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Evolution often follows similar trajectories in replicate populations, suggesting that it may be predictable. However, populations are naturally embedded in multispecies communities, and the extent to which evolution is contingent on the specific species interacting with the focal population is still largely unexplored. Here, we study adaptations in strains of 11 different species, experimentally evolved both in isolation and in various pairwise co-cultures. Although partner-specific effects are detectable, evolution was mostly shared between strains evolved with different partners; similar changes occurred in strains' growth abilities, in community properties, and in about half of the repeatedly mutated genes. This pattern persisted even in species pre-adapted to the abiotic conditions. These findings indicate that evolution may not always depend strongly on the biotic environment, making predictions regarding coevolutionary dynamics less challenging than previously thought. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids and chromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified β-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the β-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP β-hairpin as a highly specific target for cancer therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Studying cholesterol biology in the brain has been greatly hindered by the lack of adequate cholesterol visualization techniques. Here, we present a protocol for using a high-affinity cholesterol probe D4H ∗ -mCherry as a histology reagent in mouse or human brain tissue. We describe steps for D4H ∗ tissue treatment and crosslinking leading to stable labeling of intracellular membrane cholesterol. Furthermore, co-labeling with Rab5 endosomal marker and optimized buffers to reduce background enable punctate cholesterol visualization within the organelle membranes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Iron overload, characterized by accumulation of iron in tissues, induces a multiorgan toxicity whose mechanisms are not fully understood. Using cultured cell lines, Caenorhabditis elegans, and mice, we found that ferroptosis occurs in the context of iron-overload-mediated damage. Exogenous oleic acid protected against iron-overload-toxicity in cell culture and Caenorhabditis elegans by suppressing ferroptosis. In mice, oleic acid protected against FAC-induced liver lipid peroxidation and damage. Oleic acid changed the cellular lipid composition, characterized by decreased levels of polyunsaturated fatty acyl phospholipids and decreased levels of ether-linked phospholipids. The protective effect of oleic acid in cells was attenuated by GW6471 (PPAR-α antagonist), as well as in Caenorhabditis elegans lacking the nuclear hormone receptor NHR-49 (a PPAR-α functional homologue). These results highlight ferroptosis as a driver of iron-overload-mediated damage, which is inhibited by oleic acid. This monounsaturated fatty acid represents a potential therapeutic approach to mitigating organ damage in iron overload individuals., Competing Interests: Declaration of interests B.R.S. is an inventor on patents and patent applications involving ferroptosis, holds equity in and serves as a consultant to Exarta Therapeutics, and ProJenX Inc, holds equity in Sonata Therapeutics, and serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer & Feld LLP., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Genome-wide association studies (GWASs) associate phenotypes and genetic variants across a study cohort. GWASs require large-scale cohorts with both phenotype and genetic sequencing data, limiting studied phenotypes. The Human Phenotype Project is a longitudinal study that has measured a wide range of clinical and biomolecular features from a self-assignment cohort over 5 years. The phenotypes collected are quantitative traits, providing higher-resolution insights into the genetics of complex phenotypes., Methods: We present the results of GWASs and polygenic risk score phenome-wide association studies with 729 clinical phenotypes and 4,043 molecular features from the Human Phenotype Project. This includes clinical traits that have not been previously associated with genetics, including measures from continuous sleep monitoring, continuous glucose monitoring, liver ultrasound, hormonal status, and fundus imaging., Findings: In GWAS of 8,706 individuals, we found significant associations between 169 clinical traits and 1,184 single-nucleotide polymorphisms. We found genes associated with both glycemic control and mental disorders, and we quantify the strength of genetic signals in serum metabolites. In polygenic risk score phenome-wide association studies for clinical traits, we found 16,047 significant associations., Conclusions: The entire set of findings, which we disseminate publicly, provides newfound resolution into the genetic architecture of complex human phenotypes., Funding: E.S. is supported by the Minerva foundation with funding from the Federal German Ministry for Education and Research and by the European Research Council and the Israel Science Foundation., Competing Interests: Declaration of interests H.R., D.W.-V., T.S., and A.D. are employees of Pheno.AI, Ltd, a biomedical data science company from Tel-Aviv, Israel. A.W., A.K., and E.S. are paid consultants to Pheno.AI, Ltd., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. Escherichia coli adhesion is mediated by appendages, including type-1 fimbriae and curli amyloid fibers. Antifouling surfaces prevent the adhesion of bacteria to combat biofilm formation. Here, we used single-cell force-spectroscopy to study the interaction between E. coli and glass or two antifouling surfaces: the tripeptide DOPA-Phe(4F)-Phe(4F)-OMe and poly(ethylene glycol) polymer-brush. Our results indicate that both antifoulants significantly deter E. coli initial adhesion. By using two mutant strains expressing no type-1 fimbriae or curli amyloids, we studied the adhesion mechanism. Our results suggest that the bacteria adhere to different antifoulants via separate mechanisms. Finally, we show that some bacteria adhere much better than others, illustrating how the variability of bacterial cultures affects biofilm formation. Our results emphasize how additional study at the single-cell level can enhance our understanding of bacterial adhesion, thus leading to novel antifouling technologies., Competing Interests: The authors declare that they have no conflict of interest., (© 2024 The Author(s).)

