Spider venoms are a complex concoction of enzymes, polyamines, inorganic salts and disulfide-rich peptides (DRPs). Although DRPs are widely distributed and abundant, their evolutionary origin has remained elusive. This knowledge gap stems from the extensive molecular divergence of DRPs and a lack of sequence and structural data from diverse lineages. By evaluating DRPs under a comprehensive phylogenetic, structural and evolutionary framework, we have not only identified 78 novel spider toxin superfamilies but also provided the first evidence for their common origin. We trace the origin of these toxin superfamilies to a primordial knot - which we name 'Adi Shakti', after the creator of the Universe according to Hindu mythology - 375 MYA in the common ancestor of Araneomorphae and Mygalomorphae. As the lineages under evaluation constitute nearly 60% of extant spiders, our findings provide fascinating insights into the early evolution and diversification of the spider venom arsenal. Reliance on a single molecular toxin scaffold by nearly all spiders is in complete contrast to most other venomous animals that have recruited into their venoms diverse toxins with independent origins. By comparatively evaluating the molecular evolutionary histories of araneomorph and mygalomorph spider venom toxins, we highlight their contrasting evolutionary diversification rates. Our results also suggest that venom deployment (e.g., prey capture or self-defense) influences evolutionary diversification of DRP toxin superfamilies.

Methylation is a widely occurring modification that requires the methyl donor S-adenosylmethionine (SAM) and acts in regulation of gene expression and other processes. SAM is synthesized from methionine, which is imported or generated through the 1-carbon cycle (1CC). Alterations in 1CC function have clear effects on lifespan and stress responses, but the wide distribution of this modification has made identification of specific mechanistic links difficult. Exploiting a dynamic stress-induced transcription model, we find that two SAM synthases in Caenorhabditis elegans, SAMS-1 and SAMS-4, contribute differently to modification of H3K4me3, gene expression and survival. We find that sams-4 enhances H3K4me3 in heat shocked animals lacking sams-1, however, sams-1 cannot compensate for sams-4, which is required to survive heat stress. This suggests that the regulatory functions of SAM depend on its enzymatic source and that provisioning of SAM may be an important regulatory step linking 1CC function to phenotypes in aging and stress.

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.

Cone rod dystrophy (CRD) is a genetically inherited retinal disease that can be associated with male infertility, while the specific genetic mechanisms are not well known. Here we report CEP78 as a causative gene of a particular syndrome including CRD and male infertility with multiple morphological abnormalities of sperm flagella (MMAF) both in human and mouse. Cep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced electroretinogram amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted connecting cilia elongation and abnormal structures. Cep78 deletion also caused male infertility and MMAF, with disordered '9 + 2' structure and triplet microtubules in sperm flagella. Intraflagellar transport (IFT) proteins IFT20 and TTC21A are identified as interacting proteins of CEP78. Furthermore, CEP78 regulated the interaction, stability, and centriolar localization of its interacting protein. Insufficiency of CEP78 or its interacting protein causes abnormal centriole elongation and cilia shortening. Absence of CEP78 protein in human caused similar phenotypes in vision and MMAF as Cep78-/- mice. Collectively, our study supports the important roles of CEP78 defects in centriole and ciliary dysfunctions and molecular pathogenesis of such multi-system syndrome.

Many microbiota-based therapeutics rely on our ability to introduce a microbe of choice into an already-colonized intestine. In this study, we used genetically barcoded Bacteroides thetaiotaomicron (B. theta) strains to quantify population bottlenecks experienced by a B. theta population during colonization of the mouse gut. As expected, this reveals an inverse relationship between microbiota complexity and the probability that an individual wildtype B. theta clone will colonize the gut. The polysaccharide capsule of B. theta is important for resistance against attacks from other bacteria, phage, and the host immune system, and correspondingly acapsular B. theta loses in competitive colonization against the wildtype strain. Surprisingly, the acapsular strain did not show a colonization defect in mice with a low-complexity microbiota, as we found that acapsular strains have an indistinguishable colonization probability to the wildtype strain on single-strain colonization. This discrepancy could be resolved by tracking in vivo growth dynamics of both strains: acapsular B .theta shows a longer lag-phase in the gut lumen as well as a slightly slower net growth rate. Therefore, as long as there is no niche competitor for the acapsular strain, this has only a small influence on colonization probability. However, the presence of a strong niche competitor (i.e., wildtype B. theta, SPF microbiota) rapidly excludes the acapsular strain during competitive colonization. Correspondingly, the acapsular strain shows a similarly low colonization probability in the context of a co-colonization with the wildtype strain or a complete microbiota. In summary, neutral tagging and detailed analysis of bacterial growth kinetics can therefore quantify the mechanisms of colonization resistance in differently-colonized animals.

The human gut microbiome contains a diversity of microbial species that varies in composition over time and across individuals. These species (and strains within species) can migrate across hosts and evolve by mutation and recombination within hosts. How the ecological process of community assembly interacts with intra-species diversity and evolutionary change is a longstanding question. Two contrasting hypotheses have been proposed based on ecological observations and theory: Diversity Begets Diversity (DBD), in which taxa tend to become more diverse in already diverse communities, and Ecological Controls (EC), in which higher community diversity impedes diversification within taxa. Previously, using 16S rRNA gene amplicon data across a range of environments, we showed a generally positive relationship between taxa diversity and community diversity at higher taxonomic levels, consistent with the predictions of DBD (Madi et al., 2020). However, this positive 'diversity slope' reaches a plateau at high levels of community diversity. Here we show that this general pattern holds at much finer genetic resolution, by analyzing intra-species strain and nucleotide variation in static and temporally sampled shotgun-sequenced fecal metagenomes from cohorts of healthy human hosts. We find that both intra-species polymorphism and strain number are positively correlated with community Shannon diversity. This trend is consistent with DBD, although we cannot exclude abiotic drivers of diversity. Shannon diversity is also predictive of increases in polymorphism over time scales up to ~4-6 months, after which the diversity slope flattens and then becomes negative-consistent with DBD eventually giving way to EC. Also supporting a complex mixture of DBD and EC, the number of strains per focal species is positively associated with Shannon diversity but negatively associated with richness. Finally, we show that higher community diversity predicts gene loss in a focal species at a future time point. This observation is broadly consistent with the Black Queen Hypothesis, which posits that genes with functions provided by the community are less likely to be retained in a focal species' genome. Together, our results show that a mixture of DBD, EC, and Black Queen may operate simultaneously in the human gut microbiome, adding to a growing body of evidence that these eco-evolutionary processes are key drivers of biodiversity and ecosystem function.

Hepatitis B virus (HBV) infection represents a significant global health threat, accounting for 300 million chronic infections and up to 1 million deaths each year. HBV disproportionately affects people who are under-served by health systems due to social exclusion, and can further amplify inequities through its impact on physical and mental health, relationship with stigma and discrimination, and economic costs. The 'inclusion health' agenda focuses on excluded and vulnerable populations, who often experience barriers to accessing healthcare, and are under-represented by research, resources, interventions, advocacy, and policy. In this article, we assimilate evidence to establish HBV on the inclusion health agenda, and consider how this view can inform provision of better approaches to diagnosis, treatment, and prevention. We suggest approaches to redress the unmet need for HBV interventions among excluded populations as an imperative to progress the global goal for the elimination of viral hepatitis as a public health threat.

SARS-CoV-2 has adapted in a stepwise manner, with multiple beneficial mutations accumulating in a rapid succession at origins of VOCs, and the reasons for this are unclear. Here, we searched for coordinated evolution of amino acid sites in the spike protein of SARS-CoV-2. Specifically, we searched for concordantly evolving site pairs (CSPs) for which changes at one site were rapidly followed by changes at the other site in the same lineage. We detected 46 sites which formed 45 CSP. Sites in CSP were closer to each other in the protein structure than random pairs, indicating that concordant evolution has a functional basis. Notably, site pairs carrying lineage defining mutations of the four VOCs that circulated before May 2021 are enriched in CSPs. For the Alpha VOC, the enrichment is detected even if Alpha sequences are removed from analysis, indicating that VOC origin could have been facilitated by positive epistasis. Additionally, we detected nine discordantly evolving pairs of sites where mutations at one site unexpectedly rarely occurred on the background of a specific allele at another site, for example on the background of wild-type D at site 614 (four pairs) or derived Y at site 501 (three pairs). Our findings hint that positive epistasis between accumulating mutations could have delayed the assembly of advantageous combinations of mutations comprising at least some of the VOCs.

