Reviewer #1 (Public Review):

In this study, Romero, Prosper, and colleagues have investigated the differential gene expression and regulation in hematopoietic stem and progenitor cells (HSPCs) in young or elderly healthy individuals. With the use of single-cell RNA sequencing (scRNA seq), the authors identified that the stem/progenitor repertoire is changed in elderly individuals, which is accompanied by changes in cell differentiation. The authors additionally compare HSPCs from patients with myelodysplastic syndrome (MDS) and found that MDS patients exhibit specific alterations in erythroid differentiation gene regulatory networks in MDS HSPCs. Overall, this study deals with a valuable resource of HSPC profiles in healthy individuals and proves the biased hematopoietic landscape over aging at a transcriptome level. It will serve as a valuable resource for understanding the molecular basis for hematopoietic aging, which will be useful for future therapeutics and applications.

Reviewer #1 (Public Review):

The authors of this manuscript aimed at demonstrating the hypothesis that hyperacusis is triggered by increased sensitivity in mid-range frequency following high-frequency cochlear trauma. The study combines a large variety of careful physiological and behavioral measurements that converge toward the above-mentioned interpretation, which was proposed in an earlier report. This will likely boost the development of hyperacusis mouse models which is beneficial for future treatments.

Reviewer #1 (Public Review):

This is a carefully-conducted fMRI study looking at how neural representations in the hippocampus, entorhinal cortex, and ventromedial prefrontal cortex change as a function of local and global spatial learning. Collectively, the results from the study provide valuable additional constraints on our understanding of representational change in the medial temporal lobes and spatial learning. The most notable finding is that representational similarity in the hippocampus post-local-learning (but prior to any global navigation trials) predicts the efficiency of subsequent global navigation.

Strengths:

The paper has several strengths. It uses a clever two-phase paradigm that makes it possible to track how participants learn local structure as well as how they piece together global structure based on exposure to local environments. Using this paradigm, the authors show that - after local learning - hippocampal representations of landmarks that appeared within the same local environment show differentiation (i.e., neural similarity is higher for more distant landmarks) but landmarks that appeared in different local environments show the opposite pattern of results (i.e., neural similarity is lower for more distant landmarks); after participants have the opportunity to navigate globally, the latter finding goes away (i.e., neural similarity for landmarks that occurred in different local environments is no longer influenced by the distance between landmarks). Lastly, the authors show that the degree of hippocampal sensitivity to global distance after local-only learning (but before participants have the opportunity to navigate globally) negatively predicts subsequent global navigation efficiency. Taken together, these results meaningfully extend the space of data that can be used to constrain theories of MTL contributions to spatial learning.

Weaknesses:

1. The study has an exploratory feel, in the sense that - for the most part - the authors do not set forth specific predictions or hypotheses regarding the results they expected to obtain. When hypotheses are listed, they are phrased in a general way (e.g., "We hypothesized that we would find evidence for both integration and differentiation emerging at the same time points across learning, as participants build local and global representations of the virtual environment", and "We hypothesized that there would be a change in EC and hippocampal pattern similarity for items located on the same track vs. items located on different tracks" - this does not specify what the change will be and whether the change is expected to be different for EC vs. hippocampus). I should emphasize that this is not, unto itself, a weakness of the study, and it appears that the authors have corrected for multiple comparisons (encompassing the range of outcomes explored) throughout the paper. However, at times it was unclear what "denominator" was being used for the multiple comparisons corrections (i.e., what was the full space of analysis options that was being corrected for) - it would be helpful if the authors could specify this more concretely, throughout the paper.

2. Some of the analyses featured prominently in the paper (e.g., interactions between context and scan in EC) did not pass multiple comparisons correction. I think it's fine to include these results in the paper, but it should be made clear whenever they are mentioned that the results were not significant after multiple comparisons correction (e.g., in the discussion, the authors say "learning restructures representations in the hippocampus and in the EC", but in that sentence, they don't mention that the EC results fail to pass multiple comparisons correction).

3. The authors describe the "flat" pattern across the distance 2, 3, and 4 conditions in Figure 4c (post-global navigation) and in Figure 5b (in the "more efficient" group) as indicating integration. However, this flat pattern across 2, 3, and 4 (unto itself) could simply indicate that the region is insensitive to location - is there some other evidence that the authors could bring to bear on the claim that this truly reflects integration? Relatedly, in the discussion, the authors say "the data suggest that, prior to Global Navigation, LEs had integrated only the nearest landmarks located on different tracks (link distance 2)" - what is the basis for this claim? Considered on its own, the fact that similarity was high for link distance 2 does not indicate that integration took place. If the authors cannot get more direct evidence for integration, it might be useful for them to hedge a bit more in how they interpret the results (the finding is still very interesting, regardless of its cause).

Reviewer #1 (Public Review):

The manuscript is likely of interest to cryo-electron microscopists working on cellular samples. It details a data-acquisition scheme for mapping large areas at a fine pixel size by cryo-electron microscopy for the purpose of macromolecular identification by high-resolution 2D template matching (2DTM). The authors succinctly describe the methodology, as well as detail the apparent effects of microscope aberrations on 2DTM results.

While other montaging approaches have been described recently, the one presented here differs in its approach to controlling defocus and avoids the need to sacrifice a biologically meaningful region of a sample. The authors investigate the compatibility of the data acquisition with their 2DTM method using cryoFIB-milled mouse neutrophil-like cells and the 60S ribosome as an example case. In order to minimize unnecessary exposures, the authors restrict illumination to a circle inscribed on the detector and use beam image-shift in lieu of stage shift. This approach introduces several optical aberrations for which the authors investigate the effects on the 2DTM results. The results of the investigated aberration effects may be of general interest to the cryoEM community, not just those using montaging methods.

Reviewer #1 (Public Review)

The aim of the authors was to clarify the function of pinnae, forming part of katydid ears in the forelegs. Previous work suggested a protective function for the thin tympana or a device for directional hearing. A major strength of the paper is the combination of methods, such as experimental biophysical measurements with Laser-Doppler-Vibrometry and numerical modelling. In addition, detailed morphological data were obtained by scanning ears with a µCT-scanner, which formed the basis for producing 3D-printed models of the ear. These methods were combined with audiograms of sensory units in the ear, and measurements of behavioral sensitivity.

Using experimental ablation of the pinnae, the authors can convincingly show that the cavities formed by the pinnae produce resonances at very high ultrasonic frequencies and that these resonances boost the perception of sound by about 20 - 30 decibels, i.e. make the ear more sensitive for these frequencies. By contrast, the data do not support the hypothesis that pinnae serve in directional hearing.

To my knowledge, the method of performing acoustic measurements using synthetic 3D-printed scaled ear models is completely new in the field. It offers great advantages for studying the often-minute structures in insects.

Reviewer #1 (Public Review):

Using single cell analysis, Paxman et al observe protein aggregation in aging yeast that is specific to cells with deregulated rDNA silencing. This is confirmed in sir2 mutant cells. The authors then investigate the mechanism by which silencing defects of the rDNA locus might be linked to a decline in protein homeostasis. Through a screen for aggregation of RNA binding proteins, they find that disruption of rDNA silencing leads to aggregation of those RNA binding proteins that are involved in rRNA processing. Overexpression of a subset of these rRNA binding genes consistently shorten the lifespan of mode 1 cells, presumably by contributing to their defects in protein homeostasis. This suggests that age dependent changes in rDNA silencing lead to the aberrant expression of rRNAs and the formation of rDNA circles. Deletion of fob1 (resulting in a loss of rDNA recombination) indeed suppresses aggregation of Nop15 that is used for in-depth analysis. Of note, enhancing rRNA transcription or Nop15 expression leads to enhanced protein aggregation even in the absence of rDNA circles.

In all this study addresses an interesting and exciting question and is well executed. Importantly, it contributes to the understanding of distinct aging trajectories and raises important questions how these processes might be relevant in multicellular organisms. Given the fact that the paper focuses on rDNA silencing, I think that using the term "chromatin stability" is too broad and should be replaced with "rDNA silencing".

Reviewer #1 (Public Review):

In this manuscript the authors set out to characterise the process of differentiation of inner cell mass cells within the mouse blastocyst into either epiblast or primitive endoderm, which is a binary fate choice, using various models. To this end, they made use of well-established reporter cell lines previously generated in their lab as well as a widely used fluorescent system (FUCCI) that allows stages in the cell cycle to be visualised and sorted. The experimental output was compared with computational models and published data generated from mouse embryos during the process of primitive endoderm and epiblast segregation. Their data uncovered interesting mechanistic insight into the dynamics of the cell cycle and how these correlate with lineage choice and amplification. The methods have been carefully considered and validated in previous work by the group and the analysis is thorough. The single cell profiling is particularly well presented, and backed up by immunofluorescence data using well-characterised lineage reporters with appropriate statistical analysis. Probably the most interesting finding, which the authors identify as unexpected, is the considerable lengthening of the G1 part of the cell cycle in cells differentiating into PrE, but coinciding with a reduction in overall cell cycle length. Also, cell cycle length from mother to daughter cells in all conditions appears not to be inherited, yet sister cells, and to a lesser extent, cousins, appear to retain similar cell cycle dynamics. This feature is attributed to differential levels of FGF, suggested by the use of PD03 or PD17 as downstream inhibitors. Not surprisingly, levels of the PrE-associated factor Hex could predict the likelihood of differentiation to PrE, but also higher levels of Hex correlated with a shorter cell cycle. Also, blocking MEK/ERK signalling increased cell cycle duration as well as reducing differentiation to PrE in the culture conditions designed to promote differentiation to epiblast. The aims of the paper appear to be achieved and the results adequately support the authors' conclusions. A similar system to the one established here could be envisaged for downstream developmental processes, such as those involving binary decisions for specific tissue formation in organogenesis, but would require the generation and validation of different reporter cell lines.

Reviewer #1 (Public Review):

Selecting appropriate Bioinformatics approaches to arrive at a consensus classification of SNVs can be labor intensive and misleading due to discordance in results from different programs. The authors evaluated 31 Bioinformatic or computational tools used for in silico prediction of single nucleotide variants (SNVs). They selected a filtered list of SNVs at the HBA1, HBA2, and HBB genes, and compared in silico prediction results with annotations based on evidence in literature and databases curated by an expert panel comprising co-authors of this study. They found both specificity and concordance among different tools lacking in certain aspects when thresholds are chosen to maximize the Matthews correlation (MCC) and thus proposed an improved strategy. For this, the authors focused on the top prediction algorithms and varied their decision thresholds separately for pathogenic and benign variant classification and optimized the predictive power of these tools by choosing thresholds that generated at least supporting strength likelihood ratios (LRs) to achieve balanced classification.

The authors have likely spent significant effort annotating the list of pathogenic or benign SNVs in adult globin genes by iteratively evaluating independent annotations submitted by experts and arriving at a consensus. These annotations when added to the database of SNVs might improve the breadth of knowledge on the pathogenicity of adult globin SNVs and likely lead to an improved prediction by the existing tools. Further, setting non-overlapping thresholds for pathogenic and benign variants seems to improve the balance in the prediction of some of the tools (with certain tradeoffs) in the context of the gene and the variant class. This is consistent with the findings of Wilcox et. al., while at the same time the authors have focused on globin variants and compared many more programs. Thus, while not a novel approach, the scale is expansive, and might guide future studies with the improved ACMG/AMP framework.

However, there are certain caveats from my perspective and these need to be explained or improved.

• The authors' approach relies heavily on the revised consensus annotations which, from my understanding, is essentially being considered as a "truth dataset", whereas variants are classified in silico according to existing annotations in the databases. The binary classification metrics compare the in silico predictions to the authors' annotations and these showed low specificity but higher sensitivity and accuracy indicating that many benign variants were misclassified as pathogenic. The authors have not clearly mentioned whether the "observed_pathogenecity" information in the input dataset in supplementary file 2 is from the Ithagenes database or the authors' reannotations. Hence, if a significant number of pathogenic variants were reannotated as benign by the expert panel, that will likely result in the tools misclassifying them as pathogenic since the tools rely on database annotations.

• The results and measure of success focus on different benchmarks for the two major analyses the authors performed. While they generated a lot of data, they have not attempted to explore and present all facets of the data for each analysis. For instance, to assess the predictive power of the 31 tools initially, the authors focus on benchmark metrics for binary classification such as Accuracy, Sensitivity, Specificity and MCC. However, in the later improved approach, the focus is on LRs but the effect of separate thresholds for pathogenic and benign classification on accuracy, sensitivity, and specificity and MCC are not explored in the results instead just mentioning PPV for certain variant types, tools, and genes.

• There is a general trade-off to altering thresholds to increase specificity which leads to reduced accuracy and sensitivity. Thus, in this case, the improved approach suggested by the authors increases specificity but there is a simultaneous reduction in accuracy and sensitivity thus leading to the potentially higher misclassification of pathogenic variants as benign. One has to consider then, whether this is ideal in the case of globins where an in silico misclassification of pathogenicity can be easily verified by subsequent diagnostic testing to confirm whether the variant actually affects hemoglobin. Overclassification of pathogenicity in the case of globins is thus not necessarily a major problem since they will not directly lead to patients receiving treatment before additional confirmatory tests. However, misclassification of pathogenic variants as benign will pose greater harm to individuals at risk of disease.

• This is a largely descriptive study of the performance of various programs, but the authors did not attempt to explain why according to them the various tools performed a certain way in their analysis. Thus, their rationale for proposing the improved approach of separate thresholds for pathogenic and benign variants was unclear. Attempting to understand whether there is a correlation between the type of data the tool uses, and its performance could explain the tools' prediction power and how to improve it. For instance, some of the tools are metapredictors that take as input scores from various other tools also tested in this study. Thus, there will be some redundancy in the final classification.

• Expanding on the previous point, the reason for discordance in HBA genes but concordance in HBB was unclear. It might be a result of the bigger HBB dataset compared to HBA although the authors did not explore or mention whether the size of the dataset correlates with concordance. They also did not test for concordance or discordance after the separate thresholds were applied so it is not clear whether their proposed approach improves concordance for the HBA variant predictions of the top tools.

Reviewer #1 (Public Review):

Mating influences many behaviours such as enhanced oviposition, suppressed mating, and a change in dietary preference. In this study, Boehm et al explore the circuit basis of the mated female's enhanced preference for polyamines.