Achieving complex behavior in soft-bodied animals is a hard task, because their body morphology is not constrained by a fixed number of jointed elements, as in skeletal animals, and thus the control system has to deal with practically an infinite number of control variables (degrees of freedom). Almost 30 years of research on Octopus vulgaris motor control has revealed that octopuses efficiently control their body with strategies that emerged during the adaptive coevolution of their nervous system and body morphology. In this minireview, we highlight principles of embodied organization that were revealed by studying octopus motor control, and that are used as inspiration for soft robotics. We describe the evolved solutions to the problem, implemented from the lowest level, the muscular system, to the network organization in higher motor control centers of the octopus brain. We show how the higher motor control centers, where the sensory-motor interface lies, can control and coordinate limbs with large degrees of freedom without using body-part maps to represent sensory and motor information, as they do in vertebrates. We demonstrate how this unique control mechanism, which allows efficient control of the body in a large variety of behaviors, is embodied within the animal's body morphology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications., Competing Interests: Declaration of interests The authors state that no competing financial interests exist., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification., Competing Interests: Declaration of interests The authors have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The notion that psychological stress can deteriorate our health is widely accepted. However, the mechanisms at play are poorly understood. In this issue of Cell, Schneider et al. identify the impact of glucocorticoids on enteric glia and neurons and elucidate the underlying mechanisms that link psychological stress to the exacerbation of gut inflammation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Here, we provide a step-by-step protocol for the collection and intracerebroventricular infusion of cerebrospinal fluid (CSF) in mice. We describe steps to withdraw CSF quickly and abundantly while avoiding blood contamination. Using the Lynch coil technique, we gain functional insights into the collected CSF by slowly infusing minimal amounts of CSF directly to the lateral ventricles of aged mice. This protocol is versatile and can be used to infuse drugs, antibodies, or scarce biological compounds. For complete details on the use and execution of this protocol, please refer to Iram et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Viral reproduction is contingent on viral protein synthesis that relies on the host ribosomes. As such, viruses have evolved remarkable strategies to hijack the host translational apparatus in order to favor viral protein production and to interfere with cellular innate defenses. Here, we describe the approaches viruses use to exploit the translation machinery, focusing on commonalities across diverse viral families, and discuss the functional relevance of this process. We illustrate the complementary strategies host cells utilize to block viral protein production and consider how cells ensure an efficient antiviral response that relies on translation during this tug of war over the ribosome. Finally, we highlight potential roles mRNA modifications and ribosome quality control play in translational regulation and innate immunity. We address these topics in the context of the COVID-19 pandemic and focus on the gaps in our current knowledge of these mechanisms, specifically in viruses with pandemic potential., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The transfer of endocytosed cargoes to lysosomes (LYSs) requires HOPS, a multiprotein complex that tethers late endosomes (LEs) to LYSs before fusion. Many proteins interact with HOPS on LEs/LYSs. However, it is not clear whether these HOPS interactors localize to LEs or LYSs or how they participate in tethering. Here, we biochemically characterized endosomes purified from untreated or experimentally manipulated cells to put HOPS and interacting proteins in order and to establish their functional interdependence. Our results assign Rab2a and Rab7 to LEs and Arl8 and BORC to LYSs and show that HOPS drives LE-LYS fusion by bridging late endosomal Rab2a with lysosomal BORC-anchored Arl8. We further show that Rab7 is absent from sites of HOPS-dependent tethering but promotes fusion by moving LEs toward LYSs via dynein. Thus, our study identifies the topology of the machinery for LE-LYS tethering and elucidates the role of different small GTPases in the process., Competing Interests: Declaration of interests The authors declare that there is no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Embryonic stem cells (ESCs) are self-renewing and pluripotent. In recent years, factors that control pluripotency, mostly nuclear, have been identified. To identify non-nuclear regulators of ESCs, we screened an endogenously labeled fluorescent fusion-protein library in mouse ESCs. One of the more compelling hits was the cell-cycle-associated protein 1 (CAPRIN1). CAPRIN1 knockout had little effect in ESCs, but it significantly altered differentiation and gene expression programs. Using RIP-seq and SLAM-seq, we found that CAPRIN1 associates with, and promotes the degradation of, thousands of RNA transcripts. CAPRIN1 interactome identified XRN2 as the likely ribonuclease. Upon early ESC differentiation, XRN2 is located in the nucleus and colocalizes with CAPRIN1 in small RNA granules in a CAPRIN1-dependent manner. We propose that CAPRIN1 regulates an RNA degradation pathway operating during early ESC differentiation, thus eliminating undesired spuriously transcribed transcripts in ESCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-β signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification., Competing Interests: Conflict of interests B.E.R. is a member of the journal’s Editorial Board. He is a founder, holds shares, and is the Chief Scientific Officer of CellCure Neuroscience Ltd. The company did not fund the study presented in this manuscript and has no interest in its results. A patent application related to the data presented in this manuscript has been submitted., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu 2+ and the other for Cu 1+ , the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The dietary factor vitamin K has been found to protect against ferroptosis, a form of cell death driven by lipid peroxidation. This reveals new dietary links to cancers and degenerative conditions and a key factor involved in warfarin poisoning., Competing Interests: Declaration of interests B.R.S. is an inventor on patents and patent applications involving small-molecule drug discovery and ferroptosis; has co-founded and serves as a consultant to Inzen Therapeutics, Exarta Therapeutics, and ProJenX, Inc.; serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer & Feld LLP; and receives sponsored research support from Sumitomo Dainippon Pharma Oncology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts., Competing Interests: Declaration of interests H. Sokol received consultancy or lecture fees from Carenity, Abbvie, Astellas, Danone, Ferring, Mayoly Spindler, MSD, Novartis, Roche, Tillots, Enterome, Maat, BiomX, Biose, and Takeda and is a co-founder of Exeliom Bioscience. N.M. received consultancy or lecture fees from BiomX, Pfizer, Takeda, Janssen, Ferring, Nestle, and BMS and grant support from Takeda, Janssen, Abbott, Pfizer, Abbvie, Neopharm, Corundum Innovation Ltd, Mycolivia, and Nestle. S.K.-R., E.W., Y.M, Y.S., I.W., E.K., N.B.-I., D.I., H.B.-D., J.N., N.K., E.K., T.C., E.F.-G., L.Z., A.C., U.R., I.G.-S., M.G., V.L., N.Z., S.P., and M.S. are paid BiomX employees. R.S. is a scientific cofounder of Ecophage and BiomX. E.E. is a scientific cofounder of DayTwo and BiomX and an advisor to Hello Inside and Aposense. E.E. serves as a scientific advisory board member in Cell. A patent proposal has been submitted by the Weizmann Institute of Science and BiomX., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The development of new vaccines, as well as our understanding of key processes that shape viral evolution and host antibody repertoires, relies on measuring multiple antibody responses against large panels of viruses. Given the enormous diversity of circulating virus strains and antibody responses, comprehensively testing all antibody-virus interactions is infeasible. Even within individual studies with limited panels, exhaustive testing is not always performed, and there is no common framework for combining information across studies with partially overlapping panels, especially when the assay type or host species differ. Prior studies have demonstrated that antibody-virus interactions can be characterized in a vastly simpler and lower dimensional space, suggesting that relatively few measurements could predict unmeasured antibody-virus interactions. Here, we apply matrix completion to several large-scale influenza and HIV-1 studies. We explore how prediction accuracy evolves as the number of measurements changes and approximates the number of additional measurements necessary in several highly incomplete datasets (suggesting ∼250,000 measurements could be saved). In addition, we show how the method can combine disparate datasets, even when the number of available measurements is below the theoretical limit that guarantees successful prediction. This approach can be readily generalized to other viruses or more broadly to other low-dimensional biological datasets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Disease recovery