Altruism is critical for cooperation and productivity in human societies but is known to vary strongly across contexts and individuals. The origin of these differences is largely unknown, but may in principle reflect variations in different neurocognitive processes that temporally unfold during altruistic decision making (ranging from initial perceptual processing via value computations to final integrative choice mechanisms). Here, we elucidate the neural origins of individual and contextual differences in altruism by examining altruistic choices in different inequality contexts with computational modeling and electroencephalography (EEG). Our results show that across all contexts and individuals, wealth distribution choices recruit a similar late decision process evident in model-predicted evidence accumulation signals over parietal regions. Contextual and individual differences in behavior related instead to initial processing of stimulus-locked inequality-related value information in centroparietal and centrofrontal sensors, as well as to gamma-band synchronization of these value-related signals with parietal response-locked evidence-accumulation signals. Our findings suggest separable biological bases for individual and contextual differences in altruism that relate to differences in the initial processing of choice-relevant information.

Spinally-projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies and Tph2-Cre transgenic mice, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on sensory perception. Our tracing experiments revealed that serotonergic neurons of the LPGi were much more susceptible to transduction with spinally injected AAV2retro vectors than medial hindbrain serotonergic neurons. Taking advantage of this difference, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and their impact on nociceptive sensitivity. The LPGi neurons that give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord appear to be ideally posed to contribute to widespread systemic pain control.

CRISPR-based diagnostics (CRISPRDx) have improved clinical decision-making, especially during the COVID-19 pandemic, by detecting nucleic acids and identifying variants. This has been accelerated by the discovery of new and engineered CRISPR effectors, which have expanded the portfolio of diagnostic applications to include a broad range of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline necessitates customized detection schemes based on the fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is especially relevant for variant detection, a low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver, CriSNPr (CRISPR-based SNP recognition), which provides the user with the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. Furthermore, we provide a database of curated pre-designed gRNAs as well as target/off-target for all human and SARS-CoV-2 variants reported thus far. CriSNPr has been validated on multiple Cas proteins, demonstrating its broad and immediate applicability across multiple detection platforms. CriSNPr can be found at http://crisnpr.igib.res.in/.

Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

During respiratory viral infections, the precise roles of monocytes and dendritic cells (DCs) in the nasopharynx in limiting infection and influencing disease severity are incompletely described. We studied circulating and nasopharyngeal monocytes and DCs in healthy controls (HC) and in patients with mild to moderate infections (primarily influenza A virus, IAV). As compared to HCs, patients with acute IAV infection displayed reduced DC but increased intermediate monocytes frequencies in blood, and an accumulation of most monocyte and DC subsets in the nasopharynx. IAV patients had more mature monocytes and DCs in the nasopharynx, and higher levels of TNFα, IL-6 and IFNα in plasma and the nasopharynx than HCs. In blood, monocytes were the most frequent cellular source of TNFα during IAV infection and remained responsive to additional stimulation with TLR7/8L. Immune responses in older patients skewed towards increased monocyte frequencies rather than DCs, suggesting a contributory role for monocytes in disease severity. In patients with other respiratory virus infections, we observed changes in monocyte and DC frequencies in the nasopharynx distinct from IAV patients, while differences in blood were more similar across infection groups. Using SomaScan, a high-throughput aptamer-based assay to study proteomic changes between patients and HCs, we found differential expression of innate immunity-related proteins in plasma and nasopharyngeal secretions of IAV and SARS-CoV-2 patients. Together, our findings demonstrate tissue-specific and pathogen-specific patterns of monocyte and DC function during human respiratory viral infections and highlight the importance of comparative investigations in blood and the nasopharynx.

The ability to simultaneously modulate a set of genes for lineage-specific development has made microRNA an ideal master regulator for organogenesis. However, most microRNA deletions do not exhibit obvious phenotypic defects possibly due to functional redundancy. MicroRNAs are known to regulate skeletal lineages as the loss of their maturation enzyme Dicer impairs bone remodeling processes. Therefore, it is important to identify specific microRNA essential for bone homeostasis. We report the loss of MIR27a causing severe osteoporosis in mice. MIR27a affects osteoclast-mediated bone resorption but not osteoblast-mediated bone formation during skeletal remodeling. Gene profiling and bioinformatics further identify the specific targets of MIR27a in osteoclast cells. MIR27a exerts its effects on osteoclast differentiation through modulation of Squstm1/p62 whose mutations have been linked to Paget's disease of bone. Our findings reveal a new MIR27a-p62 axis necessary and sufficient to mediate osteoclast differentiation and highlight a therapeutic implication for osteoporosis.

Comment on doi: 10.7554/eLife.81892.

Addressing the climate crisis requires radical and urgent action at all levels of society. Universities are ideally positioned to lead such action but are largely failing to do so. At the same time, many academic scientists find their work impeded by bureaucracy, excessive competitiveness, and a loss of academic freedom. Here, drawing on the framework of "Doughnut Economics," developed by Kate Raworth, we suggest seven new principles for rethinking the norms of scientific practice. Based on these, we propose a call to action, and encourage academics to take concrete steps towards the creation of a flourishing scientific enterprise that is fit for the challenges of the 21st century.

The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallet et. al, 2015) reported that AMPK in yeast, i.e. Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.

One challenge in neuroscience is to understand how information flows between neurons in vivo to trigger specific behaviors. Granger causality (GC) has been proposed as a simple and effective measure for identifying dynamical interactions. At single-cell resolution however, GC analysis is rarely used compared to directionless correlation analysis. Here, we study the applicability of GC analysis for calcium imaging data in diverse contexts. We first show that despite underlying linearity assumptions, GC analysis successfully retrieves non-linear interactions in a synthetic network simulating intracellular calcium fluctuations of spiking neurons. We highlight the potential pitfalls of applying GC analysis on real in vivo calcium signals, and offer solutions regarding the choice of GC analysis parameters. We took advantage of calcium imaging datasets from motoneurons in embryonic zebrafish to show how the improved GC can retrieve true underlying information flow. Applied to the network of brainstem neurons of larval zebrafish, our pipeline reveals strong driver neurons in the locus of the mesencephalic locomotor region (MLR), driving target neurons matching expectations from anatomical and physiological studies. Altogether, this practical toolbox can be applied on in vivo population calcium signals to increase the selectivity of GC to infer flow of information across neurons.

Healthy neuronal networks rely on homeostatic plasticity to maintain stable firing rates despite changing synaptic drive. These mechanisms, however, can themselves be destabilizing if activated inappropriately or excessively. For example, prolonged activity deprivation can lead to rebound hyperactivity and seizures. While many forms of homeostasis have been described, whether and how the magnitude of homeostatic plasticity is constrained remains unknown. Here we uncover negative regulation of cortical network homeostasis by the PARbZIP family of transcription factors. In cortical slice cultures made from knockout mice lacking all three of these factors, the network response to prolonged activity withdrawal measured with calcium imaging is much stronger, while baseline activity is unchanged. Whole cell recordings reveal an exaggerated increase in the frequency of miniature excitatory synaptic currents reflecting enhanced upregulation of recurrent excitatory synaptic transmission. Genetic analyses reveal that two of the factors, Hlf and Tef, are critical for constraining plasticity and for preventing life-threatening seizures. These data indicate that transcriptional activation is not only required for many forms of homeostatic plasticity but is also involved in restraint of the response to activity deprivation.

Microglia, the resident immune cells of the brain, play a complex role in health and disease. They actively survey the brain parenchyma by physically interacting with other cells and structurally shaping the brain. Yet the mechanisms underlying microglial motility and significance for synapse stability, especially in the hippocampus during adulthood, remain widely unresolved. Here we investigated the effect of neuronal activity on microglial motility and the implications for the formation and survival of dendritic spines on hippocampal CA1 neurons in vivo. We used repetitive two-photon in vivo imaging in the hippocampus of awake and anesthetized mice to simultaneously study the motility of microglia and their interaction with dendritic spines. We found that CA3 to CA1 input is sufficient to modulate microglial process motility. Simultaneously, more dendritic spines emerged in mice after awake compared to anesthetized imaging. Interestingly, the rate of microglial contacts with individual dendritic spines and dendrites was associated with the stability, removal, and emergence of dendritic spines. These results suggest that microglia might sense neuronal activity via neurotransmitter release and actively participate in synaptic rewiring of the hippocampal neural network during adulthood. Further, this study has profound relevance for hippocampal learning and memory processes.