A previous study from this group had identified a mechanism by which mating reduced sensitivity of the olfactory sensory neurons resulting in a preference for higher concentrations of polyamines after mating. However, the preference for polyamines outlasts this mechanism by many days. So, in this study, the authors explore central brain circuits that might encode this persistent behavioural switch. Briefly, they identify neurons within the mushroom body - intrinsic neurons, output neurons and dopaminergic neurons (DAN) - that are involved in this behaviour. They also identify output neurons of the lateral horn that are involved in it.

The behaviour itself consists of two phases: 1) the mating experience, and 2) the subsequent expression of the polyamine preference. The authors use behavioural assays and neurogenetics to demonstrate that:

1. The ability to detect odours via the OR67d neurons at the time of mating is necessary to bring about the behavioural switch.

2. Activity of the intrinsic neurons of the mushroom body is necessary at both times - during the mating and the expression - to bring about the behavioural switch.

3. They identify one set of dopaminergic neurons - B1 DANs - that are sufficient but not necessary ***at the time of mating*** to induce the switch in virgin females.

4. They identify a second set of dopaminergic neurons - B2 DANs - that are necessary to ***at the time of expression*** to demonstrate the increased polyamine preference ******in mated females.

5. They identify a set of mushroom body output neurons (MBONs) downstream of the B1-DANs and show that output from the B1 region is necessary and sufficient at the time of mating for the expression of polyamine preference.

6. They identify MBONs downstream of the B2 DANs and find that they play no role at the time of mating, but that they are necessary and sufficient at the time of expression to suppress the polyamine preference.

7. They identify a set of output neurons of the lateral horn and find that they are necessary at the time of expression of polyamine preference.

The authors also use functional imaging to show that there are no brain-wide changes upon mating in the encoding of one of the polyamines. They explore how cVA (an odour they believe is relevant at the time of mating) is represented in the neurons they have identified. They find that the B1 DANs show enhanced representation of cVA post mating, however, their MBONs do not alter their response to cVA post mating. The B2-MBONs respond to both putrescine and cVA and show no alteration in their response post mating.

In summary, the authors have identified a mechanism similar to associative learning that operates across the mushroom body and lateral horn, to 'learn' the experience of mating and express it as an enhanced preference to a nutritionally rich food source.

Reviewer #1 (Public Review):

In this study, Zheng and Zhao identified the unannotated open reading frames (ORFs) in Drosophila, termed utORF, mainly based on proteomics datasets. The authors extended their analyses to the birth and the evolutionary heterogeneity of utORF. These analyses uncovered several types of utORFs that bear different feature, including transcription, age, distribution, and evolutionary conservation.

The origin of de novo protein-coding genes is interesting. The authors' attempts to uncover utORFs from proteomics datasets are much appreciated, but crucial cross-validation is missing. Given a high potential of false positives in MS datasets, it is difficult to evaluate the evolutionary aspects of the identified ORFs. Some experimental validation is needed to confirm the translational potential of utORFs with or without start codons.

Reviewer #1 (Public Review):

In this manuscript, Goering et al. investigate subcellular RNA localization across different cell types focusing on epithelial cells (mouse C2bbe1 and human HCA-7 enterocyte monolayers, canine MDCK epithelial cells) as well as neuronal cultures (mouse CAD cells). They use their recently established Halo-seq method to investigate transcriptome-wide RNA localization biases in C2bbe1 enterocyte monolayers and find that 5'TOP-motif containing mRNAs, which encode ribosomal proteins (RPs), are enriched on the basal side of these cells. These results are supported by smFISH against endogenous RP-encoding mRNAs (RPL7 and RPS28) as well as Firefly luciferase reporter transcripts with and without mutated 5'TOP sequences. Furthermore, they find that 5'TOP-motifs are not only driving localization to the basal side of epithelial cells but also to neuronal processes. To investigate the molecular mechanism behind the observed RNA localization biases, they reduce expression of several Larp proteins and find that RNA localization is consistently Larp1-dependent. Additionally, the localization depends on the placement of the TOP sequence in the 5'UTR and not the 3'UTR. To confirm that similar RNA localization biases can be conserved across cell types for other classes of transcripts, they perform similar experiments with a GA-rich element containing Net1 3'UTR transcript, which has previously been shown to exhibit a strong localization bias in several cell types. In order to determine if motor proteins contribute to these RNA distributions, they use motor protein inhibitors to confirm that the localization of individual members of both classes of transcripts, 5'TOP and GA-rich, is kinesin-dependent and that RNA localization to specific subcellular regions is likely to coincide with RNA localization to microtubule plus ends that concentrate in the basal side of epithelial cells as well as in neuronal processes.

In summary, Goering et al. present an interesting study that contributes to our understanding of RNA localization. While RNA localization has predominantly been studied in a single cell type or experimental system, this work looks for commonalities to explain general principles. I believe that this is an important advance, but there are several points that should be addressed.

Comments:

1. The Mili lab has previously characterized the localization of ribosomal proteins and NET1 to protrusions (Wang et al, 2017, Moissoglu et al 2019, Crisafis et al., 2020) and the role of kinesins in this localization (Pichon et al, 2021). These papers should be cited and their work discussed. I do not believe this reduces the novelty of this study and supports the generality of the RNA localization patterns to additional cellular locations in other cell types.

2. The 5'TOP motif begins with an invariant C nucleotide and mutation of this first nucleotide next to the cap has been shown to reduce translation regulation during mTOR inhibition (Avni et al, 1994 and Biberman et al 1997) and also Lapr1 binding (Lahr et al, 2017). Consequently, it is not clear to me if RPS28 initiates transcription with an A as indicated in Figure 3B. There also seems to be some differences in published CAGE datasets, but this point needs to be clarified. Additionally, it is not clear to me how the 5'TOP Firefly luciferase reporters were generated and if the transcription start site and exact 5'-ends of these constructs were determined. This is again essential to determine if it is a pyrimidine sequence in the 5'UTR that is important for localization or the 5'TOP motif and if Larp1 is directly regulating the localization by binding to the 5'TOP motif or if the effect they observe is indirect (e.g. is Larp1 also basally localized?). It should also be noted that Larp1 has been suggested to bind pyrimidine-rich sequences in the 5'UTR that are not next to the cap, but the details of this interaction are less clear (Al-Ashtal et al, 2021)

3. In figure 1A, they indicate that mRNA stability can contribute to RNA localization, but this point is never discussed. This may be important to their work since Larp1 has also been found to impact mRNA half-lives (Aoki et al, 2013 and Mattijssen et al 2020, Al-Ashtal et al 2021). Is it possible the effect they see when Larp1 is depleted comes from decreased stability?

4. Also Moor et al, 2017 saw that feeding cycles changed the localization of 5'TOP mRNAs. Similarly, does mTOR inhibition or activation or simply active translation alter the localization patterns they observe? Further evidence for dynamic regulation of RNA localization would strengthen this paper

5. For smFISH quantification, is every mRNA treated as an independent measurement so that the statistics are calculated on hundreds of mRNAs? Large sample sizes can give significant p-values but have very small differences as observe for Firefly vs. OSBPL3 localization. Since determining the biological interpretation of effect size is not always clear, I would suggest plotting RNA position per cell or only treat biological replicates as independent measurements to determine statistical significance. This should also be done for other smFISH comparisons

6. F: How was the segmentation of soma vs. neurites performed? It would be good to have a larger image as a supplemental figure so that it is clear the proximal or distal neurites segments are being compared

Joint Public Review:

While the presence of fascin in the nucleus, and its function at the cytoplasmic side of the nuclear envelope, have been shown previously, the role of fascin in the nucleus is not known. This important new study reveals that nuclear fascin regulates nuclear actin, likely actin bundling, DNA damage response, and too much nuclear fascin promotes apoptosis. The authors begin by using biochemical fractionation and imaging (a strength of this group) to show that fascin can localise to the nucleus of two human cancer cell lines. Mutation of a putative nuclear export sequence in fascin, or treatment with an exportin-1 inhibitor, results in nuclear accumulation of fascin, demonstrating that it shuttles between the cytoplasm and the nucleus. Imaging experiments clearly show the colocalisation of fascin with tagged nuclear actin; in combination with fascin-knockdown cells and expression of a non-bundling fascin mutant, this implies a requirement of fascin for nuclear actin bundling.

To explore the molecular complexes that may be regulating nuclear fascin function, the authors examined potential nuclear fascin-interacting proteins using mass spectrometry (MS)-based affinity proteomics. A smart approach exploited GFP-tagged fascin-specific nanobodies that contained nuclear localisation or nuclear export signals, which targeted fascin to the nucleus or cytoplasm, respectively. Proteomic analysis identified histones H3 and H4 as hits enriched in nuclear fascin nanobody pull-downs over non-nuclear fascin nanobody pull-downs. There are some deficiencies in the reporting of the MS data that would benefit from expansion to ensure the results of these experiments are clear, such as hit selection threshold criteria and any statistical analyses used. The potential interaction of fascin with histone H3 was suggested further using FRET between GFP-tagged histone H3 and mCherry-tagged nuclear fascin nanobody, although additional controls would improve interpretation of these data. While they are clearly present in the same complex, the imaging and FRET experiments stop short of showing the interaction is direct. While the use of FRET can be a very powerful means to show interaction, the authors require further controls, for example, a negative control would be important.

The authors identified reduced focal staining of the DNA damage response factor γH2AX in the first hour after DNA damage induction in fascin-knockdown cells. The role of fascin in the DDR is interesting, but the way the images are presented/analysed - the data are not as convincing as they might be. The differences look quite subtle due to relatively large variance and/or heterogeneity. Chromatin compaction was then tested using histone H2B-H2B FRET. Some statistical tests need to be clarified to ensure that comparisons between groups were tested appropriately, particularly for the interpretation of the chromatin compaction results upon the addition of DNA damaging agents to fascin-knockdown cells. Perhaps for discussion, but what role do the authors propose for fascin in chromatin organisation?

Driving fascin to the nucleus using the nuclear-targeted fascin nanobody resulted in substantially reduced filopodia formation, 2D migration speed, and invasion into 3D collagen gel. The alignment of representative confocal z-stacks in the presentation of the invasion assay (nuclear nanobody and fascin-knockdown cells compared to the other conditions) should be clarified. Longer-term nuclear targeting of fascin with the nanobody induced cell cycle arrest and caspase-3 cleavage, implicating nuclear fascin dynamics in loss of cancer cell viability. The phenotypic screening was well performed, including a dose-response analysis of hits and a secondary screen, to identify compounds that could induce nuclear localisation of fascin and promote apoptosis. Very useful supplementary tables have dose-response curves built in to enable interrogation of the screening datasets. The screening identified three compounds that regulate histone phosphorylation; interestingly, two of the compounds reduced histone phosphorylation and reduced histone pulldown in nuclear fascin nanobody affinity purifications in the cancer cells tested. The most potent histone H3 phosphorylation inhibitor also increased γH2AX staining, which appeared to correlate with fascin localisation in the nucleus. Can the authors make, or comment on, further evidence that Haspin-induced effects, for example, increased γH2X (was this at DNA-damage-associated foci in the nucleus?), are due to nuclear localization of fascin and/or resultant F-actin polymerization? Some follow-up data on Haspin could help to enhance the impact of the final part of the paper.

Although further delineation of the role of phospho-histone H3 in modulating nuclear fascin function would help to corroborate the ideas derived from the final figure of the paper, particularly to distinguish correlation from causation, this study demonstrates that nuclear fascin associates with histone H3, promotes nuclear actin, likely bundling, promotes DNA damage response and can induce apoptosis in cancer cell lines. The subcellular localisation of fascin, and its dynamic nuclear localisation, therefore appear important for regulating cancer cell behaviour. The idea that previously described nuclear envelope-localised fascin could serve as a pool of fascin for rapid nuclear import in response to cellular stress, discussed here, is very interesting. Given that fascin is upregulated in many solid tumours, questions around whether the spatiotemporal dynamics of fascin can inform prognostic assessments or can be targeted/modulated therapeutically in tumours will be exciting to discuss or address later. Overall, the quantitative characterisation of nuclear fascin functions will be of interest to cancer cell biologists, particularly those curious about the regulation of nuclear actin and its role in controlling cell behaviour.

Reviewer #1 (Public Review):

Langerhans cells are immunogenic and tolerogenic immune cells (part of the dendritic cell family) in the epidermis. They are therefore crucial in all immune responses that originate in the skin (e.g., allergic hypersensitivities, vaccine administration, immune surveillance against skin cancer/melanoma, etc.). The authors have previously detected the expression of this novel molecule, RETICULON 1A (RTN1A) in Langerhans cells - both in human and mouse epidermis. This manuscript is now the first evidence for a function of RTN1A in human Langerhans cells.

Langerhans cells are of dendritic shape and they need to migrate through connective tissues to lymph nodes in order to fulfil their immunologic functions. RTN1A (and other members of this protein family) are known from other dendritically-shaped cells in the nervous system. This led the authors to aim at elucidating whether RTN1A somehow regulated dendrites, migration and activation of Langerhans cells. Indeed, they find a link between RTN1A and morphology and function in Langerhans cells. The experiments described in this manuscript lead the authors to conclude that RTN1A regulates dendrite movement and morphology.<br /> - RTN1A promotes extension of dendrites and maintenance of dendritic shape in situ (determined in antibody inhibition experiments);<br /> - RTN1A does not allow or promote migration of Langerhans cells from the epidermis;<br /> - RTN1A inhibits calcium flux (determined in a model cell line);<br /> - RTN1A regulates cell adhesion and cell size (determined in a model cell line);<br /> - RTN1A in Langerhans cells is down-regulated by Toll-like receptor stimulation - allowing activation and migration;<br /> - likewise, this TLR-induced RTN1A down-regulation leads to the formation of large clusters of Langerhans cells in the epidermis.<br /> Overall the authors find that RTN1A maintains and regulates LC residency and homeostasis within the epidermis.<br /> Notably, all this work has been performed with healthy HUMAN skin.

A major strength of this work is its novelty. The authors delineate a well-defined function for RTN1A in human Langerhans cells for the first time. Their work also highlights some cell biological features (regulation of dendrite properties) that appear similar across dendritically shaped cells of very different origins (Langerhans cells, Purkinje cells, neurons). Another strength is the fact that the authors worked with primary human cells and tissues (skin, epidermal explants) ex vivo as much as possible. It should be emphasised that Langerhans cells are rare within the epidermis, therefore, large quantities of skin are needed for large experimental setups - a logistical challenge. Only for a few experiments did the authors resort to an established human cell line (e.g. to transfect it with RTN1A). Moreover, the paper contains outstanding fluorescence microscopy. Informative pictures, excellent photographic resolution!