dynamics are often difficult to assess, as patients display heterogeneous recovery courses. To model recovery dynamics, exemplified by severe COVID-19, we apply a computational scheme on longitudinally sampled blood transcriptomes, generating recovery states, which we then link to cellular and molecular mechanisms, presenting a framework for studying the kinetics of recovery compared with non-recovery over time and long-term effects of the disease. Specifically, a decrease in mature neutrophils is the strongest cellular effect during recovery, with direct implications on disease outcome. Furthermore, we present strong indications for global regulatory changes in gene programs, decoupled from cell compositional changes, including an early rise in T cell activation and differentiation, resulting in immune rebalancing between interferon and NF-κB activity and restoration of cell homeostasis. Overall, we present a clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues., Competing Interests: Declaration of interests F.J.T. reports receiving consulting fees from ImmunAI and ownership interest in Dermagnostix. S.S.S.-O. holds equity and is a consultant of CytoReason., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

γ-oscillatory activity is ubiquitous across brain areas. Numerous studies have suggested that γ-synchrony is likely to enhance the transmission of sensory information. However, direct causal evidence is still lacking. Here, we test this hypothesis in the mouse olfactory system, where local GABAergic granule cells (GCs) in the olfactory bulb shape mitral/tufted cell (MTC) excitatory output from the olfactory bulb. By optogenetically modulating GC activity, we successfully dissociate MTC γ-synchronization from its firing rates. Recording of odor responses in downstream piriform cortex neurons shows that increasing MTC γ-synchronization enhances cortical neuron odor-evoked firing rates, reduces response variability, and improves odor ensemble representation. These gains occur despite a reduction in MTC firing rates. Furthermore, reducing MTC γ-synchronization without changing the MTC firing rates, by suppressing GC activity, degrades piriform cortex odor-evoked responses. These findings provide causal evidence that increased γ-synchronization enhances the transmission of sensory information between two brain regions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The study of cell cycle progression and regulation is important to our understanding of fundamental biophysics, aging, and disease mechanisms. Local chromatin movements are generally considered to be constrained and relatively consistent during all interphase stages, although recent advances in our understanding of genome organization challenge this claim. Here, we use high spatiotemporal resolution, 4D (x, y, z and time) localization microscopy by point-spread-function (PSF) engineering and deep learning-based image analysis, for live imaging of mouse embryonic fibroblast (MEF 3T3) and MEF 3T3 double Lamin A Knockout (LmnaKO) cell lines, to characterize telomere diffusion during the interphase. We detected varying constraint levels imposed on chromatin, which are prominently decreased during G0/G1. Our 4D measurements of telomere diffusion offer an effective method to investigate chromatin dynamics and reveal cell-cycle-dependent motion constraints, which may be caused by various cellular processes., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation., Competing Interests: Declaration of interests J.M.C. reports an advisory role in VantAI. A.Q.-V. reports honoraria from AstraZeneca. M.O. reports advisory roles for PharMar, Novartis, and Targeted Oncology, and reports honoraria from Bristol-Myers Squibb and Merck Sharp & Dohme. C.M.R. has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, AstraZeneca, Bicycle, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Jazz, Lilly, Pfizer, PharmaMar, Syros, and Vavotek. C.M.R. serves on the scientific advisory boards of Bridge Medicines, Earli, and Harpoon Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This protocol describes the use of a biolayer interferometry platform for assessing antibody-antigen interactions. The protocol focuses on affinity determination and epitope binning, although the system can be utilized for measuring any protein-protein interaction. Readings are collected in real time, allowing the use of unlabeled molecules, and data can thus be obtained in a fast and easy manner. Experiments should be carefully designed, taking into consideration the tested interaction, available sensors, and suitable controls. For complete details on the use and execution of this protocol, please refer to Noy-Porat et al. (2021)., Competing Interests: Patent application for the described antibodies in Noy-Porat et al. (2021) was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (© 2021 The Author(s).)