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15's abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases' abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Background: An increasing number of observational studies have reported the persistence of symptoms following recovery from acute COVID-19 disease in non-cancer patients. The long-term consequences of COVID-19 are not fully understood particularly in the cancer patient population. The purpose of this study is to assess post-acute sequelae of SARS-CoV-2 infection (PASC) in cancer patients following acute COVID-19 recovery. Methods: We identified cancer patients at MD Anderson Cancer Center who were diagnosed with COVID-19 disease between March 1, 2020 and Sept 1, 2020 and followed them till May 2021. To assess PASC, we collected patients reported outcomes through questionnaires that were sent to patients daily for 14 days after COVID-19 diagnosis then weekly for 3 months, and then monthly thereafter. We also reviewed patients' electronic medical records to capture the persistence or emergence of new COVID19-related symptoms reported during any clinic or hospital encounter beyond 30 days of the acute illness and up to 14 months. Results: We included 312 cancer patients with a median age of 57 years (18-86). The majority of patients had solid tumors (75%). Of the 312 patients, 188 (60%) reported long COVID-19 symptoms with a median duration of 7 months and up to 14 months after COVID-19 diagnosis. The most common symptoms reported included fatigue (82%), sleep disturbances (78%), myalgias (67%) and gastrointestinal symptoms (61%), followed by headache, altered smell or taste, dyspnea (47%) and cough (46%). A higher number of females reported a persistence of symptoms compared to males (63% vs 37%; p=0.036). Cancer type, neutropenia, lymphocytopenia, and hospital admission during acute COVID-19 disease were comparable in both groups. Among the 188 patients with PASC, only 16 (8.5%) were readmitted for COVID-related reasons. Conclusions: More than one out of two cancer patients, and more likely females, report PASC that may persist beyond 6 months and even one year. The most common symptoms are non-respiratory and consist of fatigue, sleep disturbance, myalgia and gastro-intestinal symptoms. Most of the cancer patients with PASC were managed on outpatient basis with only 8,5% requiring a COVID-19 related re-admission. Funding: This research is supported by the National Institutes of Health/National Cancer Institute under award number P30CA016672, which supports the MD Anderson Cancer Center Clinical Trials Office. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

During development, retinal progenitors navigate a complex landscape of fate decisions to generate the major cell classes necessary for proper vision. Transcriptional regulation is critical to generate diversity within these major cell classes. Here, we aim to provide the resources and techniques required to identify transcription factors necessary to generate and maintain diversity in photoreceptor subtypes, which are critical for vision. First, we generate a key resource: a high-quality and deep transcriptomic profile of each photoreceptor subtype in adult zebrafish. We make this resource openly accessible, easy to explore, and have integrated it with other currently available photoreceptor transcriptomic datasets. Second, using our transcriptomic profiles, we derive an in-depth map of expression of transcription factors in photoreceptors. Third, we use efficient CRISPR-Cas9 based mutagenesis to screen for null phenotypes in F0 larvae (F0 screening) as a fast, efficient, and versatile technique to assess the involvement of candidate transcription factors in the generation of photoreceptor subtypes. We first show that known phenotypes can be easily replicated using this method: loss of S cones in foxq2 mutants and loss of rods in nr2e3 mutants. We then identify novel functions for the transcription factor Tbx2, demonstrating that it plays distinct roles in controlling the generation of all photoreceptor subtypes within the retina. Our study provides a roadmap to discover additional factors involved in this process. Additionally, we explore four transcription factors of unknown function (Skor1a, Sall1a, Lrrfip1a, and Xbp1), and find no evidence for their involvement in the generation of photoreceptor subtypes. This dataset and screening method will be a valuable way to explore the genes involved in many other essential aspects of photoreceptor biology.

The proprioceptive system is essential for the control of coordinated movement, posture and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

In developing rats, behavioral state exerts a profound modulatory influence on neural activity throughout the sensorimotor system, including primary motor cortex (M1). We hypothesized that similar state-dependent modulation occurs in prefrontal cortical areas with which M1 forms functional connections. Here, using 8- and 12-day-old rats cycling freely between sleep and wake, we record neural activity in M1, secondary motor cortex (M2), and medial prefrontal cortex (mPFC). At both ages in all three areas, neural activity increased during active sleep (AS) compared with wake. Also, regardless of behavioral state, neural activity in all three areas increased during periods when limbs were moving. The movement-related activity in M2 and mPFC, like that in M1, is driven by sensory feedback. Our results, which diverge from those of previous studies using anesthetized pups, demonstrate that AS-dependent modulation and sensory responsivity extend to prefrontal cortex. These findings expand the range of possible factors shaping the activity-dependent development of higher-order cortical areas.

Female Aedes aegypti mosquitoes impose a severe global public health burden as vectors of multiple viral pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges like intense droughts and intermittent precipitation, which create unpredictable, suboptimal conditions for egg-laying. Here we show that under drought-like conditions simulated in the laboratory, females retain mature eggs in their ovaries for extended periods, while maintaining the viability of these eggs until they can be laid in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes named tweedledee and tweedledum, each encoding a small, secreted protein that both show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. CRISPR-Cas9 deletion of both tweedledee and tweedledum demonstrates that they are specifically required for extended retention of viable eggs. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with 'insurance' to flexibly extend their reproductive schedule without losing reproductive capacity, thus allowing this species to exploit unpredictable habitats in a changing world.

Schooling in fish is linked to a number of factors such as increased foraging success, predator avoidance, and social interactions. In addition, a prevailing hypothesis is that swimming in groups provides energetic benefits through hydrodynamic interactions. Thrust wakes are frequently occurring flow structures in fish schools as they are shed behind swimming fish. Despite increased flow speeds in these wakes, recent modeling work has suggested that swimming directly in-line behind an individual may lead to increased efficiency. However, only limited data are available on live fish interacting with thrust wakes. Here we designed a controlled experiment in which brook trout, Salvelinus fontinalis, interact with thrust wakes generated by a robotic mechanism that produces a fish-like wake. We show that trout swim in thrust wakes, reduce their tail-beat frequencies, and synchronize with the robotic flapping mechanism. Our flow and pressure field analysis revealed that the trout are interacting with oncoming vortices and that they exhibit reduced pressure drag at the head compared to swimming in isolation. Together, these experiments suggest that trout swim energetically more efficiently in thrust wakes and support the hypothesis that swimming in the wake of one another is an advantageous strategy to save energy in a school.

BACKGROUND: More than half of Chinese patients with hormone receptor positive (HR+) ductal carcinoma in situ (DCIS) are treated with mastectomy, and usually subjected to postoperative endocrine therapy (ET). Given that long-term ET can cause severe adverse effects it is important to determine the beneficial effect and safety of post-mastectomy ET on the disease-free survival (DFS) and adverse events in patients with HR+ DCIS. METHODS: To explore beneficial effect and safety of post-mastectomy ET in patients with HR+ DCIS, we performed a multicenter, population-based study. This retrospective study analyzed the DFS and adverse events in 1037 HR+ DCIS Chinese patients with or without post-mastectomy ET from eight breast centers between 2006 and 2016. The median follow-up time period was 86 months. RESULTS: There were 791 DCIS patients receiving ET (ET group). Those patients were followed up for a median of 86 months (range, 60-177 months). There were 23 cases with tumor recurrence or distant metastasis. There were similar 5-year DFS rates and DFS between the ET and non-ET groups, even for those with high-risk factors. Conversely, 37.04% of patients suffered from adverse events after ET, which were significantly higher than those in the non-ET group. CONCLUSIONS: ET after mastectomy did not benefit patients with HR+ DCIS for their DFS, rather increased adverse events in those patients. Therefore, ET after mastectomy may not be recommended for patients with HR+ DCIS, even for those with high-risk factors, such as multifocal, microinvasive, and higher T stage. FUNDING: This study was supported by grants from Outstanding Scientific Fund of Shengjing Hospital (201803) and Outstanding Young Scholars of Liaoning Province (2019-YQ-10).

Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. We find that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. Our findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, our work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system.