There are no major weaknesses in this work. The methods are appropriate, results are sound.

Definitely, the authors achieved their aims, namely to find out what the novel molecule RTN1A does in human Langerhans cells. The data presented indeed support the conclusion that this molecule regulates the maintenance of the epidermis and, inversely, when missing or blocked, the immunologic migration of Langerhans cells out of the epidermis.

This is a valuable contribution to the topic of how Langerhans cells can remain within the epidermis and what allows them to migrate when immunologically needed. Langerhans cells are key immunostimulatory or tolerogenic (depending on context) cells in the body, and therefore this work will be of interest to the immunological, dermatological, and cell biology community.

Reviewer #1 (Public Review):

In this manuscript, Hansen and coworkers make use of the powerful, single-molecule assay CoSMoS to study the recognition of the 5' splice site by the U1 snRNP. Specifically, they investigate how 5' splice site oligos interacts with purified U1 snRNP to isolate 5' splice site-binding from other factors, including the CBC, BBP, and any other factors in whole cell extract that may impact binding; previous studies have investigated binding in vivo or in cellular extracts or with limited quantitative capabilities. The authors find evidence for a reversible, two-step, binding reaction in which a short-lived interaction precedes a long-lived interaction and in which binding depends on the 5' splice site sequence and the 5' end of U1. The data further suggests a compelling kinetic framework for how U1 surveys nascent transcripts for a bona fide 5'SS; specifically, both authentic and inauthentic 5' splice sites form the short-lived complexes but whereas the inauthentic complex preferentially dissociates, the authentic complex preferentially proceeds to a stable complex. Using oligos with different mutations to limit base-pairing they find that at least six potential base-pairs are required for association but that a stretch of seven base-pairs, with a maximum of one mismatch, is required for the long-lived interaction, with residues near the 5' splice site playing more important roles and with length being a stronger predictor of complex lifetime than thermodynamics, with implications for splice site predictions.

The work focuses on the determinants and mechanism of the first and a pivotal step in splicing, in a manner that completes recent structural advances. The work extends findings presented in a previous publication from the lab (Larson and Hoskins, 2017) studying binding of U1 snRNP to the 5' splice site in extract. In that study, the authors provided early evidence of two-step U1 snRNP binding in the absence of the cap binding complex or the branch point binding protein, with a more stable state following a weaker state; although factors in the extract may have influenced binding, the results are not qualitatively different here. The authors also showed some evidence in the previous study that longer binding depended on crossing a threshold and did not increase further with greater stabilization. Still, this new work is of high quality with conclusions justified by the data and of significant interest to the splicing field and of general interest to those investigating binding of snRNPs to nucleic acid.

Specific Points:

1. To test and define the role of protein in the snRNP, the authors need to investigate the roles of Yhc1 and Luc7 in 5' splice site binding in this assay, particularly with respect to defining the basis of asymmetry and snRNP destasbilization.

2. The similarity or difference of the two-step recognition mechanism described here to the recognition mechanisms of other nucleic acids by other RNP complexes is unclear. The authors need to put their findings into a larger context, relating their findings to studies of analogous systems described in the literature.

3. It is important that the authors address whether they can rule out that the exclusively long-lived complexes skip the short-lived conformation.

4. Given the co-transcriptional nature of many splicing events, the authors should discuss how recruitment by RNAP II might impact the two-step process. For example, fast dissociation by short duplexes might be countered by retention of U1 locally via RNP II.

Reviewer #1 (Public Review):

The manuscript focuses on an important question, how early life trauma causes aggression later in life. As aggression may ruin the life of both the aggressor and the victim and the life of their families, this question influences the life of a relatively large population. Uncovering the mechanisms of this behavior may provide options for treatment.

Based on transcriptome analysis, the authors suggest that epigenetic downregulation of TTR and the resultant hypothalamic decrease of thyroid hormone availability are responsible for the long lasting effects of early life trauma on the behavior. Using virus mediated gene knock down, the authors replicated the behavioral effects of the early life trauma demonstrating the involvement of decreased TTR expression in the development of aggression.

Strengths

The well defined experimental model and the selection of extreme phenotypes helps to identify the genes that are involved in the development of phenotype. The examination of females where the PPS does not cause aggression also helped to identify the important genes.

The suggested role of TTR in the development of aggression is proved by virus mediated gene knock down.

Weaknesses

However, the authors clearly demonstrated that both the TTR knock down and the early life trauma result in a decrease of hypothalamic thyroid hormone availability, they did not examine whether this local hypothalamic hypothyroidism is involved in the development of aggression. This question is important as in humans, hypothyroidism is not associated with aggression, rather increased T3 level was found in association with aggression. Therefore, it is possible that the decreased TTR expression causes the aggressive phenotype independently from its effect on the hypothalamic thyroid hormone availability. This could be tested by examining whether local hypothalamic T3 administration can reverse the aggressive phenotype of the used mouse models.

There is a discrepancy in the data. Despite of the large increase of hypothalamic TRH expression, the circulating thyroid hormone levels are not influenced. There are many TRH neuron populations in the hypothalamus and only a small portion of the hypothalamic TRH neurons are involved in the regulation of the circulating thyroid hormone levels. Therefore, it would be necessary to perform in situ hybridization to determine which TRH neuron population is regulated in the experimental model. Because of the unchanged circulating thyroid hormone levels, it is unlikely that the TRH expression is increased in the hypophysiotropic TRH neurons of the PVN. The in situ hybridization data could help to understand which cell populations of the hypothalamus could be involved in the development of aggression. For example, there is a TRH neuron population in the lateral hypothalamic attack area (PMID: 15908131) that could be involved in this behavior.

The authors measured serum total T4 and T3 levels. This could be misleading as the thyroid hormone binding capacity of blood may highly influence these data. Thus, measurement of free thyroid hormone levels would be far more informative.<br /> The quality of the images illustrating immunocytochemistry is very weak.

Reviewer #1 (Public Review):

Charpentier et al. use facial recognition technology to show that mothers in a group of mandrills lead their offspring to associate with phenotypically similar offspring. Mandrills are a species of primate that live in large, matrilineal troops, with a single, dominant male that fathers the majority of the offspring. Male breeder turnover and extra-pair mating by females can lead to variation in relatedness between group members and the potential for kin-selected benefits from preferentially cooperating with closer relatives within the group. The authors argue that the strategy of influencing the social network of their offspring could be favoured by "second-order kin selection", a mechanism by which inclusive fitness benefits are accrued to female actors through kin-selected benefits to their offspring. This interpretation is supported by a theoretical model.

The paper highlights a previously unappreciated mechanism for favouring association between non-kin in social groups and also contributes a nice insight into the complexity of social interactions in a relatively understudied wild primate species. The conclusions are strengthened by data showing associations between mothers were not influenced by the facial similarity of their offspring -- this suggests that mothers are making decisions based on the appearance of offspring and not their mothers.

Some remaining questions regarding the strength of the authors' interpretation exist:<br /> Given the challenges of studying mandrills in the field, the fact that the study reports data from a single group is understandable but potential issues remain with the independence of data points. There may be an additional issue arising from the fact that this troop is semi-captive.

The number of genotyped offspring is relatively small (n = 15) and paternity is inferred from the identity of the dominant male. However, the authors also refer to the fact that it's normal for female mandrills to mate with several males during ovulation.

What evidence is there to support a beneficial effect of nepotism in this species? What form could nepotism take and does it necessarily have to involve full sibs? If a female did not associate with offspring as shown here, would nepotistic interactions simply arise between her offspring and offspring that were less facially similar?

Joint Public Review:

While prime editing has been successfully implemented for hPSCs, its use for the generation of disease models is comparatively less explored. In this manuscript, Hanqin Li et al. set out to identify the most efficient methodology for correcting heterozygous mutations in human iPSC. For this purpose, the authors tested several known gene editing methods, including TALENs, conventional CRISPR/Cas9, and prime editing (PE) and, not surprisingly, found that PE resulted in the best balance of correct versus unwanted editing events.

In this process, the authors noted a lower editing efficiency of hPSCs, compared with tumour cell lines, and explored ways to improve it. Nucleofection of in vitro-transcribed mRNA-based delivery approach significantly increased the editing efficiency, without the need to select for targeted clones. The authors optimise the delivery of prime editing components and demonstrate that their optimised method can achieve >60% editing efficiency in hPSCs and be used for Parkinson's disease modelling.

Finally, they demonstrate that multiple rounds of mRNA-based prime editing can yield near complete editing of hPSCs, and extend their findings to disease-causing mutations.

Perhaps the major weakness of the manuscript is the relative lack of perceived novelty, since the different gene editing and delivery methods used in these studies have all been reported and tested in contexts that are not so distant to the one explored here. As a matter of fact, most findings in the paper (with the notable exception of mRNA delivery outperforming RNPs -but then again, the specific activity of the homemade recombinant nCas9-RT protein could be an issue and is not appropriately benchmarked) would have arguably been the best guess by researchers familiar with the literature on the topic.

At any rate, the study methodology is sound and the results are presented in a clear manner and strongly support the authors' conclusions. In combination with a streamlined workflow (or 'platform'), the optimized PE protocol described in this manuscript could very well be the go-to reference for editing heterozygous mutations in human iPSC. Additional strengths of this paper include having validated the most critical findings across genomic loci (4 different loci in 3 different genes) and 2 independent iPSC lines.

Although the utility of this method for more complex genetic editing needs to be investigated, the current platform paves the way for future prime editing methods for hPSCs.

Reviewer #1 (Public Review):

The stated goal of this research was to look for interactions between metabolism, (manipulated by glucose starvation) and the circadian clock. This is a hot topic currently, as bi-directional links between metabolism and rhythmicity are found in several organisms and this connection has important implications for human health. The authors work with the model organism Neurospora crassa, a filamentous fungus that has many advantages for this type of research.

The authors' first approach was to assay the effects of glucose starvation on the levels of the RNA and protein products of the key clock genes frq, wc-1, and wc-2. The WC-1 and WC-2 proteins form a complex, WCC, that activates frq transcription. The surprising finding was that WC-1 and WC-2 protein levels and WCC transcriptional activity were drastically reduced but frq RNA and protein levels remained the same. Under conditions where rhythmicity is expressed, the rhythms of frq RNA, FRQ protein, and expression of clock-driven "output" genes were also unaffected by starvation. The standard model for the molecular clock is a transcription/translation feedback loop dependent on the levels and activity of these clock gene products, so this disconnect between the starvation-induced changes in the stoichiometry of the loop components and the lack of effects of starvation on rhythmicity calls into question our understanding of the molecular mechanism of the clock. This is yet another example of the inadequacy of the TTFL model to explain rhythmicity. For me, the most significant sentence in the paper was this: "...an unknown mechanism must recalibrate the central clockwork to keep frq transcript levels and oscillation glucose-compensated despite the decline in WCC levels."

The author's second approach was to try to identify mechanisms for the response to starvation by focussing on frq and its regulators, using mutations in the frq gene and strains with alterations in the activity of kinases and phosphatases known to modify FRQ protein. The finding that all of these manipulations have some effect on the starvation-induced changes in WC protein level is taken by the authors to indicate a role for FRQ itself in the response to starvation. This conclusion is subject to the caveat that manipulations of the activity of multifunctional kinases and phosphatases will certainly have pleiotropic effects on many cellular processes beyond FRQ protein activity.

The third section of the paper is a major transcriptomic study of the effects of starvation on global gene expression. Two strains are compared under two conditions: wc wild-type and the wc-1 knockout strain, under fed and starved conditions. The hypothesis is that WCC has a role in the starvation response. The results of starvation on the wild-type are unsurprising and predictable: the expression of many genes involved in metabolic processes is affected. There are no new insights that come from these results and no new testable hypotheses are generated by the data.

The authors refer to the wc-1 mutant strain as "clockless" and discuss its effects on the transcriptome only in terms of WC-1's function in the clock mechanism. However, WCC is known to be a major transcriptional regulator, controlling a number of genes beyond the TTFL. As acknowledged earlier in the paper, WC-1 is also the major light receptor in Neurospora. The transcriptomics experiments were carried out in a light/dark cycle, with cultures harvested at the end of the light period, when "an adapted state for light-dependent genes can be expected" according to the authors. However, wc-1 mutants are essentially blind, and so those samples are equivalent to being harvested in the dark. The multifunctional nature of WCC complicates the interpretation of the transcriptomics data. The differences in the transcriptome between wild-type and wc-1 may not be due to loss of clock function, but rather the loss of a major multifunctional transcription factor, or the difference between light and "dark".

In the final set of experiments, the authors tested the hypothesis that the changes in the transcriptome between wild type and wc-1 might make wc-1 less competent to recover growth after starvation. They also test the recovery of frq9, a "clockless" mutant. The very surprising result is that the growth rates of these two mutants are slower than the wild type after transfer from starvation media to high glucose. This is surprising because there will be several generations of nuclear division and doublings of mass within a few hours and the transcriptome should have recovered fully fairly rapidly. A mechanism for this apparent "after-effect" is suggested with evidence concerning differences in expression of a glucose transporter, but it is not clear why this expression should not change rapidly with re-feeding on high glucose. As with previous experiments, the cultures were grown in light/dark cycles, which results in different conditions for the mutants, both of which have very low or absent WC-1 and are therefore blind to light. The potential effects of light have been disregarded.

The title of the paper refers to a "flexible circadian clock" but this concept of flexibility is not developed in the paper. I would substitute "the White Collar Complex" for this phrase: "Adaptation to starvation requires a functional White Collar Complex in Neurospora crassa" would be more accurate. Some experiments are also conducted using an frq null "clockless" strain, but because WC expression is very low in frq null mutants, any effects of frq null could also be attributed to WC depletion.

The major conclusion I took away from this paper is the multifunctional nature of the WCC as a transcription factor complex. It has been known for a long time that WCC controls the expression of many genes beyond the frq gene at the core of the circadian transcription/translation feedback loop. WC-1 is also the major blue light photoreceptor in Neurospora, controlling the expression of light-regulated genes, and this fact is barely touched on in the paper. These new data now extend the role of WCC in the regulation of metabolic networks as well.

