sleep-wake and feeding separation. The authors also show that glucose metabolic genes are required in Dh44 neurons to develop sleep rhythm and that DH44 neurons respond differently in malnutrition or younger larvae.

Strengths:

Previous studies from the same lab have shown the sleep is required for LTM formation in the larvae, and that this requires DN1a and DH44 neurons. The current work builds upon this observation and addresses in more detail when and how this might develop. The authors can show that low quality food exposure and enhanced feeding during larval stage of Drosophila affects the formation of sleep rhythm and long-term memory. This suggests that the development of sleep and LTM are only possible under well fed and balanced nutrition in fly larvae. Non-sleep larvae were fed in low sugar conditions and indeed, the authors also find glucose metabolic genes to be required for a proper sleep rhythm. The paper presents precise genetic manipulations of individual classes of neurons in fly larvae followed by careful behavioural analysis. The authors also combine thermogenetic or peptide bath application experiments with direct calcium imaging of specific neurons.

Weaknesses:

The authors tried to induce sleep in younger L2 larvae, however the behavioral results suggest that they were not able to induce proper sleep behaviour as in normal L3 larvae. Thus, they cannot show that sleep during L2 stage would be sufficient to form LTM.<br /> The authors suggest that larval Dh44 neurons may integrate "information about the nutritional environment through the direct sensing of glucose levels to modulate sleep-wake rhythm development". They identify glucose metabolism genes (e.g., Glut1) in the downstream DH44 neurons as being required for the organization of the sleep-wake-feeding rhythm, and that CCHa signaling in DN1a signaling to the DH44 cells via the receptor. However, how this is connected is not well explained. Do the authors think that the nutrient sensing is only occurring in the DH44 neurons and not in DN1a or other neurons? Would not knocking down glucose metabolism in any neuron lead to a functional defect? What is the evidence that Dh44 neurons are specific sensors of nutritional state? For example, do the authors think that e.g. the overexpression of Glut1 in Dh44 neurons, a manipulation that can increase transport of glucose into cells, would rescue the effects of low-sugar food?<br /> Some of the genetic controls seem to be inconsistent suggesting some genetic background effects. In Figure 2B, npf-gal4 flies without the UAS show no significant circadian change in sleep duration, whereas UAS-TrpA flies do. The genetic control data in Figure 2D are also inconsistent. Npf-Gal4 seems to have some effect by itself without the UAS. The same is not seen with R76G11-Gal4. Suppl Fig 2: Naïve OCT and AM preference in L3 expressing various combinations of the transgenes show significant differences. npf-Gal4 alone seems to influence preference.<br /> The sleep duration and bout number/length data are highly variable.

Reviewer #1 (Public Review):

Summary:

The manuscript gives a broad overview of how to write NeuroML, and a brief description of how to use it with different simulators and for different purposes - cells to networks, simulation, optimization, and analysis. From this perspective, it can be an extremely useful document to introduce new users to NeuroML.

However, the manuscript itself seems to lose sight of this goal in many places, and instead, the description at times seems to target software developers. For example, there is a long paragraph on the board and user community. The discussion on simulator tools seems more for developers, not users. All the information presented at the level of a developer is likely to be distracting to readers..

Strengths:

The modularity of NeuroML is indeed a great advantage. For example, the ability to specify the channel file allows different channels to be used with different morphologies without redundancy. The hierarchical nature of NeuroML also is commendable, and well illustrated in Figures 2a through c.

The number of tools available to work with NeuroML is impressive.

The abstract, beginning, and end of the manuscript present and discuss incorporating NeuroML into research workflows to support FAIR principles.

Having a Python API and providing examples using this API is fantastic. Exporting to NeuroML from Python is also a great feature.

Weaknesses:

Though modularity is a strength, it is unclear to me why the cell morphology isn't also treated similarly, i.e., specify the morphology of a multi-compartmental model in a separate file, and then allow the cell file to specify not only the files containing channels, but also the file containing the multi-compartmental morphology, and then specify the conductance for different segment groups. Also, after pynml_write_neuroml2_file, you would not have a super long neuroML file for each variation of conductances, since there would be no need to rewrite the multi-compartmental morphology for each conductance variation.

This would be especially important for optimizations, if each trial optimization wrote out the neuroML file, then including the full morphology of a realistic cell would take up excessive disk space, as opposed to just writing out the conductance densities. As long as cell morphology must be included in every cell file, then NeuroML is not sufficiently modular, and the authors should moderate their claim of modularity (line 419) and building blocks (551). In addition, this is very important for downloading NeuroML-compliant reconstructions from NeuroMorpho.org. If the cell morphology cannot be imported, then the user has to edit the file downloaded from NeuroMorpho.org, and provenance can be lost. Also, Figure 2d loses the hierarchical nature by showing ion channels, synapses, and networks as separate main branches of NeuroML.