Skeletal muscles support the stability and mobility of the skeleton but differ in biomechanical properties and physiological functions. The intrinsic factors that regulate muscle-specific characteristics are poorly understood. To study these, we constructed a large atlas of RNA-seq profiles from six leg muscles and two locations from one muscle, using biopsies from 20 healthy young males. We identified differential expression patterns and cellular composition across the seven tissues using three bioinformatics approaches confirmed by large-scale newly developed quantitative immune-histology procedures. With all three procedures, the muscle samples clustered into three groups congruent with their anatomical location. Concomitant with genes marking oxidative metabolism, genes marking fast- or slow-twitch myofibers differed between the three groups. The groups of muscles with higher expression of slow-twitch genes were enriched in endothelial cells and showed higher capillary content. In addition, expression profiles of Homeobox (HOX) transcription factors differed between the three groups and were confirmed by spatial RNA hybridization. We created an open-source graphical interface to explore and visualize the leg muscle atlas (https://tabbassidaloii.shinyapps.io/muscleAtlasShinyApp/). Our study reveals the molecular specialization of human leg muscles, and provides a novel resource to study muscle-specific molecular features, which could be linked with (patho)physiological processes.

Comment on doi: 10.7554/eLife.82595.

The locus coeruleus (LC) houses the vast majority of noradrenergic neurons in the brain and regulates many fundamental functions including fight and flight response, attention control, and sleep/wake cycles. While efferent projections of the LC have been extensively investigated, little is known about its local circuit organization. Here, we performed large-scale multi-patch recordings of noradrenergic neurons in adult mouse LC to profile their morpho-electric properties while simultaneously examining their interactions. LC noradrenergic neurons are diverse and could be classified into two major morpho-electric types. While fast excitatory synaptic transmission among LC noradrenergic neurons was not observed in our preparation, these mature LC neurons connected via gap junction at a rate similar to their early developmental stage and comparable to other brain regions. Most electrical connections form between dendrites and are restricted to narrowly spaced pairs or small clusters of neurons of the same type. In addition, more than two electrically coupled cell pairs were often identified across a cohort of neurons from individual multi-cell recording sets that followed a chain-like organizational pattern. The assembly of LC noradrenergic neurons thus follows a spatial and cell type-specific wiring principle that may be imposed by a unique chain-like rule.

Computational models starting from large ensembles of evolutionarily related protein sequences capture a representation of protein families and learn constraints associated to protein structure and function. They thus open the possibility for generating novel sequences belonging to protein families. Protein language models trained on multiple sequence alignments, such as MSA Transformer, are highly attractive candidates to this end. We propose and test an iterative method that directly employs the masked language modeling objective to generate sequences using MSA Transformer. We demonstrate that the resulting sequences score as well as natural sequences, for homology, coevolution and structure-based measures. For large protein families, our synthetic sequences have similar or better properties compared to sequences generated by Potts models, including experimentally-validated ones. Moreover, for small protein families, our generation method based on MSA Transformer outperforms Potts models. Our method also more accurately reproduces the higher-order statistics and the distribution of sequences in sequence space of natural data than Potts models. MSA Transformer is thus a strong candidate for protein sequence generation and protein design.

Drug metabolism by the microbiome can influence anti-cancer treatment success. We previously suggested that chemotherapies with antimicrobial activity can select for adaptations in bacterial drug metabolism that can inadvertently influence the host's chemoresistance. We demonstrated that evolved resistance against fluoropyrimidine chemotherapy lowered its efficacy in worms feeding on drug-evolved bacteria (Rosener et al., 2020). Here we examine a model system that captures local interactions that can occur in the tumor microenvironment. Gammaproteobacteria colonizing pancreatic tumors can degrade the nucleoside-analog chemotherapy gemcitabine and, in doing so, can increase the tumor's chemoresistance. Using a genetic screen in Escherichia coli, we mapped all loss-of-function mutations conferring gemcitabine resistance. Surprisingly, we infer that one third of top resistance mutations increase or decrease bacterial drug breakdown and therefore can either lower or raise the gemcitabine load in the local environment. Experiments in three E. coli strains revealed that evolved adaptation converged to inactivation of the nucleoside permease NupC, an adaptation that increased the drug burden on co-cultured cancer cells. The two studies provide complementary insights on the potential impact of microbiome adaptation to chemotherapy by showing that bacteria-drug interactions can have local and systemic influence on drug activity.

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.

Newborns are unable to reach the adult-level humoral immune response partly due to the potent immunoregulatory role of IL-10. Increased IL-10 production by neonatal B cells has been attributed to the larger population of IL-10-producting CD43+ B-1 cells in neonates. Here, we show that neonatal mouse CD43- non-B-1 cells also produce substantial amounts of IL-10 following B cell antigen receptor (BCR) activation. In neonatal mouse CD43- non-B-1 cells, BCR engagement activated STAT5 under the control of phosphorylated forms of signaling molecules Syk, Btk, PKC, FAK and Rac1. Neonatal STAT5 activation led to IL-6 production, which in turn was responsible for IL-10 production in an autocrine/paracrine fashion through the activation of STAT3. In addition to the increased IL-6 production in response to BCR stimulation, elevated expression of IL-6Rα expression in neonatal B cells rendered them highly susceptible to IL-6 mediated STAT3 phosphorylation and IL-10 production. Finally, IL-10 secreted from neonatal mouse CD43- non-B-1 cells was sufficient to inhibit TNF-α secretion by macrophages. Our results unveil a distinct mechanism of IL-6-dependent IL-10 production in BCR-stimulated neonatal CD19+CD43- B cells.

Background: In Italy, Regions have the mandate to implement population-based screening programs for breast, cervical, and colorectal cancer. From March to May 2020, a severe lockdown was imposed due to the COVID-19 pandemic by the Italian Ministry of Health, with the suspension of screening programs. This paper describes the impact of the pandemic on Italian screening activities and test coverage in 2020 overall and by socio-economic characteristics. Methods: The regional number of subjects invited and of screening tests performed in 2020 were compared with those in 2019. Invitation and examination coverage were also calculated. PASSI surveillance system, through telephone interviews, collects information about screening test uptake by test provider (public screening and private opportunistic). Test coverage and test uptake in the last year were computed, by educational attainment, perceived economic difficulties, and citizenship. Results: A reduction of subjects invited and tests performed, with differences between periods and geographic macro areas, was observed in 2020 vs. 2019. The reduction in examination coverage was larger than that in invitation coverage for all screening programs. From the second half of 2020, the trend for test coverage showed a decrease in all the macro areas for all the screening programs. Compared with the pre-pandemic period, there was a greater difference according to the level of education in the odds of having had a test last year vs. never having been screened or not being up to date with screening tests. Conclusions: The lockdown and the ongoing COVID-19 emergency caused an important delay in screening activities. This increased the pre-existing individual and geographical inequalities in access. The opportunistic screening did not mitigate the impact of the pandemic. Funding: This study was partially supported by Italian Ministry of Health - Ricerca Corrente Annual Program 2023.

Peroxiredoxin 5 (Prdx5) is involved in pathophysiological regulation via the stress-induced cellular response. However, its function in the bone remains largely unknown. Here, we show that Prdx5 is involved in osteoclast and osteoblast differentiation, resulting in osteoporotic phenotypes in Prdx5 knockout (Prdx5Ko) male mice. To investigate the function of Prdx5 in the bone, osteoblasts were analyzed through immunoprecipitation (IP) and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) methods, while osteoclasts were analyzed through RNA-sequencing. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as a potential binding partner of Prdx5 during osteoblast differentiation in vitro. Prdx5 acts as a negative regulator of hnRNPK-mediated osteocalcin (Bglap) expression. In addition, transcriptomic analysis revealed that in vitro differentiated osteoclasts from the bone marrow-derived macrophages of Prdx5Ko mice showed enhanced expression of several osteoclast-related genes. These findings indicate that Prdx5 might contribute to the maintenance of bone homeostasis by regulating osteoblast differentiation. This study proposes a new function of Prdx5 in bone remodeling that may be used in developing therapeutic strategies for bone diseases.

The absence of evidence to assess treatment efficacy partially underpins the unsustainable expenditure of the US healthcare system, a challenge exacerbated by a limited understanding of the factors influencing the translation of clinical research into practice. Leveraging a dataset of >10,000 UpToDate articles, sampled every 3 months between 2011 and 2020, we trace the path of research (37,050 newly added articles from 887 journals) from initial publication to the point-of-care, compared to the 2.4 million uncited studies published during the same time window across 18 medical specialties. Our analysis reveals substantial variation in how specialties prioritize/adopt research, with regards to a fraction of literature cited (0.4-2.4%) and quality-of-evidence incorporated. In 9 of 18 specialties, less than 1 in 10 clinical trials are ever cited. Furthermore, case reports represent one of the most cited article types in 12 of 18 specialties, comprising nearly a third of newly added references for some specialties (e.g. dermatology). Anesthesiology, cardiology, critical care, geriatrics, internal medicine, and oncology tended to favor higher-quality evidence. By modeling citations as a function of National Institutes of Health (NIH) department-specific funding, we estimate the cost of bringing one new clinical citation to the point-of-care as ranging from thousands to tens of thousands of dollars depending on specialty. The success of a subset of specialties in incorporating a larger proportion of published research, as well as high(er) quality of evidence, demonstrates the existence of translational strategies that should be applied more broadly. In addition to providing a baseline for monitoring the efficiency of research investments, we also describe new 'impact' indices to assess the efficacy of reforms to the clinical scientific enterprise.