Reviewer #1 (Public Review):

This manuscript attempts to explain the well-known difference in DNA mutation rates between father vs. mother (paternal mutation is 4 times higher than maternal mutation in humans). Although the mutation rate difference was believed to arrive from the number of cell divisions (male germ cells undergo many more divisions compared to female germ cells), recent studies suggested that most mutations arise from DNA damage (which will be proportional to the absolute time) rather than DNA replication-induced mutations (which will be proportional to the number of cell divisions). The authors thus revisited the question as to why the paternal mutation rate is higher (if absolute time is more important than the number of cell divisions in causing mutations). They used 'taxonomic approaches' comparing paternal/maternal mutation rates of mammals, birds, and reptiles, correlating them to specifics of reproductive mode in these species. To measure paternal vs. maternal mutation rate, they compared the mutation rates of neutrally evolving DNA sequences between the X chromosome vs. autosomes, as well as the Z chromosome (utilizing the fact that the X chromosome will spend twice more generations in females than males, while autosomes spend equal time. Likewise, the Z chromosome will spend twice more time in males than in females, while autosomes spend equal time).

They first confirm the paternal bias across a broad range of species (amniotes), eliminating many species-specific parameters (longevity, sex chromosome karyotype (XY vs. ZW), etc) as a contributor to the paternal bias. This implies that something common in males in these broad species causes paternal bias. They show that in mammals, the paternal bias correlates with a generation time. They propose that the total mutation is determined by the combination of the mutation rate during early embryogenesis (when both male and female have the same mutation rate) and the later mutation rate when two sexes exhibit different mutation rates. This model seems to explain why generation time correlates well with the extent of paternal bias in mammals. However, this does not explain at all why birds do not exhibit any correlation with a generation time. The speculation on this feels rather weak (although there is nothing they can do about this. Fact is fact).

The logic behind their analysis is well laid out and seems mostly sound. Their finding is of broad interest in the field.

- I am confused by this statement (the last sentence in the result section): 'If indeed the developmental window when both sexes have a similar mutation rate is short in birds then, under our model, generation times are expected to have little to no influence on α." Based on their model, if the early period is gone, when the mutation rates are similar between sexes are similar, intuitively it feels that generation time influences α even more. Am I missing something? (if the period with the same mutation rate is gone, then females and males are mutating at different rates the whole time).

- The authors state that this paper provides a simple explanation as to why paternal biases arise without relying on the number of cell divisions. However, it seems to me that the entire paper relies on the recent findings that mutation arises based on absolute time (instead of cell division number), and the novelty in this paper is the idea of 'two-phase mutation rates' to explain the observed numbers of paternal bias in various species. Yet it fails to explain the mutation rate difference in birds. There is not enough speculation or explanation as to what determines different mutation rates in males of various species. Although the modeling seems to be sound and there is nothing that can be done experimentally, I felt somewhat unsatisfied at the end of the manuscript.

Reviewer #1 (Public Review):

In Drosophila germline, most piRNA loci use a non-canonical mechanism to transcribe piRNA precursors at the presence of H3K9me3, which depends on an HP1a paralog called Rhino/HP1d that specifically binds piRNA loci. How does Rhino find the right loci to bind? The current model in the field posits that maternally deposited piRNAs provide a specificity cue for Rhino. Now, Baumgartner et al. from Brennecke Group described a novel factor, the ZAD zinc-finger protein CG2678/Kipferl, that appears to provide another key specificity input to a subset of Rhino's chromatin binding, specifically in differentiated female germline (but not in males or stem/progenitor cell types in the female germline). Using genetics, genomics, genome editing, microscopy and biochemical approaches, Baumgartner et al. propose that Kipferl binds a G-rich DNA motif and, at the presence of local H3K9me3, recruits and/or stabilizes the binding of Rhino to these loci and then convert them from transcriptionally inert heterochromatin to piRNA-producing loci. Overall, the text is well written, the figure is clear, and the data is of high quality. With some additional experiments and text edits, this work represents a significant contribution to the field and should attract readers working on piRNA, transposon, satellite DNA, zinc-finger proteins, HP1 and heterochromatin.

Specific concerns

1. The genetic hierarchy between Kipferl and Rhino requires further clarification. Authors seem to propose a simple model where Kipferl acts genetically upstream of Rhino. This simple hierarchy is at odds with several observations. First, the center of Kipferl binding generally has less Kipferl binding without Rhino (Fig 5D). In some cases, Kipferl binding is completely gone without Rhino (Fig 7E middle, bottom). The text describes the loss of Kipferl spreading without Rhino but should also mention this reduction/loss in Kipferl binding. The effect of rhino-/- on Kipferl's chromatin binding should be shown along with wildtype level of Kipferl enrichment in Fig 5C for proper comparison. How should readers understand the effect of Rhino on Kipferl? What is the prominent Kipferl domain in rhino-/- in Fig 5B? Second, the broad binding of Kipferl is gone in rhino-/-, does it mean Kipferl requires Rhino to spread? Or, could Rhino (that is recruited by maternally deposited Piwi/piRNA) recruit Kipferl to neighboring sites, which look like a spreading phenomenon? Most importantly, the argument of Kipferl recruiting Rhino should be directly demonstrated by a sufficiency test in addition to the presented evidence of necessity. Could authors tether Kipferl in H3K9me3-decorated regions to see if Rhino is recruited and vice versa? Observations like 42AB in Fig 5E make one wonder if Rhino also recruits Kipferl, so their relationship is not simply Kipferl recruiting or acting upstream of Rhino, as described throughout this manuscript. Clarifying the relationship between Kipferl and Rhino is essential as it is a central claim made.

2. DNA binding of Kipferl remains putative. Since the 4th zinc-finger is shown to impact Kipferl localization via interaction with Rhino, it remains formally possible that the first three zinc-fingers control Kipferl localization via protein-protein interaction rather than direct DNA binding. Unless direct biochemical evidence of Kipferl binding DNA is available, the DNA binding of Kipferl should be toned down and described as putative and requires further investigation in text.

3. The relative contribution of maternally deposited piRNAs versus Kipferl in recruiting Rhino is unaddressed. Prior work from multiple groups including Mohn et al. 2014 Cell from the same group of this manuscript suggested a role of maternally deposited piRNAs in determining a subset of H3K9me3 domains as Rhino binding sites. Is Kipferl or maternally deposited piRNA a better predictor of Rhino binding? This manuscript proposes that Kipferl binds a simple G-rich motif and at the presence of H3K9me3 recruits Rhino binding. The readers are left wondering where maternally deposited piRNAs fit in the model of Rhino recruitment, which should be tested or discussed in text, as maternally deposited piRNA is seen as the key determinant of Rhino binding before this work.

Reviewer #1 (Public Review):

This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. HOPS has been characterized extensively through biochemical studies, which indicate that HOPS cooperates with SNAREs to facilitate membrane fusion. The authors conclude that HOPS is not a highly flexible structure as has been proposed, but instead has a stiff backbone to which the SNARE-binding Vps33 subunit is tightly anchored. Because the ends of HOPS bind to opposing membranes, the implication is that HOPS acts as a lever and membrane stressor, thereby amplifying the effects of SNARE assembly and catalyzing fusion.

The structural biology analysis was based on an improved purification protocol and appears to be well done. An atomic-level structure is always valuable, and this contribution will undoubtedly guide further research involving HOPS. Initial steps in this direction are presented in the form of functional studies of structure-guided mutants.

Structures are most useful when they help to define mechanisms, and the authors argue that the HOPS structure explains how HOPS catalyzes membrane fusion. The key conclusion is that the antiparallel association of the Vps11 and Vps18 subunits create a rigid core for the complex, leaving flexible ends that bind the Ypt7 GTPase to anchor the two membranes. This model is inconsistent with earlier suggestions that HOPS bends to bring the two membranes together. Instead, the inferred rigidity of the HOPS core, combined with the central location of the SNARE-binding module, suggests that HOPS acts as a lever that exerts a force on the membranes to promote SNARE-driven membrane fusion.

This interpretation is interesting and potentially exciting, but I question why the authors are certain that the Vps11-Vps18 core is truly rigid. Proteins can undergo all sorts of rearrangements. Is there evidence that Vps11 and Vps18 interact strongly and in a unique configuration? Portions of a protein that have a consistent structure in vitro might nevertheless rearrange during functional interactions in vivo. If there is any flexibility of the Vps11-Vps18 core, this property combined with the evident flexibility of the Ypt7-binding portions and the low affinity of Vps41 for Ypt7 would make HOPS anything but a rigid membrane stressor. If the authors wish to make a strong point about the functional implications of the HOPS structure, these points need to be addressed.

Reviewer #1 (Public Review):

The authors used a combination of biochemical assays and cryoEM to investigate the role of PME-1 in regulating PP2A, which revealed that PME-1 uses its unstructured loops to associate with the B-domain of the PP2A holoenzyme to regulate the function of the C-domain. This is a high quality work. This reviewer finds the later work involving p53 to be a helpful step in explaining the role the PME-1:PP2A interaction can have on important phosphorylation pathways, but I consider this aspect of the work to be very preliminary, especially given its rather minor effects. That said, the authors do not make claims that extend beyond the scope of the evidence they provide and thus I find the connection and discussion of PME-1, PP2A and p53 to be suitable on the whole.

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to discover mechanism(s) that would allow the bacteriophage T4 to overcome the phage defense exerted by the toxIN toxin-antitoxin system, which itself was engineered into an E. coli strain (in trans on a medium copy plasmid). To identify ToxN-resistant phages, experimental evolution was used as a method of choice. The resistant phages obtained after ~5-25 rounds of propagation on toxIN -/+ cells were subsequently sequenced. The depth of sequencing reads thereby revealed the amplification of a two-gene operon for which the authors show causality for ToxN resistance (precisely, for one of the two genes, namely tifA). Through an elegant series of experiments, the authors further demonstrate that the evolutionary benefit of the phages with respect to the toxIN defense system occurred an at evolutionary cost, namely the loss of other accessory genes. These (large) gene deletions differed between the parallel evolved phages, showing a different solution to the same problem, namely phage genome reducing - likely to keep compatibility with the headful capsid packing approach of the phage.<br /> Importantly, the authors also demonstrate that the loss of these accessory genes narrowed the phages' host range given that those lost genes encode anti-defense proteins against other phage defense modules (including unidentified systems in well-studied E. coli strains).

Collectively, this work recapitulates the arms race between phages and their host and showed how adaptation to one host and overcoming its anti-phage barrier can compromise future infection of other hosts. Importantly, while the selected gain-of-function was based on a similar strategy in the parallel evolution lines (that is, amplification of the antitoxin-encoding gene tifA), the lost accessory genes differed amongst the independently evolved phages. These different solutions to the packaging problem likely benefit the phage on a population level once the phages encounter a new host.

The major strength of the study lies in the impressive combination of experimental evolution, genomics, and genetics, which allows the authors to identify genomes changes and demonstrate their causality. Very well executed work for which the authors should be congratulated.

There are only very minor weaknesses, which are solely related to the presentation of the data and the discussion.

Overall, this study is likely to impact significantly future research, given the findings (e.g., host switch based on genomic rearrangements) but also the methodology. Importantly, this study also demonstrates once again the power of experimental evolution with respect to pinpointing new anti-defense elements (such as IPIII in this study), which will help to uncover new anti-phage defense systems in the future (as, for instance, the unknown system in strain ECOR17, as mentioned in the manuscript).

Reviewer #1 (Public Review):

This paper presents a detailed molecular investigation into the behavior of PIP5K kinase, a membrane associated enzyme that catalyzes the production of PIP2 from PIP in cell membranes. Building on previous work on this system, the researchers use single-molecule fluorescence microscopy to study how the oligomeric state of PIP5K impacts membrane binding, PIP phosphorylation, and compositional patterning of lipid domains leading to stochastic bistability in membranes.

A highlight of this study is the extensive experimental approaches that combine various single-molecule analyses, including diffusion and residence time distributions, as well as macroscopic measurements of membrane binding isotherms and PIP2 production. With this, it becomes evident that PIP5K exists in both monomeric and higher-order oligomeric forms, with the latter potentiating catalytic activity. This coupled to cooperative binding to the membrane linked to PIP2 production leads to a positive feedback system where patterning of the lipid composition emerges with stochastic bistable behavior, with oligomerization of the kinase acting as a modulating factor. This aspect of the research is interesting as it connects the higher-order oligomerization of the protein kinase to a means of modulating self-organization of the lipids within the cell membrane, a phenomenon that may be important for optimizing cellular signalling in biology.

The majority of the studies are carried out carefully and with exquisite single-molecule approaches. However, a weakness of the study is that the ultimate conclusion of the activity linked specifically to dimerization is not clearly supported by the data. The results presented reflect a comparison of monomers vs. oligomers, without a clear identification of conditions where dimers persist. The mutation constructed to disrupt dimerization shifts the system to monomers with an associated decrease in catalytic activity. However, this finding does not provide a strong connection to the dimer state, but rather the loss of the effect when oligomerization is disrupted. Other properties of the protein may be impacted, such as stability and fold, as well as the overall binding propensity to the membrane. The catalytic activity measured per PIP5K molecule does indicate that an increased density for the wild-type protein leads to an increase in the rate of PIP2 production, providing evidence that oligomerization increases function. Yet, many of the results throughout the paper provide support for general oligomerization rather than dimerization, and so further investigation is needed in order to clarify the interpretation in what is otherwise an interesting system and study.

Reviewer #1 (Public Review):

Here, the authors have followed up their prior work on the structures of individual domains of the cyclic GMP-dependent protein kinase Iα/β (PKG Iα/β) and have generated a crystal structure of residues 71-686 of PKGIβ bound to AMP-PNP:Mn2+ in the absence of cGMP, representing a nearly full-length protein lacking only the N-terminal leucine zipper dimerization domain. In general, the individual domain structures resemble those determined previously by the authors and other groups. The AI motif in the R domain occupies the active site, as expected, and is part of an extended interface involving the helical subdomain of CNB-A with αG-helix of the C-domain. In addition, the αA helix of CNB-B interacts with the activation loop, and the αB helix of CNB-B contacts a loop in the C-lobe of the C-domain, with the CNB-A and CNB-B interdomain helix interacting with pT532 in the activation loop. Together these four R:C contacts allow the R-domain to grip the C-domain between its two CNB domains, ensuring complete inhibition of catalytic activity. The structure of the autoinhibited C-domain is similar to that of the isolated PKG C-domain and that of the PKA C-domain, but there are differences that could prevent unwanted crosstalk between cGMP and cAMP, for instance by preventing PKA RIα from binding to the PKG Iβ C-domain. Subtle changes in the individual CNB-A/B structures as well as their relative orientation upon cGMP binding suggest a mechanism for releasing the autoinhibited C-domain allowing its activation, in which cGMP binding to the CNB-A phosphate-binding cassette alters its conformation so that it cannot bind to the C-domain but instead promotes R:R interactions. Finally, the authors solved the crystal structure of the isolated CNB-A domain of PKG Iα bearing an activating R177Q disease mutation, whose structure reveals that the mutant would not be able to adopt a closed conformation and instead closely mimics the cGMP-bound WT CNB-A, suggesting a mechanistic basis for the constitutive activation of the R177Q PKG Iα, which is linked to TAAD syndrome.