In Figure 5, the difference between the core and native simulator is unclear. What is involved in helper scripts? I thought neurons could read NeuroML? If so, why do you need the export simulator-specific scripts? In addition, it seems strange to call something the "core" simulation engine, when it cannot support multi-compartmental models. It is unclear why "other simulators" that natively support NeuroML cannot be called the core. It might be more helpful to replace this sort of classification with a user-targeted description. The authors already state which simulators support NeuroML and which ones need code to be exported. In contrast, lines 369-370 mention that not all NeuroML models are supported by each simulator. I recommend expanding this to explain which features are supported in each simulator. Then, the unhelpful separation between core and native could be eliminated.

The body of the manuscript has so much other detail that I lose sight of how NeuroML supports FAIR. It is also unclear who is the intended audience. When I get to lines 336-344, it seems that this description is too much detail for the audience. The paragraph beginning on line 691 is a great example of being unclear about who is the audience. Does someone wanting to develop NeuroML models need to understand XSD schema? If so, the explanation is not clear. XSD schema is not defined and instead explains NeuroML-specific aspects of XSD. Lines 734-735 are another example of explaining to code developers (not model developers).

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.<br /> In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:

In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Following the revision of the original manuscript, I can clearly state that my concerns have been addressed and the article has been improved.

Reviewer #1 (Public Review):

Summary:

The work from Petazzi et al. aimed at identifying novel factors supporting the differentiation of human hematopoietic progenitors from induced pluripotent stem cells (iPSCs). The authors developed an inducible CRISPR-mediated activation strategy (iCRISPRa) to test the impact of newly identified candidate factors on the generation of hematopoietic progenitors in vitro. They first compared previously published transcriptomic data of iPSC-derived hemato-endothelial populations with cells isolated ex vivo from the aorta-gonad-mesonephros (AGM) region of the human embryo and they identified 9 transcription factors expressed in the aortic hemogenic endothelium that were poorly expressed in the in vitro differentiated cells. They then tested the activation of these candidate factors in an iPSC-based culture system supporting the differentiation of hematopoietic progenitors in vitro. They found that the IGF binding protein 2 (IGFBP2) was the most upregulated gene in arterial endothelium after activation and they demonstrated that IGFBP2 promotes the generation of functional hematopoietic progenitors in vitro.

Strengths:

The authors developed an extremely useful doxycycline-inducible system to activate the expression of specific candidate genes in human iPSC. This approach allows us to simultaneously test the impact of 9 different transcription factors on in vitro differentiation of hematopoietic cells, and the system appears to be very versatile and applicable to a broad variety of studies.

The system was extensively validated for the expression of 1 transcription factor (RUNX1) in both HeLa cells and human iPSC, and a detailed characterization of this test experiment was provided.

The authors exhaustively demonstrated the role of IGFBP2 in promoting the generation of functional hematopoietic progenitors in vitro from iPSCs. Even though the use of IGFBP2-interacting proteins IGF1 and IGF2 have been previously reported in human iPSC-derived hematopoietic differentiation in vitro (Ditadi and Sturgeon, Methods 2016; Ng et al., Nature Biotechnology 2016), and IGFBP-2 itself has been shown to promote adult HSC expansion ex vivo (Zhang et al., Blood 2008), its role on supporting in vitro hematopoiesis was demonstrated here for the first time.

Weaknesses:

Although the authors performed a very thorough characterization of the system in proof-of-principle experiments activating a single transcription factor, the data provided when 9 independent factors were used is not sufficient to fully validate the experimental strategy. Indeed, in the current version of the manuscript, it is not clear whether the results presented in both the scRNAseq analysis and the functional assays are the consequence of the simultaneous activation of all 9 TF or just a subset of them. This is essential to establish whether all the proposed factors play a role during embryonic hematopoiesis, and a more complete analysis of the scRNAseq dataset could help clarify this aspect.

Similarly, the data presented in the manuscript are not sufficient to clarify at what stage of the endothelial-to-hematopoietic transition (EHT) the TF activation has an impact. Indeed, even though the overall increase of functional hematopoietic progenitors is fully demonstrated, the assays proposed in the manuscript do not clarify whether this is due to a specific effect at the endothelial level or to an increased proliferation rate of the generated hematopoietic progenitors. Similar conclusions can be applied to the functional validation of IGFBP2 in vitro.

The overall conclusions are sometimes vague and not always supported by the data. For instance, the authors state that the CRISPR activation strategy resulted in transcriptional remodeling and a steer in cell identity, but they do not specify which cell types are involved and at what level of the EHT process this is happening. In the discussion, the authors also claim that they provided evidence to support that RUNX1T1 could regulate IGFBP2 expression. However, this is exclusively based on the enrichment of RUNX1T1 gRNA in cells expressing higher levels of IGFBP2 and it does not demonstrate any direct or indirect association of the two factors.