Update of doi: 10.1101/2022.08.01.502322.

Update of doi: 10.1101/2022.07.22.501123.

The survival prognosis of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) is largely different, and little is known about the anti-tumor mechanism of tumor-infiltrated exhausted CD8+ T cells (Tex) in HNSCC. We performed cell-level multi-omics sequencing on human HNSCC samples to decipher the multi-dimensional characteristics of Tex cells. A proliferative exhausted CD8+ T cell cluster (P-Tex) which was beneficial to survival outcomes of patients with HPV-positive HNSCC was identified. Interestingly, P-Tex cells expressed CDK4 genes as high as cancer cells, which could be simultaneously inhibited by CDK4 inhibitors and might be a potential reason for the ineffectiveness of CDK4 inhibitors in treating HPV-positive HNSCC. P-Tex cells could aggregate in the antigen-presenting cell niches and activate certain signaling pathways. Together, our findings suggest a promising role for P-Tex cells in the prognosis of patients with HPV-positive HNSCC by providing modest but persistent anti-tumor effects.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

There has been debate about whether addition of an irrelevant distractor option to an otherwise binary decision influences which of the two choices is taken. We show that disparate views on this question are reconciled if distractors exert two opposing but not mutually exclusive effects. Each effect predominates in a different part of decision space: (1) a positive distractor effect predicts high-value distractors improve decision-making; (2) a negative distractor effect, of the type associated with divisive normalisation models, entails decreased accuracy with increased distractor values. Here, we demonstrate both distractor effects coexist in human decision making but in different parts of a decision space defined by the choice values. We show disruption of the medial intraparietal area (MIP) by transcranial magnetic stimulation (TMS) increases positive distractor effects at the expense of negative distractor effects. Furthermore, individuals with larger MIP volumes are also less susceptible to the disruption induced by TMS. These findings also demonstrate a causal link between MIP and the impact of distractors on decision-making via divisive normalisation.

Dynamic nucleocytoplasmic transport of E-M factors regulates cellular E-M states; yet, it remains unknown how simultaneously trapping these factors affects epithelia at the macroscale. To explore this question, we performed nuclear export inhibition (NEI) via leptomycin B and Selinexor treatment, which biases nuclear localization of CRM1-associated E-M factors. We examined changes in collective cellular phenotypes across a range of substrate stiffnesses. Following NEI, soft substrates elevate collective migration of MCF10A cells for up to 24 hr, while stiffer substrates reduce migration at all time points. Our results suggest that NEI disrupts migration through competition between intercellular adhesions and mechanoactivation, generally causing loss of cell-cell coordination. Specifically, across substrate stiffnesses, NEI fosters an atypical E-M state wherein MCF10A cells become both more epithelial and more mesenchymal. We observe that NEI fosters a range of these concurrent phenotypes, from more epithelial shYAP MCF10A cells to more mesenchymal MDCK II cells. α-Catenin emerges as a potential link between E-M states, where it maintains normal levels of intercellular adhesion and transmits mechanoactive characteristics to collective behavior. Ultimately, to accommodate the concurrent states observed here, we propose an expanded E-M model, which may help further understand fundamental biological phenomena and inform pathological treatments.

The Omicron BA.1 variant of SARS-CoV-2 escapes convalescent sera and monoclonal antibodies that are effective against earlier strains of the virus. This immune evasion is largely a consequence of mutations in the BA.1 receptor binding domain (RBD), the major antigenic target of SARS-CoV-2. Previous studies have identified several key RBD mutations leading to escape from most antibodies. However, little is known about how these escape mutations interact with each other and with other mutations in the RBD. Here, we systematically map these interactions by measuring the binding affinity of all possible combinations of these 15 RBD mutations (215=32,768 genotypes) to 4 monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10987, and S309) with distinct epitopes. We find that BA.1 can lose affinity to diverse antibodies by acquiring a few large-effect mutations and can reduce affinity to others through several small-effect mutations. However, our results also reveal alternative pathways to antibody escape that does not include every large-effect mutation. Moreover, epistatic interactions are shown to constrain affinity decline in S309 but only modestly shape the affinity landscapes of other antibodies. Together with previous work on the ACE2 affinity landscape, our results suggest that the escape of each antibody is mediated by distinct groups of mutations, whose deleterious effects on ACE2 affinity are compensated by another distinct group of mutations (most notably Q498R and N501Y).

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Traditional therapeutic approaches such as chemotherapy or radiotherapy have provided only a marginal improvement in the treatment of lung carcinomas. Inhibitors targeting specific genetic aberrations present in non-small cell lung cancer (NSCLC), the most common subtype (85%), have improved the prognostic outlook, but due to the complexity of the LC mutational spectrum, only a fraction of patients benefit from these targeted molecular therapies. More recently, the realization that the immune infiltrate surrounding solid tumors can foster tumor-promoting inflammation has led to the development and implementation of anticancer immunotherapies in the clinic. In NSCLC, one of the most abundant leukocyte infiltrates is macrophages. These highly plastic phagocytes, which are part of the cellular repertoire of the innate immunity, can have a pivotal role in early NSCLC establishment, malignant progression, and tumor invasion. Emerging macrophage-targeting therapies have been focused on the re-differentiation of the macrophages toward an antitumorigenic phenotype, depletion of tumor-promoting macrophage subtypes, or combination therapies combining traditional cytotoxic treatments with immunotherapeutic agents. The most extensively used models employed for the exploration of NSCLC biology and therapy have been 2D cell lines and murine models. However, studying cancer immunology requires appropriately complex models. 3D platforms, including organoid models, are quickly advancing powerful tools to study immune cell-epithelial cell interactions within the tumor microenvironment. Co-cultures of immune cells along with NSCLC organoids allow for an in vitro observation of the tumor microenvironment dynamics closely resembling in vivo settings. Ultimately, the implementation of 3D organoid technology into tumor microenvironment-modeling platforms might facilitate the exploration of macrophage-targeted therapies in NSCLC immunotherapeutic research, thus establishing a new frontier in NSCLC treatment.

Highly fecund natural populations characterized by high early mortality abound, yet our knowledge about their recruitment dynamics is somewhat rudimentary. This knowledge gap has implications for our understanding of genetic variation, population connectivity, local adaptation, and the resilience of highly fecund populations. The concept of sweepstakes reproductive success, which posits a considerable variance and skew in individual reproductive output, is key to understanding the distribution of individual reproductive success. However, it still needs to be determined whether highly fecund organisms reproduce through sweepstakes and, if they do, the relative roles of neutral and selective sweepstakes. Here, we use coalescent-based statistical analysis of population genomic data to show that selective sweepstakes likely explain recruitment dynamics in the highly fecund Atlantic cod. We show that the Kingman coalescent (modelling no sweepstakes) and the Xi-Beta coalescent (modelling random sweepstakes), including complex demography and background selection, do not provide an adequate fit for the data. The Durrett-Schweinsberg coalescent, in which selective sweepstakes result from recurrent and pervasive selective sweeps of new mutations, offers greater explanatory power. Our results show that models of sweepstakes reproduction and multiple-merger coalescents are relevant and necessary for understanding genetic diversity in highly fecund natural populations. These findings have fundamental implications for understanding the recruitment variation of fish stocks and general evolutionary genomics of high-fecundity organisms.

Comment on doi: 10.7554/eLife.82311.