This new structure of nearly full length PKG Iβ provides a significant advance in our understanding of how the cGMP-dependent protein kinase is autoinhibited in the absence of cGMP, and provides a plausible mechanism for how it is activated upon binding cGMP, as well as defining the structural basis through which crosstalk between PKA and PKG is precluded. The main uncertainty is whether the autoinhibited PKG Iβ normally resides in a "monomeric" or "dimeric" state. The crystal structure consists of a dimer in which the linker between the R and C domains is not visible making it impossible to determine whether the R domain of one monomer interacts with the C-domain of the other monomer in the dimer or rather with its own C-domain, i.e., is autoinhibition occurring in cis or trans. Based on the results of biochemical experiments in which WT and kinase-dead mutant PKG Iβ with and without an activating KR/EE AI region mutation were co-expressed, the authors concluded that autoinhibition occur predominantly in cis, consistent with the PKG Iβ 71-686 fragment being monomeric and inactive in solution. This conclusion seems reasonable, but the issue may only be fully resolved by a structure of the intact PKG Iβ dimer. If autoinhibition does occur in cis, this leaves open the question of why PKG 1 is dimeric, and this was not really illuminated by the present structures.

The other point is that while the structure itself is a significant advance in our understanding of how PKG Iβ is autoinhibited, the paper would be strengthened by some additional analysis of the functional effects on kinase activity and cGMP regulation of mutating PKG Iβ at newly defined contact residues in the autoinhibited structure that the authors conclude are key to autoinhibition. As it stands, the only mutation that was analyzed functionally was the KR/EE AI region mutant, which as predicted was activating.

Reviewer #1 (Public Review):

They adopted a comprehensive experimental and analytic approach to understand molecular and cellular mechanisms underlying tissue-specific responses against 3-CePs. They used two cell lines - BxPC-3 and HCT-15 - as example models for responsive and non-responsive cell lines, respectively. Although mutation rates didn't differ by the drug treatment, they observed changes in cell cycle and expression of genes involved in DNA damage, repair and so on. Furthermore, they combined RNA-seq and ATAC-seq data and applied two approaches, pairwise and crosswise, to identify a number of gene groups that are altered in each cell line upon the drug treatment. Finally, they calculated enrichment of up/down genes in different cell lines, tumor types and samples to estimate potential responsitivity against the drug. This study is unique in in-depth analysis of RNA-seq and ATAC-seq data in identifying genetic signature underlying drug treatment. This study has the potential to be applied to different drugs and cell lines.

However, several major concerns need to be resolved. First of all, the biological and clinical performance of 3-CePs is not clearly described. They referenced several papers but they seem to have focused on the chemical properties of the drug. Without proven activity of 3-CePs against cancers in vitro and in vivo, the rationale of the study would be compromised.

Their RNA-seq analysis was focused on discovering differentially expressed genes between cell lines, time points, etc. Interestingly, they found that DNA damage and repair signal was specifically increased in HCT-15. But is this approach capable of finding signals that are constitutively expressed in different cell lines? In other words, what if the differential responsiveness to 3-CePs was already there even before the drug was introduced?

Are there any overlapping signals between pairwise vs crosswise approaches?

Probably a similar question with the above: is this methodology applicable to other drugs in addition to 3-CePs?

Reviewer #1 (Public Review):

The manuscript provides a dataset of single-cell transcriptomics of several adult mice ovaries and performs computational analysis to determine the molecular signatures of the cells isolated.

Strengths:<br /> - Provide data from different estrous stages and lactating.<br /> - Many markers are validated.<br /> - Several estrous cycle-specific biomarkers are revealed.

Weaknesses:<br /> - It does not stratify or provided trajectories of the data according to the different estrous stages and lactation periods.<br /> - Only single markers are validated, making it difficult to see the population.<br /> - The population of peri-ovulatory GC could be better characterised.<br /> - There is no mention of specific populations or states in the lactation sample.<br /> - Monocle analysis could be made more robust.<br /> - Specific populations of theca cells (interna and externa) are not named.<br /> - Differences between stroma 1 and stroma 2 are not found.<br /> - OSE is only mentioned in the Discussion.

Reviewer #1 (Public Review):

This manuscript reports a new analytical method (rhapsodi) to impute genotypes on human gamete data. The authors characterize the specificity and sensitivity of the approach and benchmark it against the current tool to analyze gamete data. rhapsodi is more efficient and versatile than the current approach, and thus represents an important technical feat. The last analysis of the manuscript is a reanalysis of the SpermSeq dataset, a massive sequencing effort to characterize recombination in human sperm haplotype data. rhapsodi fails to find any deviations from random segregation and challenges the notion that there are distorters in the human genome. In general, the manuscript represents an important technical piece but the results could be better contextualized to provide a perspective of what are the implications of the findings for our understanding of human recombination and segregation distortion.

Reviewer #1 (Public Review):

In an effort to better understand the regulatory switch controlled by distinct phosphorylation sites in the C-tail of AKT (pS473 versus pS477/pT497) the authors set out to mutate basic sites on the AKT N-terminal PH domain. In so doing, they found that mutation of one basic residue (R86) reduced AKT enzymatic activity. This result prompted the hypothesis that loss of the R86 side chain might stabilize the autoinhibitory form of AKT (specifically the PH/Kinase interaction). Consistent with this hypothesis the authors report that the R86A mutation increases the binding of the PH domain to the AKT kinase domain. Subsequent solution NMR data suggest stabilization of the PH domain occurs upon mutation of R86 to Ala. The authors also make excellent use of segmental labeling combined with NMR spectroscopy to gain additional insight into how mutation of R86 to Ala affects the PH/kinase domain interaction. The findings underscore the importance of solution measurements in understanding the effects of dynamics, conformational heterogeneity, and structural pre-organization in regulating function (crystal structures of the wildtype and R86A PH domains are nearly identical).

Given the oncogenic mutant E17K drives AKT function, it is interesting that the only differences (outside of the site of mutation) between the crystal structures of WT and R86A mutant PH domains are differences in side chain orientations for E17 and Y18. This difference led the authors to further investigate the role of positions 17 and 18 in autoinhibition and to explore the connection to the R86 side chain. The findings support the conclusions that Y18 in particular is involved in autoinhibitory interactions with the AKT kinase domain and that loss of the R86 sidechain leads to preorganization of the PH domain (and the Y18 sidechain) for enhanced autoinhibition. These results, therefore, advance our understanding of AKT autoinhibition and extend previous reports of AKT regulation and the role of E17. Specifically, the authors suggest (based on the findings reported here and the previously determined crystal structure of the AKT E17K PH domain) that the E17K mutation drives AKT activity by engaging with Y18 in a manner that restricts the Y18 sidechain from mediating autoinhibitory contacts with the kinase domain. Moreover, their findings provide more detailed insight into the interplay between C-tail phosphorylation status and the effects of E17K on AKT catalytic activity. The new insights into the mechanism by which E17K drives AKT activity add to the previously described roles of E17K in augmenting affinity for phospholipid (Carpten et al. 2007, Landgraf et al. 2008, Truebestein et al. 2021).

Crystal structures of autoinhibited AKT have thus far only been obtained in the presence of exogenous ligands, either small molecule inhibitors or a covalently linked nanobody. While these structures are valuable, the field lacks high-resolution structural information for autoinhibited AKT without ligands and it is thought that bound ligands shift the conformational preferences and obscure the physiologically relevant autoinhibitory interfaces. The work presented in this manuscript makes process toward a better understanding of the physiologically relevant contacts between PH and kinase domains that control activity. Another gap in the information provided by existing crystal structures is the lack of electron density for the important activation loop in the kinase domain. Interestingly, the authors note that the AlphaFold-predicted AKT structure reveals a potential interaction between Y18 and F309 on the activation loop. This prompted experimental approaches (kinase assays and 19F NMR) the results of which largely support a regulatory role for F309. Thus, the solution-based experiments presented here provide significant insight beyond avail crystal structures.

Overall, the manuscript is very interesting - in particular the allosteric connections between R86 and the E17-Y18 dipeptide segment. The resulting insights into the role of the PH domain residue Y18 in autoinhibition and a mechanism by which the oncogenic E17K mutation might alter the side chain conformation of Y18 to interfere with autoinhibition add significantly to our understanding of AKT regulation.

Reviewer #1 (Public Review):

Using data from a tetraplegic individual, the authors first addressed whether the neural representations for attempted single finger movements would still be organized in a way that is typical for healthy participants. They did this by comparing the distances between attempted finger movements in the implanted area to fMRI measures in healthy participants in M1 and ROI that mostly encompassed BA5 (SPLa). The representational structure was more similar to M1 than to SPLa. One weakness in the current posted version is that a) the comparison RDM differs strongly in their reliability and b) the SPLa RDM is likely not very well matched for the implanted location.

Secondly, they test how the representational structure would change during task training on a simple finger classification task. The authors convincingly demonstrate the stability of the representational structure of the finger movements despite ongoing training and continued confusion between middle, ring and pinkie finger.

Finally, they demonstrate that the representational structure in the recorded area switches from a more muscle-like representation to a representation that is better explained by an orderly sensory mapping, even though the central-peripheral exchange of sensory-motor signals was completely disrupted in the tetraplegic individual.

Together the results have potentially important practical implications for the placement of BCI implants, as well as theoretical implications for the role of the implanted region in sensory-motor control of the fingers.

Reviewer #1 (Public Review):

Okawa et al show that topical oral application of an agent used in SPECT imaging, hydroxymethylene diphosphonate (HMDP-DNV), displaces pre-existing nitrogen-containing bisphosphonate (N-BP) from the jawbone of mice and prevents the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ), a devastating complication that rarely occurs after invasive dental procedures in N-BP treated patients. They further demonstrate pro-inflammatory genomic signaling in gingival cells of N-BP treated mice, which reverses with HMDP-DNV. The methods are well-described overall and the results are potentially important. However, limitations include the short study period and the lack of multiple time points. Additional data to address these limitations would help to strengthen the authors' conclusions. If these results are added, this work could have a high impact in the field and the data could set the stage for further testing. The significance lies in the unmet need for therapeutic options to prevent this complication, which is widely dreaded and impedes the use of often needed bisphosphonate therapy.

Reviewer #1 (Public Review):

In this paper, Chaudhary et al assessed 143 children with AML, and out of 20 mitochondria-related DEGs that were chosen for validation, 16 were found to be significantly dysregulated. They show that upregulation of SDHC and CLIC1 and downregulation of SLC25A29 are independently predictive of lower survival, which was included in developing a prognostic risk score. They also show that this risk score model is independently predictive of survival better than ELN risk categorization, and high-risk patients had significantly inferior OS and event-free survival. The authors demonstrated that high-risk patients are associated with poor-risk cytogenetics, ELN intermediate/poor risk group, absence of RUNX1-RUNX1T1, and not attaining remission (p=0.016). The risk score also predicted survival in the TCGA dataset. They concluded that they have "identified and validated mitochondria-related DEGs with prognostic impact in pediatric AML and also developed a novel 3-gene based externally validated gene signature predictive of survival."

Although this paper is interesting, it lacks novelty and does not advance the field significantly. The authors have used a similar approach in their recent paper in Mitochondrion where they showed that PGC1A driven increased mitochondrial DNA copy number predicts outcome in pediatric AML patients. Additionally, the authors have a small number of patients and chose only 20 genes for their analysis.

Reviewer #1 (Public Review):

In the current manuscript, the authors developed a general framework to study the evolution of multicellular life cycles and the investigated evolutionary advantage of certain life cycles and multicellular structures over other ones. Simple multicellular life cycles are comprised of growth of the propagule into a colony and its fragmentation to give rise to new propagule. For the evolution of multicellularity, a multicellular trait is not only identified by the genotype of individuals inside each propagule but also the life cycle it is programmed for growth and fragmentation. There is not a single fitness value but a set of fitness values, each assigned to one stage of life-cycle growth before fragmentation. The question is how natural selection chooses one life cycle over the other one. In other words how a robust life cycle is evolved from random fragmentation processes. Previous theoretical approaches mainly considered overall growth rate as a measure of advantage for a life cycle.

This work is based on an extension of the several previous works of Pichugin and Traulsen on the subject. It introduces interaction between different stages of life cycle, as well as interaction between two traits, identified with differences in life cycle patterns. For brevity and focus on life cycle patterns, possible intra-propagule genotypic heterogeneity is ignored. (This has been addressed by same authors and others in past works.) A deterministic system of ordinary differential equations is set to describe the growth and competition of different life cycle stages. Abundance of each life cycle stage is the dynamical quantity and the dynamics is reminiscent of a general replicator equation for a complex multicellular structure. The interaction terms is identified by a kernel matrix, K_ij, which is effectively fitness payoff for a group of size i when encountered with a group of size j. Interaction terms introduces effective elevation in death rates. They focus on two main scenarios, 1) a killer kernel where the kernel is only a function of and 2) a victim kernel where is only a function of. In some cases authors considered more general cases including arbitrary (random-valued).

Authors first considered the dynamics of a single life-cycles where the interaction between populations at different stages of life cycles changes the growth dynamics. They observe that the general dynamics and steady states are governed by overall growth rate of the whole lief-cycle as has been observed in the absence of group-group interactions. They suggest the modified steady states while there is no qualitative changes from no-interaction (diagonal kernel or constant-selection) case.

The second part of the work focuses of competition between two and multiple life cycles in the presence of the group-group interactions. The authors considered invasion of one rare multicellular life cycle into another resident multi-cellular life cycle. They also consider competition between multiple life cycles. They discussed the condition for ESS in this scenario. Four interaction schemes including killer and victim kernels are discussed for some examples of fragmentation. Furthermore, competition of multiple life cycle is discussed. In particular a three life cycle competition is discussed using similar kernel interactions which now resemble a rock-paper-scissor type payoff in some cases.