Background: T cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19., Methods: Antigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes., Findings: High correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [T EMRA ]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had T EMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%-38% and 20%-23%, respectively, among COVID-19 patients., Conclusions: Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19., Funding: This study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation., Competing Interests: M.L.S., B.D.-J., M.D., L.R.B., A.H.L., X.J., M.W.K., and L.J. declare that they have no competing interests to disclose. D.R.W. has a financial interest in Quanterix Corporation, a company that develops an ultra-sensitive digital immunoassay platform. He is an inventor of the Simoa technology, founder of the company, and serves on its Board of Directors. The anti-SARS-CoV-2 Simoa assays in this publication have been licensed by Brigham and Women’s Hospital to Quanterix Corporation. T.G. receives royalty payments from Brigham and Women’s Hospital for the antibody assay technology. T.N.M. has a financial interest in neuAPC Therapeutics, a company that will develop AAC for the generation of nAPCs and serves as one of its scientific advisors. V.M., X.C., and T.N.M. have a provisional patent on the generation of immunogenic nAPCs and methods of use thereof., (© 2021 The Authors.)

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants., Competing Interests: Declaration of interests Patent application for the described antibodies was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in the GNB1 gene, encoding the Gβ 1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ 1 mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to G i/o , or the Gβγ regulation of the neuronal voltage-gated Ca 2+ channel Ca V 2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ 1 protein expression levels, Gβγ binding to cytosolic segments of GIRK subunits, and in Gβγ function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by Gβγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment., Competing Interests: D.B.G. is a founder of and holds equity in Q State Biosciences and Praxis Therapeutics; holds equity in Apostle Inc., and serves as a consultant to AstraZeneca, Gilead Sciences, GoldFinch Bio and Gossamer Bio. Other authors declare no competing interests., (© 2021 The Author(s).)

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant., Competing Interests: Declaration of interests B.P. serves on the External Immunology Network of GSK and on the Scientific Advisory Board of Medicago and Boehringer Ingelheim. R.v.d.M. is an employee of the GSK group of companies and holds shares in the GSK group of companies. B.P. and F.W. are inventors on a provisional patent application (no. S20-530 63/138,163 [STAN-1821PRV]) submitted by the Board of Trustees of the Leland Stanford Junior University, Stanford, CA, that covers the use of “Modulating The Epigenome With Adjuvants To Stimulate Broad And Persistent Innate Immunity Against Diverse Viruses.” The remaining authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STING V155M was most effective at maximizing CD8 + T cell responses at an antigen/adjuvant mass ratio of 5:1. STING V155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STING V155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV + TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant., Competing Interests: Declaration of interests E.H., S.-W.T., K.M., J.I., and K.H. are inventors on patent applications directed to “Messenger ribonucleic acids for enhancing immune responses and methods of use thereof” (US20180311343A1). All authors are current or previous employees of Moderna, Inc. and have received salary and stock options as compensation for their employment., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Knowledge of how leptin receptor (LepR) neurons of the mediobasal hypothalamus (MBH) access circulating leptin is still rudimentary. Employing intravital microscopy, we found that almost half of the blood-vessel-enwrapping pericytes in the MBH express LepR. Selective disruption of pericytic LepR led to increased food intake, increased fat mass, and loss of leptin-dependent signaling in nearby LepR neurons. When delivered intravenously, fluorescently tagged leptin accumulated at hypothalamic LepR pericytes, which was attenuated upon pericyte-specific LepR loss. Because a paracellular tracer was also preferentially retained at LepR pericytes, we pharmacologically targeted regulators of inter-endothelial junction tightness and found that they affect LepR neuronal signaling and food intake. Optical imaging in MBH slices revealed a long-lasting, tonic calcium increase in LepR pericytes in response to leptin, suggesting pericytic contraction and vessel constriction. Together, our data indicate that LepR pericytes facilitate localized, paracellular blood-brain barrier leaks, enabling MBH LepR neurons to access circulating leptin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal antibodies (mAbs), targeting several distinct epitopes for circumventing therapy escape mutants. Previously, we reported efficient human therapeutic mAbs recognizing epitopes on the spike receptor-binding domain (RBD) of SARS-CoV-2. Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N -glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late post-exposure, demonstrating the high potential of the mAbs for therapy of SARS-CoV-2 infection., Competing Interests: Patent application for the described antibodies was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (© 2021 The Author(s).)