Reviewer #2 (Public Review):

This preprint by Pokrovsky and coworkers is a descriptive study reporting on non-breeding itinerant behaviour of an intrapalearctic migratory raptor, the rough-legged buzzard, and relating such non-breeding movements to snow cover across the European non-breeding range. The article is based on long-term GPS tracking data from a relatively large (n=43) sample of individuals that were equipped with state-of-the-art tracking devices in the Russian Arctic during 2013-2019. The results show that, upon breeding, buzzards migrated rapidly to southern non-breeding areas, located in open areas north of the Black and Caspian seas, where they perform continuous directional movements at a slower pace, initially moving SW (Oct to Jan) and then progressively moving NE (Feb to Apr) before embarking on rapid spring migration. It is suggested that such itinerant behaviour follows variation (expansion and retreat) of snow cover across the non-breeding range.

The results are potentially useful for researchers investigating the ecological drivers of bird movement patterns. The analytical framework appears solid, although some details on the analyses (requested during the previous review round) are still unclear and have not been modified despite explicit requests. Significant weaknesses in the theoretical framework persist in the revised version, including unwarranted claiming of novelty, overselling of importance of the study, and overinterpretation of the data. Below are key points that the authors did not consider when revising their manuscript.

(1) The authors underemphasize the fact that what they term 'fox-trot' migration is actually a well-known patterns for many other migratory species, both in the Nearctic and in the Afro-Palearctic migration systems. Such behaviour has previously been identified as 'itinerant' or 'non-breeding itinerancy', involving an alternation of stopovers and movements between different short-term non-breeding residency areas, or even slow continuous movements, and it seems that the pattern the authors report for this particular species is perfectly in line with such previous evidence. For instance, this is well-documented among migratory raptors, such as the Montagu's harrier, lesser kestrels or black kites, that exploit Sahelian savannahs, where large spatio-temporal variation in greenness and hence resource availability occurs. And, besides the mentioned cuckoos and nightingales, there are studies of red-backed shrikes suggesting the same, as well as of tree swallows in the Nearctic. Therefore, the authors should avoid claiming novelty for this study and introducing unnecessary and confusing new terms in the literature (i.e. the 'fox-trot' migration patterns) when these are definitely not strictly needed as they have been previously observed and defined otherwise. Sentences such as 'We used the rough-legged buzzard as a model..." are also similarly unwarranted. This is simply a descriptive studies reporting on such behaviour in yet another migratory species. The whole introduction is pervaded by a faulty logic. The authors first introduce a new (unwarranted) term based on previous evidence from other studies (none of which felt any need to introduce and describe it); then they assume, for unclear reasons, that the species they are studying should behave in that way; even more worryingly, based on these assumptions, they make specific predictions on how this species should behave, without any sound biological reason for these predictions. I admit I hardly see any scientific logic in this approach.

(2) The species has a very standard migration for a short-distance migrant, by all means. It moves to non-breeding areas, and once there it slowly moves towards the south in autumn and back again in spring, until it departs for pre-breeding migration. This is no different from other trans-Saharan migratory raptor species that spend the non-breeding period in the Sahel. Whether the species perform any short/medium term stopover (frequenting the same are for some days) during the non-breeding stage (between end of autumn migration and onset of spring migration), as is the case of most species showing non-breeding itinerancy, is not reported. The authors only show a slower pace of movement during the non-breeding period compared to autumn and spring migration, without providing any further details. This hinders comparisons with other previous studies.

(3) The current title is unnecessarily general (it may recall rather a review or meta-analysis) and not adequately describing the content of the manuscript. In order to be informative, the title should more tightly reflect the content of the article. A valid alternative would be 'Itinerant non-breeding behaviour of an intra-Palaearctic migratory raptor', as it would be far more adequate and informative.

(4) The text, particularly the Introduction and the Discussion, would greatly benefit from profound reframing in light of the above comments. Upon reading the first sentence of the introduction, it looks surprising that the authors based their suggestion for 'fox-trot' migration based on a very outdated article on the migration of Montagu's harrier based on sparse ring recovery data which merely suggests the existence of 'movements' within the non-breeding areas (i.e. non-breeding itinerancy), while subsequent large scale satellite tracking studies of this species provided compelling evidence for non-breeding area itinerancy (and again, no mention of 'fox-trot' whatsoever). The discussion is entirely framed around potential issues related to accurate monitoring of population size and trends, which the author surprisingly refer to 'conservation implications'. As I already mentioned in my previous review, the 'conservation implications' of this study are nearly negligible. At best, it suggests that caution should be applied when interpreting population trends of migratory species based on non-breeding area counts only, a pattern that is already well known for decades (consider the long-running IWC coordinated by Wetlands International!). In addition, Christmas Bird Count, a long-term monitoring program of AOS, is mentioned without any accurate reference to what it actually is, assuming that any reader would be familiar with a very peculiar monitoring scheme of the Nearctic region.

The final paragraph epitomizes how authors are overstating the importance of this study, claiming for non-existent novelty and even 'discovery': "Our study has identified and characterized a new pattern of migratory behavior, the 'foxtrot migration', along with the associated concept of 'dynamic range'. This discovery has significant implications for conservation strategies and adequate representation of non-breeding habitats".

Reviewer #1 (Public Review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

Weaknesses:

There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analsyis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and document the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attaches if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Comments on revised version:

The authors have addressed the issues raised in the original review.

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

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

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