Background: Whether the positive associations of smoking and alcohol consumption with gastrointestinal diseases are causal is uncertain. We conducted this Mendelian randomization (MR) to comprehensively examine associations of smoking and alcohol consumption with common gastrointestinal diseases. Methods: Genetic variants associated with smoking initiation and alcohol consumption at the genome-wide significance level were selected as instrumental variables. Genetic associations with 24 gastrointestinal diseases were obtained from the UK Biobank, FinnGen study, and other large consortia. Univariable and multivariable MR analyses were conducted to estimate the overall and independent MR associations after mutual adjustment for genetic liability to smoking and alcohol consumption. Results: Genetic predisposition to smoking initiation was associated with increased risk of 20 of 24 gastrointestinal diseases, including 7 upper gastrointestinal diseases (gastroesophageal reflux, esophageal cancer, gastric ulcer, duodenal ulcer, acute gastritis, chronic gastritis and gastric cancer), 4 lower gastrointestinal diseases (irritable bowel syndrome, diverticular disease, Crohn's disease and ulcerative colitis), 8 hepatobiliary and pancreatic diseases (non-alcoholic fatty liver disease, alcoholic liver disease, cirrhosis, liver cancer, cholecystitis, cholelithiasis, acute and chronic pancreatitis), and acute appendicitis. Fifteen out of 21 associations persisted after adjusting for genetically-predicted alcohol consumption. Genetically-predicted higher alcohol consumption was associated with increased risk of duodenal cancer, alcoholic liver disease, cirrhosis, and chronic pancreatitis; however, the association for duodenal ulcer did not remain after adjustment for genetic predisposition to smoking initiation. Conclusion: This study provides MR evidence supporting causal associations of smoking with a broad range of gastrointestinal diseases, whereas alcohol consumption was associated with only a few gastrointestinal diseases. Funding: The Natural Science Fund for Distinguished Young Scholars of Zhejiang Province; National Natural Science Foundation of China; Key Project of Research and Development Plan of Hunan Province; the Swedish Heart Lung Foundation; the Swedish Research Council; the Swedish Cancer Society.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by expression of the enzyme Arginase 1 (Arg1), which we demonstrated is potently expressed in pancreatic tumor associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, Arg1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Additionally, Arg1 deletion induced compensatory mechanisms, including Arg1 overexpression in epithelial cells, namely Tuft cells, and Arg2 overexpression in a subset of macrophages. To overcome these compensatory mechanisms, we used a pharmacological approach to inhibit arginase. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Our data demonstrate that Arg1 drives immune suppression in pancreatic cancer by depleting Arginine and inhibiting T cell activation.

Human agents build models of their environment, which enable them to anticipate and plan upcoming events. However, little is known about the properties of such predictive models. Recently, it has been proposed that hippocampal representations take the form of a predictive map-like structure, the so-called successor representation (SR). Here, we used human functional magnetic resonance imaging to probe whether activity in the early visual cortex (V1) and hippocampus adhere to the postulated properties of the SR after visual sequence learning. Participants were exposed to an arbitrary spatiotemporal sequence consisting of four items (A-B-C-D). We found that after repeated exposure to the sequence, merely presenting single sequence items (e.g., - B - -) resulted in V1 activation at the successor locations of the full sequence (e.g., C-D), but not at the predecessor locations (e.g., A). This highlights that visual representations are skewed toward future states, in line with the SR. Similar results were also found in the hippocampus. Moreover, the hippocampus developed a coactivation profile that showed sensitivity to the temporal distance in sequence space, with fading representations for sequence events in the more distant past and future. V1, in contrast, showed a coactivation profile that was only sensitive to spatial distance in stimulus space. Taken together, these results provide empirical evidence for the proposition that both visual and hippocampal cortex represent a predictive map of the visual world akin to the SR.

Every decision that we make involves a conflict between exploiting our current knowledge of an action's value or exploring alternative courses of action that might lead to a better, or worse outcome. The sub-cortical nuclei that make up the basal ganglia have been proposed as a neural circuit that may contribute to resolving this explore-exploit 'dilemma'. To test this hypothesis, we examined the effects of neuromodulating the basal ganglia's output nucleus, the globus pallidus interna, in patients who had undergone deep brain stimulation (DBS) for isolated dystonia. Neuromodulation enhanced the number of exploratory choices to the lower value option in a 2-armed bandit probabilistic reversal-learning task. Enhanced exploration was explained by a reduction in the rate of evidence accumulation (drift rate) in a reinforcement learning drift diffusion model. We estimated the functional connectivity profile between the stimulating DBS electrode and the rest of the brain using a normative functional connectome derived from heathy controls. Variation in the extent of neuromodulation induced exploration between patients was associated with functional connectivity from the stimulation electrode site to a distributed brain functional network. We conclude that the basal ganglia's output nucleus, the globus pallidus interna, can adaptively modify decision choice when faced with the dilemma to explore or exploit.

Most membrane protein molecules undergo conformational changes as they transition from one functional state to another one. An understanding of the mechanism underlying these changes requires the ability to resolve individual conformational states, whose changes often occur on millisecond and angstrom scales. Tracking such changes and acquiring a sufficiently large amount of data remain challenging. Here, we use the amino-acid transporter AdiC as an example to demonstrate the application of a high-resolution fluorescence-polarization-microscopy method in tracking multistate conformational changes of a membrane protein. We have successfully resolved four conformations of AdiC by monitoring the emission-polarization changes of a fluorophore label and quantified their probabilities in the presence of a series of concentrations of its substrate arginine. The acquired data are sufficient for determining all equilibrium constants that fully establish the energetic relations among the four states. The KD values determined for arginine in four individual conformations are statistically comparable to the previously reported overall KD determined using isothermal titration calorimetry. This demonstrated strong resolving power of the present polarization-microscopy method will enable an acquisition of the quantitative information required for understanding the expected complex conformational mechanism underlying the transporter's function, as well as those of other membrane proteins.

Photosystem II (PSII) generates an oxidant whose redox potential is high enough to enable water oxidation , a substrate so abundant that it assures a practically unlimited electron source for life on earth . Our knowledge on the mechanism of water photooxidation was greatly advanced by high-resolution structures of prokaryotic PSII . Here, we show high-resolution cryogenic electron microscopy (cryo-EM) structures of eukaryotic PSII from the green alga Dunaliella salina at two distinct conformations. The conformers are also present in stacked PSII, exhibiting flexibility that may be relevant to the grana formation in chloroplasts of the green lineage. CP29, one of PSII associated light-harvesting antennae, plays a major role in distinguishing the two conformations of the supercomplex. We also show that the stacked PSII dimer, a form suggested to support the organisation of thylakoid membranes , can appear in many different orientations providing a flexible stacking mechanism for the arrangement of grana stacks in thylakoids. Our findings provide a structural basis for the heterogenous nature of the eukaryotic PSII on multiple levels.

Intracellular trafficking of AMPA receptors is a tightly regulated process which involves several adaptor proteins, and is crucial for the activity of excitatory synapses both in basal conditions and during synaptic plasticity. We found that, in rat hippocampal neurons, an intracellular pool of the tetraspanin TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalisation. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin and possibly using recycling endosomes as a delivery route. This work highlights TSPAN5 as a new adaptor regulating AMPA receptor trafficking.

Burn injuries are a leading cause of unintentional injury, associated with a dysfunctional immune response and an increased risk of infections. Despite this, little is known about the role of T cells in human burn injury. In this study, we compared the activation and function of conventional T cells and unconventional T cell subsets in skin tissue from acute burn (within 7 days from initial injury), late phase burn (beyond 7 days from initial injury), and non-burn patients. We compared T cell functionality by a combination of flow cytometry and a multi-omic single-cell approach with targeted transcriptomics and protein expression. We found a significantly lower proportion of CD8+ T cells in burn skin compared to non-burn skin, with CD4+ T cells making up the bulk of the T cell population. Both conventional and unconventional burn tissue T cells show significantly higher IFN-γ and TNF-α levels after stimulation than non-burn skin T cells. In sorted T cells, clustering showed that burn tissue had significantly higher expression of homing receptors CCR7, S1PR1, and SELL compared to non-burn skin. In unconventional T cells, including mucosal-associated invariant T (MAIT) and γδ T cells, we see significantly higher expression of cytotoxic molecules GZMB, PRF1, and GZMK. Multi-omics analysis of conventional T cells suggests a shift from tissue-resident T cells in non-burn tissue to a circulating T cell phenotype in burn tissue. In conclusion, by examining skin tissue from burn patients, our results suggest that T cells in burn tissue have a pro-inflammatory rather than a homeostatic tissue-resident phenotype, and that unconventional T cells have a higher cytotoxic capacity. Our findings have the potential to inform the development of novel treatment strategies for burns.

Update of doi: 10.1101/2022.07.07.499202.