I believe the modeling framework to address competition and natural selection between life cycles in the same framework that introduces interaction between different stages of a same life cycle is a great step forward in modeling evolution of simple multicellularity, The results are very clear and I think further analysis of the model introduced in this work can have a strong impact on our understanding of the evolution of multicellular life cycles.

Reviewer #1 (Public Review):

Richardson et al. used a multivariable Mendelian randomization framework to separate the genetically predicted effects of adiposity at two timepoints in the lifecourse, childhood and adulthood. They used data from the Avon Longitudinal Study of Parents and Children (ALSPAC). Higher childhood body size had a direct effect on lower vitamin D levels in early life, after accounting for the effect of the adult body size genetic score. However in midlife, childhood body size impacted on adult obesity to result in lower vitamin D levels. The authors conclude their findings have important clinical implications in terms of the causal influence of vitamin D deficiency on disease risk. They also serve as a proof of concept that the timepoints across the lifecourse at which exposures and outcomes are measured can impact any overall conclusions drawn by MR studies. In particular, the study underlines the significance of obesity in increasing the risk of vitamin D deficiency.

The strengths of this paper are the robustness and rigour of the methods using an established longitudinal cohort and the Mendelian randomization method.

A weakness is the lack of contrast of the authors findings from Mendelian randomization with those relevant findings from recent large randomised controlled clinical trials of vitamin D supplementation. In particular, two have shown an interaction between outcomes and BMI, a clinical measure of obesity.

Reviewer #1 (Public Review):

This manuscript describes in detail the role of glutamine in chondrocyte metabolism. The authors provide an extensive investigation into the anabolic respiratory effects of glutamine deprivation including its effect on other sources of energy such as glycolysis. Their premise is also backed up by several modes of investigation, including the use of the pharmacological inhibitor CB-839. The manuscript is decently written and there are numerous well-laid-out figures to support conclusions.

The main issue at hand is the reconciliation of the hypothesis with other recent work targeting this area. For example, Ma et a. Clin Sci (2022) recently published a paper demonstrating that glutamine supplementation, as opposed to deprivation, leads to a reduction in osteoarthritis symptoms and reduction in NF-kappaB activity. While it is possible for both mechanisms (e.g. deprivation and supplementation) to lead to similar outcomes, exploration of this topic would be of interest to the readership.

Reviewer #1 (Public Review):

This paper describes a new software tool: smartScope, for automated screening of cryo-EM grids. SmartScope can also perform automated data collection on suitable grids, including using beam-image shifts and tilted stage geometries. SmartScope uses deep-learning approaches for the selection of squares and holes of interest. The description of the software given in the paper is very promising, and as the code has not yet been made available, I cannot comment on its modularity, ease of installation, or general usability.

The convolutional neural networks for square and hole detection were trained on relatively few examples, and supposedly all from the same microscope. How easy would it be for users to re-train these detectors for their own purposes? Could a description of that be added to the paper/documentation?

The introduction makes the same point over multiple pages, and could probably be easily cut in half length-wise. This will force the authors to formulate more succinctly, and thereby more clearly. Hopefully, this would then eliminate wooly or incorrect statements like: "the beginning of each new project is fraught with uncertainty", "[The number of combinations] grows exponentially with the inclusion of each parameter" (it doesn't!), "would be an invaluable tool".

Also, the first half of the Abstract needs some rewriting. It focuses first on grid optimisation, which is not what smartScope is about. SmartScope is about grid screening. Just say that and save some lines in the Abstract too.

Lines 257-261 describe some setup in serialEM. Perhaps because I am not familiar with that software myself, but I had no clue what those lines meant. Perhaps some example setup files could be provided as supplementary information?

For the DNA polymerase data set: mention in the Results section how long the entire data collection (or 4.3k images) took. Also, the sharpened map in the validation file has a very weird distribution of greyscale values. Its inclusion of volume with varying greyscale is basically a step function, indicating that this is more or less a binary density map. I suspect that this is a result of the DeepEMhancer procedure. But given that the scattering potential of proteins is not binary, I wonder how such a map can be justified. Also, the FSC curve shown in the paper does not mention any masks, but the reported resolution of 3.4A is higher than the unmasked resolution calculated by the PDB: 3.7A. Why is the DeepEMhancer software used here? Is it hiding a slightly suboptimal map? As map quality is not what this paper is about, perhaps it would suffice to show the original map alone?

Reviewer #1 (Public Review):

This paper used two linear tracks of different cues as two contexts and tested the rate modulation of contexts during behavior and during replay events. They showed that not only sequential information, but rate information also are encoding information and that they are reinstated during replay events. This is super exciting! The data about how things change during sleep is also timely and important.

My primary criticism of this paper is that it misses the opportunity to give some key details about the statistics of neural activity during 'ripples' rather than studying identified replay events. A secondary criticism is that they limit their analyses to neurons that have place fields in both environments. I think the activity of the other 3 categories of neurons (active in Track 1 only, active in Track 2 only, and not active in either track) are also of critical interest.

Reviewer #1 (Public Review):

TRPV1-targeted therapies for pain have failed because of the effects of these drugs on thermoregulation: TRPV1 channel agonists produce acute hypothermia in animals and humans, whereas TRPV1 antagonists cause acute hyperthermia. TRPV1 activity in sensory neurons sends pain signals to the brain, but also causes the release of pro-inflammatory neuropeptides such as CGRP. TRPV1 channels are also expressed in vascular smooth muscle cells of arterioles. It is unclear which of these TRPV1-associated functions is responsible for the alterations in thermoregulation caused by TRPV1 channel agonists and antagonists. In this study it is shown that ablation of TRPV1 only in sensory neurons of transgenic mice prevents hypothermia caused by the selective channel agonist capsaicin and hyperthermia by the selective antagonist AMG 517. Conversely, ablation of TRPV1 channel expression in vascular smooth muscle cells slightly accentuated the hypo- or hyper-thermic responses caused by delivery of the agonist or antagonist, respectively. These results indicate that drug-induced changes in TRPV1 channel activity in sensory neurons are responsible for the alterations in body temperature, whereas activity of TRPV1 channels in the vasculature appear to counteract these alterations to a small extent. Importantly, transgenic mice did not show any impairments in body temperature regulation in the absence of drugs. The effects of drugs on body temperature were also eliminated in mice where central sensory terminals were ablated with capsaicin. In this setting, the sensory nerve endings can still release neuropeptides when TRPV1 channels activate, but have no electrical communication with the brain, indicating that it is the electrical signaling and not the neuroinflammatory responses which cause alterations in body temperature when TRPV1 channels are challenged by an agonist. This was further supported by results in mice deficient in the neuropeptide CGRP, which still experienced hypothermic responses when treated with capsaicin.

The data in the manuscript provide important constraints towards understanding the role of TRPV1 channels in thermoregulatory processes, and suggest that analgesic drugs that impair calcium permeability through TRPV1 channels without affecting sodium permeability could prevent pain caused by neurogenic inflammation without altering body temperature. The experimental design in this report is straightforward, adequate controls were included, and the results appear robust. However, there are also some concerns and limitations.

First, the major goal of the study is to determine whether TRPV1 channels expressed in the vasculature or in sensory neurons are responsible for the effects of drugs on body temperature. However, no clear justification is provided for how vascular TRPV1 channels could potentially give rise to the observed alterations in body temperature caused by drugs, as it would be generally expected that systemic treatment with an agonist would result in vasoconstriction and hyperthermia, and treatment with an antagonist give rise to vasodilation and hypothermia. These responses are opposite to the described effects of agonists and antagonists on body temperature, and therefore potentially rule out a contribution of vascular TRPV1 channels without necessarily requiring additional experimental testing.

Second, the effects of drugs on body temperature are shown as smoothened differences between the body temperature of control and test mice, rather than absolute body temperature in all groups of animals. This visualization obscures variability between organisms, which could contain additional relevant information, and is essential for an accurate assessment of the robustness of the results, particularly given the small numbers of animals that were tested and the high variability in the data. Statistical tests compare differences between WT and TRPV1-deficient mice after treatment with TRPV1 channel agonists and antagonists. However, these comparisons provide no information on whether there are statistically significant changes in the body temperature of TRPV1-deficient animals after treatment with drugs relative to animals treated with vehicle. This latter comparison is of higher clinical and physiological significance than what was performed in the study.

A minor third point is that experiments where TRPV1 expression was ablated in animals 8 weeks after birth appear to show opposite effects of agonists and antagonists relative to wild type mice: agonists seem to produce hyperthermia and antagonists cause hypothermia. These observations that do not align with the major conclusions of the manuscript are not discussed.

Reviewer #1 (Public Review):

This is a study linking the role of B lymphocytes to neutrophils for the achievement of LPS tolerance in an experimental setting. The manuscript is elegantly written and easy to follow. One main strength of this submission is the extensive mechanistic insights involving even transfusion of splenocytes.

Reviewer #1 (Public Review):

The manuscript by Folwer et al examines the mechanism whereby endothelial cells respond to inflammatory stimuli. Using primary Human Vascular Endothelial Cells (HUVEC) as their model, they find that upon treatment with TNF the induction and repression of hundreds of genes at both 4 and 10 hours. They found by IPA the expected inflammatory pathway and unexpectedly found that the cholesterol biosynthetic pathway was also a prominent pathway upregulated. Upon deletion of RELA, the genes that were significantly downregulated were associated with SREBP2, suggesting that TNF somehow activates SREBP2 in HUVEC. Therefore, the authors focused on understanding the mechanism of SREBP2 activation in HUVEC cells by NF-Kappa-B.

They examined TNF-induced SREBP2 cleavage over 24 hours and found that cleaved SREBP2 peaked at 10 hours. Over the same time course, they interrogated both NF-Kappa-B and SREBP2 targets and found that the expression of the NF-Kappa-B targets proceeds the SREBP2 targets, suggesting that NF-Kappa-B is somehow activating the SREBP2 pathway. Consistent with this hypothesis are studies with IKK inhibitor (which prevents NF-Kappa-B activation) and RELA knockdown that show a reduction in SREBP2 cleavage, and the inhibition of SREBP2 gene targets involved in cholesterol biosynthesis.

A series of SREBP2-processing inhibitors were used to define that the cleavage of SREBP2 by TNF-NF-Kappa-B activation was mediated by the canonical processing pathway. They then posited that TNF treatment affected the cellular cholesterol levels to activate SREPB cleavage. Whereas TNF did not change total cholesterol, they did find a change in the amount of cholesterol in the membrane both in HUVECs and in vivo by labeling the membranes with a fluorescently-labeled cholesterol-binding protein. This prompted them to look at the genes regulated by TNF-NF-Kappa-B that might be responsible for a reduction in accessible cholesterol and they focused on the lipid transporter STARD10. Interrogation of publicly available ChIP-seq of RELA from TNF-treated HUVEC cells indicates occupancy at the promoter, suggesting STARD10 is a direct RELA target. Depletion of STARD10 inhibited TNF-induced expression of cholesterol biosynthesis genes and reduced the TNF-stimulated SREBP2 cleavage and LDLR protein abundance.

Overall the data are consistent with the conclusion that NF-Kappa-B induction of STARD10 reduces cholesterol at the membrane and activates SREBP2 cleavage (model is presented in Figure 7). This illuminates the mechanism of regulation of the inflammatory response in endothelial cells. A few controls are missing and some additional analyses should be included to strengthen an already strong study.

Reviewer #1 (Public Review):

This manuscript seeks to identify the mechanism underlying priority effects in a plant-microbe-pollinator model system and to explore its evolutionary and functional consequences. The manuscript first documents alternative community states in the wild: flowers tend to be strongly dominated by either bacteria or yeast but not both. Then lab experiments are used to show that bacteria lower the nectar pH, which inhibits yeast - thereby identifying a mechanism for the observed priority effect. The authors then perform an experimental evolution experiment which shows that yeast can evolve tolerance to a lower pH. Finally, the authors show that low-pH nectar reduces pollinator consumption, suggesting a functional impact on the plant-pollinator system. Together, these multiple lines of evidence build a strong case that pH has far-reaching effects on the microbial community and beyond.

The paper is notable for the diverse approaches taken, including field observations, lab microbial competition and evolution experiments, genome resequencing of evolved strains, and field experiments with artificial flowers and nectar. This breadth can sometimes seem a bit overwhelming. The model system has been well developed by this group and is simple enough to dissect but also relevant and realistic. Whether the mechanism and interactions observed in this system can be extrapolated to other systems remains to be seen. The experimental design is generally sound. In terms of methods, the abundance of bacteria and yeast is measured using colony counts, and given that most microbes are uncultivable, it is important to show that these colony counts reflect true cell abundance in the nectar. The genome resequencing to identify pH-driven mutations is, in my mind, the least connected and developed part of the manuscript, and could be removed to sharpen and shorten the manuscript.

Overall, I think the authors achieve their aims of identifying a mechanism (pH) for the priority effect of early-colonizing bacteria on later-arriving yeast. The evolution and pollinator experiments show that pH has the potential for broader effects too. It is surprising that the authors do not discuss the inverse priority effect of early-arriving yeast on later-arriving bacteria, beyond a supplemental figure. Understandably this part of the story may warrant a separate manuscript.

I anticipate this paper will have a significant impact because it is a nice model for how one might identify and validate a mechanism for community-level interactions. I suspect it will be cited as a rare example of the mechanistic basis of priority effects, even across many systems (not just pollinator-microbe systems). It illustrates nicely a more general ecological phenomenon and is presented in a way that is accessible to a broader audience.

The 1% problem laid out clearly

5.1 Recognize that 1) the biggest threat to good decision making is harmful emotions, and 2) decision making is a two-step process (first learning and then deciding).

.

Process flow: Worldview, purpose, metric and finally design

Paper 1: Worldview and purpose Paper 2: practical implementation Paper 3: Design

https://www.higherlogic.com/blog/90-9-1-rule-online-community-engagement-data/

Realtime Notes (Incomplete) Commemorative Moment - 1st Plenary Meeting

the next few years are critical

Opening statements of the Meeting First speech civil society and the youth are critical for the climate movement but politicians are critical to make it work

First fossil fuel free car produced in Sweden Green growth can create prosperity for all The hope is that Stockholm +50 can accelerate the transition

Second speech (Hulu Kenyatta) Taking stock of the progress of the past 50 years Deepened understanding of the grave environmental threat affecting us all We stand or fall together We have made less progress on designing and implementing bold actions to address the threat We must use this opportunity to map the accelerated way forward In Kenya, we have prioritized environmental issues.

triple threat of climate change, pollution and biodiversity loss.