While decades of research have elucidated many steps of the alphavirus lifecycle, the earliest replication dynamics have remained unclear. This missing time window has obscured early replicase strand-synthesis behavior and prevented elucidation of how the first events of infection might influence subsequent viral competition. Using quantitative live-cell and single-molecule imaging, we observed the initial replicase activity and its strand preferences in situ and measured the trajectory of replication over time. Under this quantitative framework, we investigated viral competition, where one alphavirus is able to exclude superinfection by a second homologous virus. We show that this appears as an indirect phenotypic consequence of a bidirectional competition between the two species, coupled with the rapid onset of viral replication and a limited total cellular carrying capacity. Together, these results emphasize the utility of analyzing viral kinetics within single cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The gold standard for COVID-19 diagnosis is detection of viral RNA through PCR. Due to global limitations in testing capacity, effective prioritization of individuals for testing is essential., Methods: We devised a model estimating the probability of an individual to test positive for COVID-19 based on answers to 9 simple questions that have been associated with SARS-CoV-2 infection. Our model was devised from a subsample of a national symptom survey that was answered over 2 million times in Israel in its first 2 months and a targeted survey distributed to all residents of several cities in Israel. Overall, 43,752 adults were included, from which 498 self-reported as being COVID-19 positive., Findings: Our model was validated on a held-out set of individuals from Israel where it achieved an auROC of 0.737 (CI: 0.712-0.759) and auPR of 0.144 (CI: 0.119-0.177) and demonstrated its applicability outside of Israel in an independently collected symptom survey dataset from the US, UK, and Sweden. Our analyses revealed interactions between several symptoms and age, suggesting variation in the clinical manifestation of the disease in different age groups., Conclusions: Our tool can be used online and without exposure to suspected patients, thus suggesting worldwide utility in combating COVID-19 by better directing the limited testing resources through prioritization of individuals for testing, thereby increasing the rate at which positive individuals can be identified. Moreover, individuals at high risk for a positive test result can be isolated prior to testing., Funding: E.S. is supported by the Crown Human Genome Center, Larson Charitable Foundation New Scientist Fund, Else Kroener Fresenius Foundation, White Rose International Foundation, Ben B. and Joyce E. Eisenberg Foundation, Nissenbaum Family, Marcos Pinheiro de Andrade and Vanessa Buchheim, Lady Michelle Michels, and Aliza Moussaieff and grants funded by the Minerva foundation with funding from the Federal German Ministry for Education and Research and by the European Research Council and the Israel Science Foundation. H.R. is supported by the Israeli Council for Higher Education (CHE) via the Weizmann Data Science Research Center and by a research grant from Madame Olga Klein - Astrachan., Competing Interests: The study protocol was approved by the Weizmann Institute of Science review board (IRB). Informed consent was waived by the IRB, as all identifying details of the participants were removed before the computational analysis. Participants were made fully aware of the way in which the data will be stored, handled, and shared, which was provided to them and is in accordance with the privacy and data-protection policy of the Weizmann Institute of Science (https://www.weizmann.ac.il/pages/privacy-policy)., (© 2020 Elsevier Inc.)

