Erratum in PLoS Biol. 2022 Oct 14;20(10):e3001859.

Comment on PLoS Biol. 2022 Sep 6;20(9):e3001711.

Comment on PLoS Biol. 2022 Sep 6;20(9):e3001734.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Comment in PLoS Biol. 2022 Sep 7;20(9):e3001788.

The fear of environmental doom (eco-anxiety) is a new and growing source of mental distress that could particularly affect the next generation of conservation biologists. With care and empathy, educators can help to prevent worsening the mental health of students.

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have been transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources to analyze such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76∙108 read fragments were assigned to ARGs and 3.21∙109 to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs.

Sex differences in the fitness effects of genetic variants can influence the rate of adaptation and the maintenance of genetic variation. For example, "sexually antagonistic" (SA) variants, which are beneficial for one sex and harmful for the other, can both constrain adaptation and increase genetic variability for fitness components such as survival, fertility, and disease susceptibility. However, detecting variants with sex-differential fitness effects is difficult, requiring genome sequences and fitness measurements from large numbers of individuals. Here, we develop new theory for studying sex-differential selection across a complete life cycle and test our models with genotypic and reproductive success data from approximately 250,000 UK Biobank individuals. We uncover polygenic signals of sex-differential selection affecting survival, reproductive success, and overall fitness, with signals of sex-differential reproductive selection reflecting a combination of SA polymorphisms and sexually concordant polymorphisms in which the strength of selection differs between the sexes. Moreover, these signals hold up to rigorous controls that minimise the contributions of potential confounders, including sequence mapping errors, population structure, and ascertainment bias. Functional analyses reveal that sex-differentiated sites are enriched in phenotype-altering genomic regions, including coding regions and loci affecting a range of quantitative traits. Population genetic analyses show that sex-differentiated sites exhibit evolutionary histories dominated by genetic drift and/or transient balancing selection, but not long-term balancing selection, which is consistent with theoretical predictions of effectively weak SA balancing selection in historically small populations. Overall, our results are consistent with polygenic sex-differential-including SA-selection in humans. Evidence for sex-differential selection is particularly strong for variants affecting reproductive success, in which the potential contributions of nonrandom sampling to signals of sex differentiation can be excluded.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Comment in PLoS Biol. 2022 Sep 7;20(9):e3001796.

Anterior cingulate cortex (ACC) and striatum (STR) contain neurons encoding not only the expected values of actions, but also the value of stimulus features irrespective of actions. Values about stimulus features in ACC or STR might contribute to adaptive behavior by guiding fixational information sampling and biasing choices toward relevant objects, but they might also have indirect motivational functions by enabling subjects to estimate the value of putting effort into choosing objects. Here, we tested these possibilities by modulating neuronal activity in ACC and STR of nonhuman primates using transcranial ultrasound stimulation while subjects learned the relevance of objects in situations with varying motivational and cognitive demands. Motivational demand was indexed by varying gains and losses during learning, while cognitive demand was varied by increasing the uncertainty about which object features could be relevant during learning. We found that ultrasound stimulation of the ACC, but not the STR, reduced learning efficiency and prolonged information sampling when the task required averting losses and motivational demands were high. Reduced learning efficiency was particularly evident at higher cognitive demands and when subjects experienced loss of already attained tokens. These results suggest that the ACC supports flexible learning of feature values when loss experiences impose a motivational challenge and when uncertainty about the relevance of objects is high. Taken together, these findings provide causal evidence that the ACC facilitates resource allocation and improves visual information sampling during adaptive behavior.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed "TFE-Dependent ReversiblY condensing Proteins (T-DRYPs)." Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

Long-term memory formation involves the reorganization of brain circuits, termed system consolidation. Whether and how a prior fear experience influences system consolidation of new memories is poorly understood. In rats, we found that prior auditory fear learning allows the secondary auditory cortex to immediately encode new auditory memories, with these new memories purely requiring the activation of cellular mechanisms of synaptic consolidation within secondary auditory cortex. Similar results were obtained in the anterior cingulate cortex for contextual fear memories. Moreover, prior learning enabled connections from these cortices to the basolateral amygdala (BLA) to support recent memory retention. We propose that the reorganization of circuits that characterizes system consolidation occurs only in the first instance that an event is learned, subsequently allowing the immediate assimilation of new analogous events in final storage sites.

Comment on PLoS Biol. 2022 Sep 20;20(9):e3001310.

Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal fan-shaped body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation) in Drosophila. We report that dFB neurons change their physiological response-properties to dopamine (DA) and allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together, these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

Comment on PLoS Biol. 2022 Sep 27;20(9):e3001781.

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

Erratum in PLoS Biol. 2022 Oct 19;20(10):e3001869.

Following the initiation of the unprecedented global vaccination campaign against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), attention has now turned to the potential impact of this large-scale intervention on the evolution of the virus. In this Essay, we summarize what is currently known about pathogen evolution in the context of immune priming (including vaccination) from research on other pathogen species, with an eye towards the future evolution of SARS-CoV-2.

Comment on PLoS Biol. 2022 Sep 22;20(9):e3001753.

Babesia is a genus of apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to 3 Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing "just-in-time" expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian graphical model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web application to visualize and interactively explore the datasets.

Comment in PLoS Biol. 2022 Sep 23;20(9):e3001800.

Comment on PLoS Biol. 2022 Sep 20;20(9):e3001743.

Comment on PLoS Biol. 2022 Sep 20;20(9):e3001751.

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.

Comment in PLoS Biol. 2022 Sep 21;20(9):e3001793.

Comment in PLoS Biol. 2022 Sep 21;20(9):e3001795.

Comment in PLoS Biol. 2022 Sep 29;20(9):e3001830.

Wound healing in the skin is a complex physiological process that is a product of a cell state transition from homeostasis to repair. Mechanical cues are increasingly being recognized as important regulators of cellular reprogramming, but the mechanism by which it is translated to changes in gene expression and ultimately cellular behavior remains largely a mystery. To probe the molecular underpinnings of this phenomenon further, we used the down-regulation of caspase-8 as a biomarker of a cell entering the wound healing program. We found that the wound-induced release of tension within the epidermis leads to the alteration of gene expression via the nuclear translocation of the DNA methyltransferase 3A (DNMT3a). This enzyme then methylates promoters of genes that are known to be down-regulated in response to wound stimuli as well as potentially novel players in the repair program. Overall, these findings illuminate the convergence of mechanical and epigenetic signaling modules that are important regulators of the transcriptome landscape required to initiate the tissue repair process in the differentiated layers of the epidermis.

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

Translational biomedical research relies on animal experiments and provides the underlying proof of practice for clinical trials, which places an increased duty of care on translational researchers to derive the maximum possible output from every experiment performed. The implementation of open science practices has the potential to initiate a change in research culture that could improve the transparency and quality of translational research in general, as well as increasing the audience and scientific reach of published research. However, open science has become a buzzword in the scientific community that can often miss mark when it comes to practical implementation. In this Essay, we provide a guide to open science practices that can be applied throughout the research process, from study design, through data collection and analysis, to publication and dissemination, to help scientists improve the transparency and quality of their work. As open science practices continue to evolve, we also provide an online toolbox of resources that we will update continually.

In contrast to molecular changes associated with increased inflammatory responses, little is known about intracellular counter-regulatory mechanisms that control signaling cascades associated with functional responses of neutrophils. Active RHO GTPases are typically considered as effector proteins that elicit cellular responses. Strikingly, we show here that RHOH, although being constitutively GTP-bound, limits neutrophil degranulation and the formation of neutrophil extracellular traps (NETs). Mechanistically, RHOH is induced under inflammatory conditions and binds to non-muscle myosin heavy chain IIA (NMHC IIA) in activated neutrophils in order to inhibit the transport of mitochondria and granules along actin filaments, which is partially reverted upon disruption of the interaction with NMHC IIA by introducing a mutation in RhoH at lysine 34 (RhoHK34A). In parallel, RHOH inhibits actin polymerization presumably by modulating RAC1 activity. In vivo studies using Rhoh-/- mice, demonstrate an increased antibacterial defense capability against Escherichia coli (E. coli). Collectively, our data reveal a previously undefined role of RHOH as a molecular brake for actomyosin-mediated neutrophil effector functions, which represents an intracellular regulatory axis involved in controlling the strength of an antibacterial inflammatory response.

Comment on PLoS Biol. 2022 Sep 13;20(9):e3001754.