Need for legal and binding agreement for ending plastic pollution.

Next 50 years Africa is least responsible and suffering most for climate emissions Honor commitments to double climate finance Heighten ambitions

By the time we are at COP27 in Nov 2022, we should have a mature package for action.

Echoing former Swedish prime minister Our future is common and we should shape it together

Antonio Guterres Global wellbeing is being jeopordized by our inability to keep our environmental promises We are consuming 1.7 planets per year We need 3 earths if we consume at the rate of the most developed countries We face a triple crisis / threat of pollution, climate change and biodiversity loss We must end our suicidal war against nature We have the tools but lack leadership and collaboration We must act on our commitment, otherwise it is nothing but hot air and hot air is killing us New biodiversity agreement coming, as well as plastic treaty. The climate crisis threatens everything Science report that there is 50% chance of temporarily breaching 1.5Deg C in the next 5 We must cut emissions by 40%

G20 must dismantle coal in OECD countries by 2030 shift fossil fuel subsidies to support green transition and disenfranchised Transform accounting systems that support damage GDP increases when we overfish or destroy forests We must shift to a circular, regenerative economy based on trust and collaboration Everyone has a role to play Let's recommit to words and deeds enshrined in the 1972 Stockholm agreement

Abuddulah Shahed Food systems are struggling due to environmental degradation Human progress cannot progress in a degraded environment Salute small island states for pushing 1.5 deg into the limelight. Commitments must be followed by action Greater collaboration is needed more than ever Stockholm+50 provides opportunity to renew the urgency of our commitment, and to needed multi-lateralism Youth is taking matter into their own hands We need to follow their needs

July 19 environment for Nature meeting in NY

Botswana speaker

Inger Anderson, Executive Director of UNEP

We have no excuses for the inaction need to turn commitments into action The earth is our commons Nations need to protect our common home Let's unleash a paradigm shift for the benefit of future generations

President of Colombia Covid has exasperated the environmental commitments We have led the pact to protect Amazon, leading zero deforestation effort New finance targets need to achieve 100 billion dollars promised Act now and mobilize resources

"Stickers, badges, and avatar shopping can undermine the learning. Games that employ a reward system of unlocking higher learning levels tend to be more successful at keeping students on-task" I never thought of there being a heirarchy in prizes/rewards having an impact or rather correlation in success of an app meeting educational goals.

There's something here that's akin to the idea of bikeshedding? Online communities flock to the low lying ideas upon which they can proffer an opinion and play at the idea of debate. If they really cared, wouldn't they instead delve into the research and topics themselves? Do they really want Taibbi's specific take? Do they want or need his opinion on the topic? What do they really want?

Compare and cross reference this with the ideas presented by Ibram X. Kendi's article There Is No Debate Over Critical Race Theory.

Are people looking for the social equivalent of a simple "system one" conversation or are they ready, willing, and able to delve into a "system two" presentation?

Compare this also with the modern day version of the Sunday morning news (analysis) shows? They would seem to be interested in substantive policy and debate, but they also require a lot of prior context to participate. In essence, most speakers don't actually engage, but spew out talking points instead and rely on gut reactions and fear, uncertainty and doubt to make their presentations. What happened to the actual discourse? Has there been a shift in how these shows work and present since the rise of the Hard Copy sensationalist presentation? Is the competition for eyeballs weakening these analysis shows?

How might this all relate to low level mansplaining as well? What are men really trying to communicate in demonstrating this behavior? What do they gain in the long run? What is the evolutionary benefit?

All these topics seem related somehow within the spectrum of communication and what people look for and choose in what and how they consume content.

enlever les virgules des % sur le 2 ème diagramme stp ? Etablissements scolaires 11 % Autres partenaires 13 % Collectivités 9 % Syndic de rivières 8 % Agence de l'eau 30 % CR et ARS 11 % Fonds européens 1 % OFB 6 % Autres ressources 11 %

"I didn't fully understand it at the time, but throughout my time as a freshman at Boston College I've realized that I have the power to alter myself for the better and broaden my perspective on life. For most of my high school experience, I was holding to antiquated thoughts that had an impact on the majority of my daily interactions. Throughout my life, growing up as a single child has affected the way am in social interactions. This was evident in high school class discussions, as I did not yet have the confidence to be talkative and participate even up until the spring term of my senior year."

Freud considera que este ensayo busca mostrar la importancia de la vida sexual para toda actividad humana y su busqueda por ampliar el concepto de sexualidad. Esto fue motivación del autor y rechazo de sus detractores.

Post primera guerra, el psicoanálisis mantiene una salud en gradiente salvo los temas en donde lidia con la biología que aun produce tensión académica por el rechazo a la variable "factor sexual" como marcador de la vida normal y patológica.

gene name: DICER 1 PMID (PubMed ID): 33570641 HGNCID: n/a Inheritance Pattern: autosomal dominant Disease Entity: benign and malignant tumor mutation Mutation: somatic Zygosity: heterozygous Variant: n/a Family Information: n/a Case: people of all sexes, ages, ethnicities and races participated CasePresentingHPOs: individuals with DICER1-associated tumors or pathogenic germline DICER1 variants were recruited to participate CasePreviousTesting: n/a gnomAD: n/a

ReconfigBehSci. (2022, January 31). RT @fascinatorfun: Interesting as 🇩🇰 Omicron BA2 wave started sooner than 🇬🇧 “We conclude that Omicron BA.2 is inherently substantially m… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1488152457012297736

John Roberts. (2022, January 28). Some (very) early evidence that secondary attack rates of BA.2 are higher in household settings than those of its older sibling. From the latest Variant TB 35. Https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/1050999/Technical-Briefing-35-28January2022.pdf 1/ https://t.co/AFTril1jF1 [Tweet]. @john_actuary. https://twitter.com/john_actuary/status/1487086733149749251

ReconfigBehSci. (2022, January 23). RT @LauraMiers: BA.2’s growth advantage over BA.1 is jarring. Meanwhile, we are operating under the assumption that “Omicron will end the p… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1485519516914302980

I know what is it

As a future teacher, can this explain why public schools tend to receive funding whereas private religious schools are usually self funded? Is a school with any religious ties automatically unalloyed to receive government aid?

Bellesi, S., Metafuni, E., Hohaus, S., Maiolo, E., Marchionni, F., D’Innocenzo, S., La Sorda, M., Ferraironi, M., Ramundo, F., Fantoni, M., Murri, R., Cingolani, A., Sica, S., Gasbarrini, A., Sanguinetti, M., Chiusolo, P., & De Stefano, V. (2020). Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients. British Journal of Haematology, 191(2), 207–211. https://doi.org/10.1111/bjh.17034

how the enslaved kept their honor, history, martial arts

what ethnic markers mean

tell differences btwn what you know and what you dont (familiarity vs foreignness)

Haseltine, W. A. (n.d.). Birth Of The Omicron Family: BA.1, BA.2, BA.3. Each As Different As Alpha Is From Delta. Forbes. Retrieved 30 March 2022, from https://www.forbes.com/sites/williamhaseltine/2022/01/26/birth-of-the-omicron-family-ba1-ba2-ba3-each-as-different-as-alpha-is-from-delta/

DOI: 10.1016/j.isci.2022.104042

Resource: (ZFIN Cat# ZDB-ALT-111104-1,RRID:ZFIN_ZDB-ALT-111104-1)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-111104-1

What is this?

Some discussion of doing research on zettelkasten methods and workflows.

What do note taking methods and processes look like for individual people?

What questions would one ask for this sort of research in an interview setting (compared to how one would look at extant physical examples in document-based research)? #openquestions

Link this to the work of Earle Havens on commonplace books through portions of history.

1 Samuel 5 describes an event at the Temple of Dagon, the father of Baal, in Ashdod where the cult's statue is destroyed.

DOI: 10.7554/eLife.66118

Resource: (ZFIN Cat# ZDB-GENO-170907-1,RRID:ZFIN_ZDB-GENO-170907-1)

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-170907-1

What is this?

ReconfigBehSci. (2022, March 11). RT @dgurdasani1: UKHSA tech report out—TL;DR: -BA.2 now represents >80% of omicron in England -Growth rate 80% greater relative to BA.1 p… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1502598815081275393

L'association de deux sujets qui n'ont pas de corrélation vérifiéé, revient dans la conclusion en contradiction avec la conclusion de l'étude de Ramdoss, S et al.

En contradiction avec l'hypothèse :

Results suggest that CBI should not yet be considered a researched-based approach to teaching communication skills to individuals with ASD. However, CBI does seem a promising practice that warrants future research. Les résultats suggèrent que le CBI ne devrait pas encore être considéré comme un approche fondée sur la recherche pour enseigner les compétences en communication aux personnes ayant Troubles du Spectre Autistique. Cependant, le CBI semble être une pratique prometteuse qui justifie des recherches futures.

DOI: 10.7554/eLife.74821

Resource: (ZFIN Cat# ZDB-GENO-081105-1,RRID:ZFIN_ZDB-GENO-081105-1)

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-081105-1

What is this?

Deepti Gurdasani. (2022, January 10). Lots of people dismissing links between COVID-19 and all-cause diabetes. An association that’s been shown in multiple studies- whether this increase is due to more diabetes or SARS2 precipitating diabetic keto-acidosis allowing these to be diagnosed is not known. A brief look👇 [Tweet]. @dgurdasani1. https://twitter.com/dgurdasani1/status/1480546865812840450

DOI: 10.7554/eLife.67576

Resource: (ZFIN Cat# ZDB-ALT-160205-1,RRID:ZFIN_ZDB-ALT-160205-1)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-160205-1

What is this?

提到了卡片盒

What role does technology play in shaping design?

Because of the technological advances that we have made, technology has helped designers to re-imagine the impossible.

Technology has helped us to create the impossible.

What distinguishes the field, or fields, of design from other creative occupations?

I think that creativity is key for distinguishing the difference between design and other occupation.

As a designer you have to be neutral and creative in getting your clients message across.

According to this author, what role should design play in society?

I think that the author was talking about how the design community should be the new avant-garde.

I think that designers should be at the forefront of the design community.

DOI: 10.1016/j.xpro.2022.101155

Resource: (IMSR Cat# OBS_1,RRID:IMSR_OBS:1)

Curator: @scibot

SciCrunch record: RRID:IMSR_OBS:1

What is this?

Wise, J. (2022). Covid-19: One in 23 people in England had infection in early January. BMJ, 376, o222. https://doi.org/10.1136/bmj.o222

Arg1: Il est nécessaire de protéger les EM car des ressources sont surexploitées

• L'enjeu de la surpêche selon ONU: 1975 - 2021: 10% => 30% des ressources halieutiques surexploitées 90% des espèces maritimes peches ne se renouvellent pas assez vite naturellement Mer Noire, Méditerranée = Peche industrielle, technique du chalutage

• La pêche illégale et la pêche d'espèces protégées Espèces en voie d'extinction: grand requin: ailerons, chair, thon rouge Surexploitation des ressources maritimes, selon FAO 26M tonnes = 15 % prises totales.

Arg1: La militarisation des EM

• l'hégémonie américaine EU: 10 portes avions + bases militaires + 7flottes = forte capacité de projection

• le rattrapage chinois et russe CHINE: 2 portes avions -Liaoning et Shandong depuis Décembre 2019) = Sécurisation Nouvelles routes de la Soie + enjeux M de Chine méridionale et orientale RUSSIE: Forces navales car coopération militaire (Syrie) et ambitions (Arctique). Nouveau SNLE en Mer Blanche (Mai 2018)

• les puissances émergentes Iran, Inde, Turquie, Brésil (commande de 4 SNA à Naval Group - Décembre 2020)

Arg1: La délimitation des ZEE est héritée de la Convention de Montego Bay

• En 1982 est adoptée la CNUDM, réègles internationale utilisation, exploitation, circulation des espaces maritimes

• Eaux territoriales: droits souverains de l'Etat jusqu'à 22km

• La ZEE: droits souverains de l'Etat à des fins d'exploration, d'exploitation, de conservation et de gestion des ressources naturelles jusqu'à 200 miles / 370 km.==> 350 km si extension avec le plateau continental selon Convention de Genève (1958)

• Haute mer: 64% surface des MO, "bien commun de l'humanité" - Résolution 2749 de l'ONU, Arvid Pardo (1970) Exploitation= licences Autorité Internationale des Fonds Marins (AIFM) // liberté de circulation, survol, recherche sc, pose pipe line, cables, ... ==> Mare liberum car "terra nullius"

• liberté de circulation, "mare liberum" De la liberté des mers, Grotius, XVIIe Un Etat ne peut pas restreindre la circulation d'un navire étrangers hors de ses eaux territoriales. Idem Etats proches d'un passage stratégique

Arg1: Certaines façades maritimes sont inégalement intégrées au processus de mondialisation

• Concentration sur certains littoraux = pôles de M° Amérique du Nord, Europe, Asie de l'Est (16/20 ports) 11e: Rotterdam 17e: Los Angeles

• Des Etats tentent de s'intégrer dans les échanges mondiaux Inde, Vietnam, Maroc = 3e terminal construit au port de Tanger.

• certains espaces demeurent marginalisés isolation des principales routes maritimes (Amérique du Sud, Afrique) territoires enclavés = dépendance échange: PMA (République centre africaine), Afghanistan

Les bénéfices économiques de l'économie bleue

Economie bleue - Bertrand Blancheton, Introduction aux politiques économiques (2020)

Mers et océans rapportent 1500 Milliards $/an 3000 Milliards en 2030 "Qui tient la mer tiens le commerce du monde, qui tient le commerce tient la richesse, qui tient la richesse tient le monde lui-même", Walter Raleigh => Halford Mackinder

Arg1: CM + M = rôle des espaces maritimes ++

90% marchandises et matières première

• temps, - couts, + fiable
• Grandes capacités portes conteneurs (CMA CGM Megamax // Ecounter Bay)

Espaces maritimes inclus dans fluw et réseaux télécommunications = cables sous marins (1,2M km, 99% trafic intercontinental, 10T $/jour) trafics illicites: piraterie, narcotrafiquants

Kamrath, C., Rosenbauer, J., Eckert, A. J., Siedler, K., Bartelt, H., Klose, D., Sindichakis, M., Herrlinger, S., Lahn, V., & Holl, R. W. (2022). Incidence of Type 1 Diabetes in Children and Adolescents During the COVID-19 Pandemic in Germany: Results From the DPV Registry. Diabetes Care, dc210969. https://doi.org/10.2337/dc21-0969

IMDB 6,2/10 · 773

moviepilot.de ?/10

filmstarts.de 4/5

This is a very crucial topic. Fear stands among the leaders of bad decisions' motivators so when you'll grasp the depth of meaning for this subject your life will never be the same.