Making optimal decisions in the face of noise requires balancing short-term speed and accuracy. But a theory of optimality should account for the fact that short-term speed can influence long-term accuracy through learning. Here, we demonstrate that long-term learning is an important dynamical dimension of the speed-accuracy trade-off. We study learning trajectories in rats and formally characterize these dynamics in a theory expressed as both a recurrent neural network and an analytical extension of the drift-diffusion model that learns over time. The model reveals that choosing suboptimal response times to learn faster sacrifices immediate reward, but can lead to greater total reward. We empirically verify predictions of the theory, including a relationship between stimulus exposure and learning speed, and a modulation of reaction time by future learning prospects. We find that rats' strategies approximately maximize total reward over the full learning epoch, suggesting cognitive control over the learning process.

Coronary heart disease (CHD) is one of the most pressing health problems of our time and a major cause of preventable death. CHD results from complex interactions between genetic and environmental factors. Using multiplex serological testing for persistent or frequently recurring infections and genome-wide analysis in a prospective population study, we delineate the respective and combined influences of genetic variation, infections, and low-grade inflammation on the risk of incident CHD. Study participants are enrolled in the CoLaus|PsyCoLaus study, a longitudinal, population-based cohort with baseline assessments from 2003 through 2008 and follow-up visits every 5 years. We analyzed a subgroup of 3459 individuals with available genome-wide genotyping data and immunoglobulin G levels for 22 persistent or frequently recurring pathogens. All reported CHD events were evaluated by a panel of specialists. We identified independent associations with incident CHD using univariable and multivariable stepwise Cox proportional hazards regression analyses. Of the 3459 study participants, 210 (6.07%) had at least one CHD event during the 12 years of follow-up. Multivariable stepwise Cox regression analysis, adjusted for known cardiovascular risk factors, socioeconomic status, and statin intake, revealed that high polygenic risk (hazard ratio [HR] 1.31, 95% CI 1.10-1.56, p=2.64 × 10-3) and infection with Fusobacterium nucleatum (HR 1.63, 95% CI 1.08-2.45, p=1.99 × 10-2) were independently associated with incident CHD. In a prospective, population-based cohort, high polygenic risk and infection with F. nucleatum have a small, yet independent impact on CHD risk.

Antibiotic consumption and its abuses have been historically and repeatedly pointed out as the major driver of antibiotic resistance emergence and propagation. However, several examples show that resistance may persist despite substantial reductions in antibiotic use, and that other factors are at stake. Here, we study the temporal, spatial, and ecological distribution patterns of aminoglycoside resistance, by screening more than 160,000 publicly available genomes for 27 clusters of genes encoding aminoglycoside-modifying enzymes (AME genes). We find that AME genes display a very ubiquitous pattern: about 25% of sequenced bacteria carry AME genes. These bacteria were sequenced from all the continents (except Antarctica) and terrestrial biomes, and belong to a wide number of phyla. By focusing on European countries between 1997 and 2018, we show that aminoglycoside consumption has little impact on the prevalence of AME-gene-carrying bacteria, whereas most variation in prevalence is observed among biomes. We further analyze the resemblance of resistome compositions across biomes: soil, wildlife, and human samples appear to be central to understand the exchanges of AME genes between different ecological contexts. Together, these results support the idea that interventional strategies based on reducing antibiotic use should be complemented by a stronger control of exchanges, especially between ecosystems.

As we learn, dynamic memory processes build structured knowledge across our experiences. Such knowledge enables the formation of internal models of the world that we use to plan, make decisions, and act. Recent theorizing posits that mnemonic mechanisms of differentiation and integration - which at one level may seem to be at odds - both contribute to the emergence of structured knowledge. We tested this possibility using fMRI as human participants learned to navigate within local and global virtual environments over the course of 3 days. Pattern similarity analyses on entorhinal cortical and hippocampal patterns revealed evidence that differentiation and integration work concurrently to build local and global environmental representations, and that variability in integration relates to differences in navigation efficiency. These results offer new insights into the neural machinery and the underlying mechanisms that translate experiences into structured knowledge that allows us to navigate to achieve goals.

Colony stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

Humans make a number of choices when they walk, such as how fast and for how long. The preferred steady walking speed seems chosen to minimize energy expenditure per distance traveled. But the speed of actual walking bouts is not only steady, but rather a time-varying trajectory, which can also be modulated by task urgency or an individual's movement vigor. Here we show that speed trajectories and durations of human walking bouts are explained better by an objective to minimize Energy and Time, meaning the total work or energy to reach destination, plus a cost proportional to bout duration. Applied to a computational model of walking dynamics, this objective predicts dynamic speed vs. time trajectories with inverted U shapes. Model and human experiment (𝑁 = 10) show that shorter bouts are unsteady and dominated by the time and effort of accelerating, and longer ones are steadier and faster and dominated by steady-state time and effort. Individual-dependent vigor may be characterized by the energy one is willing to spend to save a unit of time, which explains why some may walk faster than others, but everyone may have similar-shaped trajectories due to similar walking dynamics. Tradeoffs between energy and time costs can predict transient, steady, and vigor-related aspects of walking.

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5'-GGGGTGACCCC-3') (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.

Chronic stress can produce reward system deficits (i.e., anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and it is required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation in the mice. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reductions in pyramidal neuron dendritic spine density, excitatory synaptic transmission, and presynaptic function, revealing a relationship between perturbation in excitatory synaptic transmission and the expression of anhedonia-like behavior in the mice. Finally, analysis of the mice mPFC tissues revealed that NPAS4 regulates the expression of numerous genes linked to glutamatergic synapses and ribosomal function, the expression of upregulated genes in CSDS-susceptible animals, and differentially expressed genes in postmortem human brains of patients with common neuropsychiatric disorders, including depression. Together, our findings position NPAS4 as a key mediator of chronic stress-induced hypofrontal states and anhedonia-like behavior.

Single spikes can trigger repeatable firing sequences in cortical networks. The mechanisms that support reliable propagation of activity from such small events and their functional consequences remain unclear. By constraining a recurrent network model with experimental statistics from turtle cortex, we generate reliable and temporally precise sequences from single spike triggers. We find that rare strong connections support sequence propagation, while dense weak connections modulate propagation reliability. We identify sections of sequences corresponding to divergent branches of strongly connected neurons which can be selectively gated. Applying external inputs to specific neurons in the sparse backbone of strong connections can effectively control propagation and route activity within the network. Finally, we demonstrate that concurrent sequences interact reliably, generating a highly combinatorial space of sequence activations. Our results reveal the impact of individual spikes in cortical circuits, detailing how repeatable sequences of activity can be triggered, sustained, and controlled during cortical computations.

Perceptual decisions are biased toward higher-value options when overall gains can be improved. When stimuli demand immediate reactions, the neurophysiological decision process dynamically evolves through distinct phases of growing anticipation, detection, and discrimination, but how value biases are exerted through these phases remains unknown. Here, by parsing motor preparation dynamics in human electrophysiology, we uncovered a multiphasic pattern of countervailing biases operating in speeded decisions. Anticipatory preparation of higher-value actions began earlier, conferring a 'starting point' advantage at stimulus onset, but the delayed preparation of lower-value actions was steeper, conferring a value-opposed buildup-rate bias. This, in turn, was countered by a transient deflection toward the higher-value action evoked by stimulus detection. A neurally-constrained process model featuring anticipatory urgency, biased detection, and accumulation of growing stimulus-discriminating evidence, successfully captured both behavior and motor preparation dynamics. Thus, an intricate interplay of distinct biasing mechanisms serves to prioritise time-constrained perceptual decisions.

M-CSF is a critical growth factor for myeloid lineage cells, including monocytes, macrophages and osteoclasts. Tissue-resident macrophages in most organs rely on local M-CSF. However, it is unclear what specific cells in the bone marrow produce M-CSF to maintain myeloid homeostasis. Here, we found that Adipoq-lineage progenitors but not mature adipocytes in bone marrow or in peripheral adipose tissue, are a major cellular source of M-CSF, with these Adipoq-lineage progenitors producing M-CSF at levels much higher than those produced by osteoblast lineage cells. Deficiency of M-CSF in bone marrow Adipoq-lineage progenitors drastically reduces the generation of bone marrow macrophages and osteoclasts, leading to severe osteopetrosis in mice. Furthermore, the osteoporosis in ovariectomized mice can be significantly alleviated by the absence of M-CSF in bone marrow Adipoq-lineage progenitors. Our findings identify bone marrow Adipoq-lineage progenitors as a major cellular source of M-CSF in bone marrow and reveal their crucial contribution to bone marrow macrophage development, osteoclastogenesis, bone homeostasis and pathological bone loss.