Introgression, endosymbiosis, and gene transfer, i.e., horizontal gene flow (HGF), are primordial sources of innovation in all domains of life. Our knowledge on HGF relies on detection methods that exploit some of its signatures left on extant genomes. One of them is the effect of HGF on branch lengths of constructed phylogenies. This signature has been formalized in statistical tests for HGF detection and used for example to detect massive adaptive gene flows in malaria vectors or to order evolutionary events involved in eukaryogenesis. However, these studies rely on the assumption that ghost lineages (all unsampled extant and extinct taxa) have little influence. We demonstrate here with simulations and data reanalysis that when considering the more realistic condition that unsampled taxa are legion compared to sampled ones, the conclusion of these studies become unfounded or even reversed. This illustrates the necessity to recognize the existence of ghosts in evolutionary studies.

Sensory pathways provide complex and multifaceted information to the brain. Recent advances have created new opportunities for applying our understanding of the brain to sensory prothesis development. Yet complex sensor physiology, limited numbers of electrodes, and nonspecific stimulation have proven to be a challenge for many sensory systems. In contrast, the vestibular system is uniquely suited for prosthesis development. Its peripheral anatomy allows site-specific stimulation of 3 separate sensory organs that encode distinct directions of head motion. Accordingly, here, we investigated whether implementing natural encoding strategies improves vestibular prosthesis performance. The eye movements produced by the vestibulo-ocular reflex (VOR), which plays an essential role in maintaining visual stability, were measured to quantify performance. Overall, implementing the natural tuning dynamics of vestibular afferents produced more temporally accurate VOR eye movements. Exploration of the parameter space further revealed that more dynamic tunings were not beneficial due to saturation and unnatural phase advances. Trends were comparable for stimulation encoding virtual versus physical head rotations, with gains enhanced in the latter case. Finally, using computational methods, we found that the same simple model explained the eye movements evoked by sinusoidal and transient stimulation and that a stimulation efficacy substantially less than 100% could account for our results. Taken together, our results establish that prosthesis encodings that incorporate naturalistic afferent dynamics and account for activation efficacy are well suited for restoration of gaze stability. More generally, these results emphasize the benefits of leveraging the brain's endogenous coding strategies in prosthesis development to improve functional outcomes.

Comment in PLoS Biol. 2022 Sep 14;20(9):e3001787.

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway.

Insulin-secreting β-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (β-GCamP6s) that show β-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of β-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder β-cell state, critical for the islet first-phase response to glucose.

Understanding the dynamics of species adaptation to their environments has long been a central focus of the study of evolution. Theories of adaptation propose that populations evolve by "walking" in a fitness landscape. This "adaptive walk" is characterised by a pattern of diminishing returns, where populations further away from their fitness optimum take larger steps than those closer to their optimal conditions. Hence, we expect young genes to evolve faster and experience mutations with stronger fitness effects than older genes because they are further away from their fitness optimum. Testing this hypothesis, however, constitutes an arduous task. Young genes are small, encode proteins with a higher degree of intrinsic disorder, are expressed at lower levels, and are involved in species-specific adaptations. Since all these factors lead to increased protein evolutionary rates, they could be masking the effect of gene age. While controlling for these factors, we used population genomic data sets of Arabidopsis and Drosophila and estimated the rate of adaptive substitutions across genes from different phylostrata. We found that a gene's evolutionary age significantly impacts the molecular rate of adaptation. Moreover, we observed that substitutions in young genes tend to have larger physicochemical effects. Our study, therefore, provides strong evidence that molecular evolution follows an adaptive walk model across a large evolutionary timescale.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1β and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

The realization that ecological principles play an important role in infectious disease dynamics has led to a renaissance in epidemiological theory. Ideas from ecological succession theory have begun to inform an understanding of the relationship between the individual microbiome and health but have not yet been applied to investigate broader, population-level epidemiological dynamics. We consider human hosts as habitat and apply ideas from succession to immune memory and multi-pathogen dynamics in populations. We demonstrate that ecologically meaningful life history characteristics of pathogens and parasites, rather than epidemiological features alone, are likely to play a meaningful role in determining the age at which people have the greatest probability of being infected. Our results indicate the potential importance of microbiome succession in determining disease incidence and highlight the need to explore how pathogen life history traits and host ecology influence successional dynamics. We conclude by exploring some of the implications that inclusion of successional theory might have for understanding the ecology of diseases and their hosts.

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a reader's ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n = 151) and cell biology (n = 400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps, and antibody labeling protocol. Important antibody identifiers like company or supplier, catalog number, or RRID were omitted frequently for primary antibodies and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers, and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish, and an antibody reporting template.