Let us consider briefly what reasons make you scared. First and foremost you must be thinking that this event or person is absolutely real. The follow up is the idea: this situation threatens you somehow. And final step to get you frightened is to assure you that you have no control.

The combo of these reasons leads you to conclusion you might become a victim so you need react preventively right now. This is a very nasty hook which you can dodge by realizing: all of those statements are equally untrue.

Take time to learn what's in the quotes related, without this solid foundation forgiveness can't be understood.

The correction of fear is your responsibility. When you ask for release from fear, you are implying that it is not. You should ask, instead, for help in the conditions that have brought the fear about. T-2.6.4

God did not create a meaningless world. W-14

I am not the victim of the world I see. W-31

I have invented the world I see. W-32

The world you see is an illusion of a world. God did not create it, for what He creates must be eternal as Himself. Yet there is nothing in the world you see that will endure forever. C-4.1

Forgive yourself the thought He wanted this for you. W-99.7

What if you recognized this world is an hallucination? What if you really understood YOU made it up? T-20.8.7

The end of dreaming is the end of fear T-28.3.4

If I defend myself I am attacked. W-135

How safe the world will look to me when I can see it! It will not look anything like what I imagine I see now. Everyone and everything I see will lean toward me to bless me. I will recognize in everyone my dearest Friend. What could there be to fear in a world that I have forgiven, and that has forgiven me? W-60.3

I thank You, Father, for Your plan to save me from the hell I made. It is not real. And You have given me the means to prove its unreality to me. The key is in my hand, and I have reached the door beyond which lies the end of dreams. W-342.1

TMDB (82%) JustWatch (90%) filmstarts.de (4,5/5) moviepilot.de (8/10) IMDB (8,3/10 · 114K · Metascore: 88)

Der unabhängige Anthony (Anthony Hopkins) lehnt auch im Alter und zunehmend von Demenz geplagt jegliche Hilfe von seiner Tochter Anne (Olivia Colman) ab. Diese Hilfe wird aber unabdingbar, als Anne beschließt, mit ihrem Mann Paul (Rufus Sewell) nach Paris zu ziehen, und Anthony somit allein in der Wohnung zurückbleiben müsste, in der Anne und Paul mit ihm leben. Dass das nicht funktionieren wird, wird schon daran deutlich, dass Anthony immer wieder sehr durcheinanderkommt. Er wundert sich etwa über den unbekannten Mann (Mark Gatiss), der auf einmal im Wohnzimmer sitzt und behauptet, sein Schwiegersohn Paul zu sein. Und selbst die Frau (Olivia Williams), die kurz darauf nach Hause kommt und behauptet seine Tochter Anne zu sein, erkennt er nicht. Die Pflegerin Laura (Imogen Poots) soll Anthony helfen, doch auch wenn er sich anfangs charmant gibt: Er hat bereits zuvor andere Pflegerinnen mit seinen Stimmungsschwankungen vergrault... filmstarts.de

Mit „The Father“ gelingt Florian Zeller eine herausragende Adaption seines eigenen Bühnenstücks. Ganz großes Kino! [filmstarts.de] (https://www.filmstarts.de/kritiken/273981/kritik.html) 4,5/5

„The Father“ zeigt aus der Sicht eines 80-Jährigen, wie sich die Wahrnehmung der Welt aufgrund von Demenz verändern kann. Das Ergebnis ist ein eindrucksvolles, ungewöhnliches und herausragend gespieltes Drama, das mit einfachsten Mitteln ein Entfremdungsgefühl entstehen lässt und verdeutlicht, was es heißt, in der Welt verloren zu gehen. Oliver Armknecht 9/10

moviepilot.de 6,1/10 IMDB 5,8/10 · 150

Muskelpaket und Einzelgänger Mosk (Thomas Sarbacher) hat alles andere als diesen süßen Labradorwelpen im Kopf: Er trainiert seit Wochen hart für die anstehenden gefängnisinternen Meisterschaften im Gewichtheben. So sträubt er sich zunächst auch vehement gegen das Projekt der neuen Gefängnisdirektorin Gloria (Clelia Sarto), bei dem ausgewählte Häftlinge Welpen zu Blindenhunden ausbilden sollen. Leider hilft weder sein abweisendes Verhalten noch ein klares „Nein“, er wird gegen seinen Willen für das Programm ausgewählt und zieht kurz darauf zusammen mit fünf weiteren Teilnehmern und Knastkumpanen in einen speziellen Trakt der Vollzugsanstalt. filmstarts.de 3,5/5

Ein Häftling findet sich gegen seinen Willen in einem Pilotprojekt wieder, bei dem er und fünf Mitgefangene Welpen zu Blindenhunden ausbilden. Unterhaltsamer Gefängnisfilm, der die Klischees des Genres umschifft. epdFilm 6/10

moviepilot.de 6,3/10 IMDB 5,7/10 · 210

Es war einmal vor langer Zeit in den endlosen Weiten des Atlasgebirges. Der Nomade Mustapha soll die besten arabischen Vollblüter und Reiter seines Stammes nach Marrakesch führen, um am ruhmreichsten aller Pferderennen teilzunehmen: dem Agdal. Aber bevor es in die Berge geht, holt er in der Stadt seine elfjährige Tochter, ein menschenscheues Mädchen mit dem Namen Zaina, von deren Existenz er erst beim Tod von Zainas Mutter erfahren hat. Denn einst musste Mustapha unter dem Druck seines Stammes diese Frau verstoßen, weil sie als Mann verkleidet an dem Agdal teilgenommen hatte. filmstarts.de (2,5/5)

moviepilot.de 6,7/10

Helge Schneider bringt es wieder einmal, wie so oft, auf den Punkt: „Die schwierigste Zeit im Leben eines Mannes ist die Pubertät, die zweitschlimmste ist die danach.“ Frauen geht es da bestimmt nicht viel besser... wenn der Körper voller Hormone gepumpt wird, die diesen verändern, das Interesse am anderen oder gleichen Geschlecht zunimmt, kurz: die Zeit, in der sich schlicht alles verändert und eine andere Bedeutung bekommt, ist wohl diejenige, die uns alle am meisten prägt. Nachdem in diesem Jahr Gus van Sant mit „Paranoid Park“ einen männlichen Jugendlichen ins Visier nimmt, kontert „Water Lilies“ von Céline Sciamma quasi von weiblicher Seite. filmstarts.de (3,5/5)

moviepilot.de 6,5/10

Miles Kendig (Walter Matthau) ist CIA-Agent und ein alter Hase in seinem Beruf. Er versteht etwas von seinem Job und schafft es so etwa, auf dem Oktoberfest einen sowjetischen Spionagering hochgehen zu lassen. Doch anstatt ihn mit einer Beförderung zu belohnen, entlässt ihn sein cholerischer Chef endgültig aus dem Dienst. Kendig macht seinem Ärger Luft, indem er seiner Geliebten Isobel (Glenda Jackson) einen Besuch in Salzburg abstattet. Dort trifft er auf den sowjetischen Agenten Yaskov (Herbert Lom), welcher ihn dazu überredet, seine Biografie zu schreiben. Kendig willigt ein und sendet jedes Kapitel sowohl an seinen ehemaligen Chef, als auch an den KGB. Schnell werden zwei CIA-Agenten ausgesendet, um den Fahnenflüchtigen zu finden, doch mit der Cleverness Kendigs hat niemand gerechnet…filmstarts.de

Godard-Doku über die Rolling Stones

moviepilot.de 6,5/10

filmstarts.de

England, 1865: Catherine (Florence Pugh) lebt gemeinsam mit ihrem Gatten Alexander (Paul Hilton) und seinem Vater Boris (Christopher Fairbank) auf dem Land. Liebe ist in dieser Beziehung nicht im Spiel und obwohl Boris beständig darauf pocht, Catherine solle ihre ehelichen Pflichten erfüllen, hat Alexander keinerlei Interesse am Körper seiner Frau. Als ihr Mann eines Tages verreist, nutzt Catherine die Möglichkeit, dem ihr auferlegten Hausarrest zu entkommen und erkundet die Gegend. So lernt sie einen der Landarbeiter, Sebastian (Cosmo Jarvis), kennen. Nach anfänglicher Unsicherheit und trotz einer priesterlichen Warnung gibt sich Catherine schließlich ihrer Leidenschaft hin und beginnt eine Affäre mit Sebastian. Doch Alexanders Rückkehr gefährdet das neu gefundene Glück und Catherine muss eine Entscheidung filmstarts.de 4,5/5

moviepilot.de 6,5/10

Aus einer eingeschüchterten jungen Frau wird eine kaltblütige Mörderin: In dieser stilsicheren, modernen und überaus spannenden neuen „Lady Macbeth“ schlägt Newcomerin Florence Pugh den Zuschauer von der ersten Szene an in ihren Bann. filmstarts.de 4,5/5

„Lady Macbeth“ nimmt die klassische russische Novelle und macht daraus einen Film, der kälter und böser kaum sein könnte. Vor allem die junge Hauptdarstellerin sorgt dafür, dass der Wandel einer unterdrückten Gattin zu einer skrupellosen Herrscherin absolut sehenswert ist. Gleichzeitig lässt sich das Drama aber auch zu allgemeinen Themen aus, gerade in Bezug auf zwischenmenschliche Machtverhältnisse. Oliver Armknecht 8/10

William Oldroyds im viktorianischen England spielende Verfilmung von Nikolai Leskows »Lady Macbeth aus Mzensk« besticht durch formale Virtuosität. epdFilm 6/10

filmstarts.de 3,5/5

filmstarts.de 2,5/5

filmstarts.de 3,5/5

filmstarts.de 4/5

TMDB (63%) JustWatch (69%) filmstarts.de (–/5) moviepilot.de (6,2/10) IMDB (6,2/10 · 2,5K · Metascore: 54)

Vor einem halben Jahrhundert war Harris Shaw (Michael Caine) mit seinem Erfolgsroman „Atomic Autumn“ alleine dafür verantwortlich, dass ein Verlag erfolgreich wurde. Doch heute sind sowohl beim Autor als auch beim Verlag der Ruhm verblasst. Shaw tippt zwar immer noch fleißig auf seiner alten Schreibmaschine, doch der Raucher und Säufer will nichts mit der Welt zu tun haben. Die junge Verlagserbin Lucy Stanbridge (Aubrey Plaza) greift derweil bei sinkenden Verkaufszahlen und einem von Influencer bestimmten Werbemarkt zum letzten Strohhalm, um ihr Unternehmen zu retten: Einsiedler Shaw soll noch einmal auf eine Lesetour gehen, um sein neues Buch zu präsentieren. Doch der hat wenig Bock... filmstarts.de

Originaltitel: Smagen af sult

TMDB (59%) | JustWatch (71%) | filmstarts.de (–/5) | moviepilot.de (–/10) | IMDB (6,4/10 · 405)

Carsten (Nikolaj Coster-Waldau) und Maggi (Katrine Greis-Rosenthal) sind ein Paar, das alles opfert, um die höchstmögliche Auszeichnung in der kulinarischen Welt zu erreichen: einen Michelin-Stern. Eigentlich haben die beiden alles, was man sich wünschen könnte. Sie führen eine liebevolle Beziehung, sie haben zwei wundervolle Söhne und ihr exklusives Restaurant in Kopenhagen, genannt Malus, läuft hervorragend. Doch eines fehlt vor allem Carsten noch zum Glück: Er möchte die offizielle Auszeichnung, die ein Michelin-Stern dem gemeinsamem Restaurant verleihen würde. Nun steht der Besuch eines Restaurantkritikers kurz bevor und die Situation droht Carsten und Maggi über den Kopf zu wachsen... filmstarts.de

TMDB (66%) JustWatch (68%) Filmstarts (4/5) Moviepilot (5,4/10) IMDB (5,7/10 · 7,6K | Metascore: 52)

Handlung

Ein gemütliches Häuschen auf dem englischen Lande: Der Weihnachtsbaum ist liebevoll geschmückt, das Feiertagsfestmahl opulent vorbereitet und nostalgische Evergreens klingen durch die Räumlichkeit. Als Nell (Keira Knightley), Simon (Matthew Goode) und ihr Sohn Art (Roman Griffin Davis) Verwandtschaft und Freunde in dem kleinen Cottage willkommen heißen, scheint dem perfekten Weihnachtsfest im Kreis der Liebsten rein gar nichts mehr im Wege zu stehen. An der Geschichte gibt es jedoch einen Haken, denn überleben wird niemand die Weihnachtsfeiertage...

Rezension(en)

Jüngstes Gericht am Heiligen Abend: Das bitterböse „Was-wäre-wenn“-Kammerspiel von Camille Griffin lässt die Generation „Fridays For Future“ unterm Tannenbaum mit der Dekadenz ihrer Eltern kollidieren – und das ist tatsächlich ein (dunkelschwarzhumoriges) Fest! (filmstarts 4/5)

„Silent Night“ beginnt wie eine typische Weihnachtskomödie rund um eine dysfunktionale Gruppe, bevor es sich nach und nach in ein Endzeitdrama verwandelt. Die Mischung der einzelnen Bestandteile geht nicht ganz auf, da fehlte dann doch ein schlüssiges Konzept. Sehenswert ist der ungewöhnliche Mix aber, zudem mitreißend gespielt. armknoli

DOI: 10.7554/eLife.72345

Resource: (ZFIN Cat# ZDB-GENO-180802-1,RRID:ZFIN_ZDB-GENO-180802-1)

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-180802-1

What is this?

missing parts of the citation: 491 U.S. 397 (1989)

this is wrong, it wasn't in 1982, the book states the case as

Zelman v. Simmons-Harris (2002)

DOI: 10.7554/eLife.69264

Resource: (ZFIN Cat# ZDB-ALT-080701-1,RRID:ZFIN_ZDB-ALT-080701-1)

Curator: @bandrow

SciCrunch record: RRID:ZFIN_ZDB-ALT-080701-1

What is this?