In the developing central nervous system, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes, which form myelin around axons. Oligodendrocytes and myelin are essential for the function of the central nervous system, as evidenced by the severe neurological symptoms that arise in demyelinating diseases such as multiple sclerosis and leukodystrophy. Although many cell-intrinsic mechanisms that regulate oligodendrocyte development and myelination have been reported, it remains unclear whether interactions among oligodendrocyte-lineage cells (OPCs and oligodendrocytes) affect oligodendrocyte development and myelination. Here, we show that blocking vesicle-associated membrane protein (VAMP) 1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development, myelination, and motor behavior in mice. Adding oligodendrocyte-lineage cell-secreted molecules to secretion-deficient OPC cultures partially restores the morphological maturation of oligodendrocytes. Moreover, we identified L-type prostaglandin D synthase as an oligodendrocyte-lineage cell-secreted protein that promotes oligodendrocyte development and myelination in vivo. These findings reveal a novel autocrine/paracrine loop model for the regulation of oligodendrocyte and myelin development.

Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on CAR-T immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1) which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial ROS release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the STC1 gene, which encodes the glycoprotein hormone staniocalcin-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo and STC1 played a critical role. These data revealed a novel function of MSC and staniocalcin-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response.

Recent studies have suggested that the human germline mutation rate and spectrum evolve rapidly. Variation in generation time has been linked to these changes, though its contribution remains unclear. We develop a framework to characterize temporal changes in polymorphisms within and between populations, while controlling for the effects of natural selection and biased gene conversion. Application to the 1000 Genomes Project dataset reveals multiple independent changes that arose after the split of continental groups, including a previously reported, transient elevation in TCC>TTC mutations in Europeans and novel signals of divergence in C>G and T>A mutation rates among population samples. We also find a significant difference between groups sampled in and outside of Africa, in old T>C polymorphisms that predate the out-of-Africa migration. This surprising signal is driven by TpG>CpG mutations, and stems in part from mis-polarized CpG transitions, which are more likely to undergo recurrent mutations. Finally, by relating the mutation spectrum of polymorphisms to parental age effects on de novo mutations, we show that plausible changes in the generation time cannot explain the patterns observed for different mutation types jointly. Thus, other factors--genetic modifiers or environmental exposures--must have had a non-negligible impact on the human mutation landscape.

Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus' impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

We analyzed changes in total costs for National Institutes of Health (NIH) awarded Research Project Grants (RPG) issued from fiscal years (FYs) 1998 to 2003. Costs are measured in 'nominal' terms, meaning exactly as stated, or in 'real' terms, meaning after adjustment for inflation. The NIH uses a data-driven price index - the Biomedical Research and Development Price Index (BRDPI) - to account for inflation, enabling assessment of changes in real (that is, BRDPI-adjusted) costs over time. The BRDPI was higher than the general inflation rate from FY1998 until FY2012; since then the BRDPI has been similar to the general inflation rate likely due to caps on senior faculty salary support. Despite increases in nominal costs, recent years have seen increases in the absolute numbers of RPG and R01 awards. Real average and median RPG costs increased during the NIH-doubling (FY1998 to FY2003), decreased after the doubling and have remained relatively stable since. Of note, though, the degree of variation of RPG costs has changed over time, with more marked extremes observed on both higher and lower levels of cost. On both ends of the cost spectrum, the agency is funding a greater proportion of solicited projects, with nearly half of RPG money going towards solicited projects. After adjusting for confounders, we find no independent association of time with BRDPI-adjusted costs; in other words, changes in real costs are largely explained by changes in the composition of the NIH-grant portfolio.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function mutations that increase DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new tools and analyses that further refine the CRISPR scanning framework.

Humans learn about the environment either directly by interacting with it or indirectly by seeking information about it from social sources such as conspecifics. The degree of confidence in the information obtained through either route should determine the impact that it has on adapting and changing behaviour. We examined whether and how behavioural and neural computations differ during non-social learning as opposed to learning from social sources. Trial-wise confidence judgements about non-social and social information sources offered a window into this learning process. Despite matching exactly the statistical features of social and non-social conditions, confidence judgements were more accurate and less changeable when they were made about social as opposed to non-social information sources. In addition to subjective reports of confidence, differences were also apparent in the Bayesian estimates of participants' subjective beliefs. Univariate activity in dorsomedial prefrontal cortex and posterior temporoparietal junction more closely tracked confidence about social as opposed to non-social information sources. In addition, the multivariate patterns of activity in the same areas encoded identities of social information sources compared to non-social information sources.

Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.

The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate. As later-specified posterior tuberal progenitors are generated, early specified anterior tuberal progenitors become progressively more distant from these BMP signals and differentiate into tuberal neurogenic cells. Gain- and loss-of-function experiments in vivo and ex vivo show that BMP signalling initiates tuberal progenitor specification, but must be eliminated for these to progress to anterior neurogenic progenitors. scRNA-Seq profiling shows that tuberal progenitors that are specified after the major period of anterior tuberal specification begin to upregulate genes that characterise radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.

A young group leader reflects on academic culture and working while going through cancer treatments.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Hyperosmolarity of the renal medulla is essential for urine concentration and water homeostasis. However, how renal medullary collecting duct (MCD) cells survive and function under harsh hyperosmotic stress remains unclear. Using RNA-Seq, we identified SLC38A2 as a novel osmoresponsive neutral amino acid transporter in MCD cells. Hyperosmotic stress-induced cell death in MCD cells occurred mainly via ferroptosis, and it was significantly attenuated by SLC38A2 overexpression but worsened by Slc38a2-gene deletion or silencing. Mechanistic studies revealed that the osmoprotective effect of SLC38A2 is dependent on the activation of mTORC1. Moreover, an in vivo study demonstrated that Slc38a2-knockout mice exhibited significantly increased medullary ferroptosis following water restriction. Collectively, these findings reveal that Slc38a2 is an important osmoresponsive gene in the renal medulla and provide novel insights into the critical role of SLC38A2 in protecting MCD cells from hyperosmolarity-induced ferroptosis via the mTORC1 signalling pathway.

Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.

Relief of ongoing pain is a potent motivator of behavior, directing actions to escape from or reduce potentially harmful stimuli. Whereas endogenous modulation of pain events is well characterized, relatively little is known about the modulation of pain relief and its corresponding neurochemical basis. Here we studied pain modulation during a probabilistic relief-seeking task (a 'wheel of fortune' gambling task), in which people actively or passively received reduction of a tonic thermal pain stimulus. We found that relief perception was enhanced by active decisions and unpredictability, and greater in high novelty-seeking trait individuals, consistent with a model in which relief is tuned by its informational content. We then probed the roles of dopaminergic and opioidergic signaling, both of which are implicated in relief processing, by embedding the task in a double-blinded cross-over design with administration of the dopamine precursor levodopa and the opioid receptor antagonist naltrexone. We found that levodopa enhanced each of these information-specific aspects of relief modulation but no significant effects of the opioidergic manipulation. These results show that dopaminergic signaling has a key role in modulating the perception of pain relief to optimize motivation and behavior.

To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3'-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3'-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.

Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3β - a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing CaV1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3β and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.

The GAL pathway of yeasts has long served as a model system for understanding of how regulatory mode of eukaryotic metabolic pathways evolves. While Gal4 mode has been well-characterized in Saccharomycetaceae clade, little is known about the regulation of the GAL pathway in other yeasts. Here, we find that Rep1, a Ndt80-like family transcription factor, serves as a galactose sensor in the commensal-pathogenic fungus Candida albicans. It is presented at the GAL gene promoters independent of the presence of galactose. Rep1 recognizes galactose via a direct physical interaction. The net result of this interaction is the recruitment of a transcriptional activator Cga1 (Candida galactose gene activator, orf19.4959) and transcription of the GAL genes proceeds. Rep1 and Cga1 are conserved across the CTG species. Rep1 itself does not possess transcriptional activity. Instead, it provides a scaffold to recruit different factors for transcriptional regulation. Rep1-Cga1 mode of regulation represents a new example of network rewiring in fungi, which provides insight into how C. albicans evolves transcriptional programs to colonize diverse host niches.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

The extreme polymorphisms of human leukocyte antigen class I (HLA class I) proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes. The canonical endoplasmic reticulum (ER) HLA class I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA class I molecules. We hypothesized variable influences of HLA class I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA class I molecules are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B*08:01 or HLA-B*35:01 being among the most resistant or sensitive, respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in accumulation of HLA-B*35:01 in LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA class I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA class I expression and increase the efficiency of cross-presentation.