Long-range movement of organelles within the cytoplasm relies on coupling to microtubule motors, a process that is often mediated by adaptor proteins. In many cases, this coupling involves organelle- or adaptor-induced activation of the microtubule motors by conformational reversal of an autoinhibited state. Herein, we show that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small guanosine triphosphatase (GTPase) ARL8 on the lysosomal membrane to couple lysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP reveal that the C-terminal region comprising three pleckstrin homology (PH) domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of lysosomes to kinesin-1 and, consequently, lysosome movement toward the cell periphery. We also find that ARL8 does not just recruit SKIP to the lysosomal membrane but also relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde lysosome transport. Finally, our analyses show that the largely disordered middle region of SKIP mediates self-association and that this self-association enhances the interaction of SKIP with kinesin-1. These findings indicate that SKIP is not just a passive connector of lysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. We anticipate that similar organelle- or GTPase-induced conformational changes could regulate the activity of other kinesin adaptors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation., Competing Interests: Declarations of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plasmids are genetic elements that colonize and replicate in prokaryotic cells (Box 1). They are considered a major driving force of prokaryote evolution, as they can migrate between populations, making them potent agents of lateral DNA transfer and microbial warfare. The importance of plasmids goes beyond microbial evolution, as they are widely used as vectors for genetic engineering in basic research (e.g., random mutagenesis) as well as applications in biotechnology (e.g., insulin production), synthetic biology, agriculture (e.g., genetic engineering of crops) and medicine (e.g., biopharmaceuticals)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extra-chromosomal genetic elements are important drivers of bacterial evolution, and their evolutionary success depends on positive selection for the genes they encode. Examples are plasmids encoding antibiotic resistance genes that are maintained in the presence of antibiotics (e.g., [1-3]). Plasmid maintenance is considered a metabolic burden to the host [4]; hence, when the cost of plasmid carriage outweighs its benefit, plasmid-free segregants are expected to outcompete plasmid-carrying cells, eventually leading to plasmid loss [5-7]. Thus, in the absence of positive selection, plasmid survival hinges upon stable persistence in the population. The ubiquity of plasmids in nature suggests that plasmids having a negligible effect on host fitness may evolve stable inheritance and thus gain a long-term persistence in the population, also in the absence of positive selection [8]. Nonetheless, the transition of plasmids into stably inherited genetic elements remains understudied. Here, we show that positive selection for a plasmid-encoded gene interferes with the evolution of plasmid stability. Evolving plasmids under different selection regimes in Escherichia coli, we find that antibiotics led to plasmid amplification, resulting in plasmid instability. Thus, under positive selection, suboptimal solutions for plasmid stability were maintained in the population hindering long-term plasmid persistence. Indeed, a survey of Escherichia plasmids confirms that antibiotic resistance genes are rarely found on small plasmids. Our results show that a plasmid-mediated advantage for the host may manifest in reduced plasmid evolutionary success. Considering plasmids as autonomously evolving entities holds promise for understanding the factors that govern their evolution., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT., Competing Interests: Declaration of Interests P.C.K. is on the Advisory Board of Novome Biotechnologies and is an ad hoc consultant for Pendulum Therapeutics, IP group, and Otsuka Pharmaceuticals. P.C.K. holds patent US20170042860A1 for use of tryptamine producing bacteria (“Methods and materials for using Ruminococcus gnavus or Clostridium sporogenes to treat gastrointestinal disorders”), and P.C.K. and Mayo Clinic have a financial interest related to this research. These interests have been reviewed and managed in accordance with Mayo Clinic Conflict-of-Interest policies. D.B.K. serves as CEO of CoreBiome, a company involved in the commercialization of microbiome analysis and a wholly owned subsidiary of OraSure Technologies. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DR hi CD11c hi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR lo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diabetic kidney disease (DKD) increases the risk for mortality and is the leading cause of end-stage renal disease. Treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of DKD, especially in patients with advanced kidney disease. Herein, we show that in diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) along with enhanced tubule-interstitial fibrosis; this is prevented by SGLT2i. Constitutive activation of mTORC1 in RPTCs induces renal fibrosis and failure and abolishes the renal-protective effects of SGLT2i in diabetes. On the contrary, partial inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function. Stimulation of mTORC1 in RPTCs turns on a pro-fibrotic program in the renal cortex, whereas its inhibition in diabetes reverses the alterations in gene expression. We suggest that RPTC mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis., Competing Interests: Declaration of Interests The Hadasit Medical Research Services and Development filed a patent application titled “Inhibitors of Amino Acids Transporters and Use Thereof,” and A.K.-L., O.M., and G.L. are listed as inventors., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)