title: Reticulospinal drive increases maximal motoneuron output in humans

abstract: Maximal rate of force development in adult humans is determined by the maximal motoneuron output, however the origin of the underlying synaptic inputs remains unclear. Here, we tested a hypothesis that the maximal motoneuron output will increase in response to a startling cue, a stimulus that purportedly activates the pontomedullary reticular formation neurons that make mono- and disynaptic connections to motoneurons via fast-conducting axons. Twenty-two men were required to produce isometric knee extensor forces "as fast and as hard" as possible from rest to 75% of maximal voluntary force, in response to visual (VC), visual-auditory (VAC), or visual-startling cue (VSC). Motoneuron activity was estimated via decomposition of high-density surface electromyogram recordings over the vastus lateralis and medialis muscles. Reaction time was significantly shorter in response to VSC compared to VAC and VC (i.e., the StartReact effect). The VSC further elicited faster neuromechanical responses including a greater number of discharges per motor unit per second and greater maximal rate of force development, with no differences between VAC and VC. We provide evidence, for the first time, that the synaptic input to motoneurons increases in response to a startling cue, suggesting a contribution of subcortical pathways to maximal motoneuron output in humans, likely originating from the pontomedullary reticular formation.

category: physiology


title: Dynamic observation of 2H labeled compounds in the human brain with 1H versus 2H magnetic resonance spectroscopy at 9.4T

abstract: The metabolic pathway of [6,6-2H2]-labeled glucose was investigated with two different techniques. The first technique used direct detection of deuterium applying Deuterium Metabolic Imaging (DMI). The second technique used the indirect detection of deuterium with proton MR spectroscopy (MRS) called Quantitative Exchanged-label Turnover (QELT) MRS. For the first time, time-resolved data was acquired for both techniques in the same healthy human subjects and directly compared. The time-curves were used in a kinetic model to estimate rates of the metabolic pathway of glucose. Two different kinetic models were compared. One included only DMI data, the second one combined DMI and QELT. For the first model, a tricarboxylic acid (TCA) cycle rate of 0.69 {+/-} 0.10 mol{middle dot}min-1{middle dot}g-1 was determined. For the second model, the estimated TCA cycle rate was 0.68 {+/-} 0.12 mol{middle dot}min-1{middle dot}g-1. In addition, the rate of glutamine synthesis from glutamate could be estimated with model 2 (0.51 {+/-} 0.15 mol{middle dot}min-1{middle dot}g-1). The sensitivity of both methods was evaluated and compared to alternative techniques.

category: neuroscience


title: A murine Wac model exhibits phenotypes relevant to DeSanto-Shinawi Syndrome

abstract: Several monogenic syndromes are associated with neurodevelopmental changes that result in cognitive impairments, including autism, attention deficit hyperactivity disorder (ADHD) and seizures. Limited studies and resources are available to make meaningful headway into the underlying mechanisms that result in these symptoms. One such example, DeSanto-Shinawi Syndrome (DESSH), is a rare disorder caused by mutations in the WAC gene. Those diagnosed with DESSH experience craniofacial alterations as well as cognitive symptoms that include autism, ADHD and seizures. However, no thorough studies from a mammalian model exist to understand how these changes occur. To overcome this, we generated constitutive murine Wac mutants and assessed phenotypes that are relevant to humans diagnosed with DESSH. Wac mutants have craniofacial, anatomical, behavioral and seizure susceptibility that are relevant to DESSH; this new model is suited to study some of the core symptoms of DESSH and the biology of Wac.


title: PSGL-1 directs early TCR signaling to repress metabolism and promote T cell exhaustion by modulating the TCF-1/TOX axis in CD8+ T cells.

abstract: Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

category: immunology


title: Modelling eDNA transport in river networks reveals highly resolved spatio-temporal patterns of freshwater biodiversity

abstract:
O_LIThe ever-increasing threats to riverine biodiversity call for the development of novel approaches for a complete assessment of biodiversity across highly resolved spatial, temporal and taxonomic scales. Past studies on riverine biodiversity patterns were often restricted to spatially scattered data, focused on specific taxonomic groups, and disregarded the temporal dimension, preventing a universal understanding of relationships between biodiversity and stream size across spatial, temporal and taxonomic scales. Recent advances in the joint use of environmental DNA (eDNA) data and novel mechanistic models for eDNA transport in river networks have the potential to uncover the full structure of riverine biodiversity at an unprecedented spatial resolution, hence providing fundamental insights into ecosystem processes and offering a basis for targeted conservation measures.
C_LIO_LIHere, we applied a mechanistic model (i.e., the eDITH model) to a metabarcoding dataset covering three taxonomic groups (fish, invertebrates and bacteria) and three seasons (spring, summer and autumn) for a 740-km2 Swiss catchment, sampled for eDNA at 73 sites.
C_LIO_LIUsing the mechanistic model, we upscaled eDNA-based biodiversity predictions to more than 1900 individual reaches, allowing an assessment of patterns of - and {beta}-diversity across seasons and taxonomic groups at a space-filling, fine scale over the whole network.
C_LIO_LIWe found that both predicted - and {beta}-diversity varied considerably depending on both season and taxonomic group. Predicted fish -diversity increased in the downstream direction at all seasons, while invertebrate and bacteria -diversity either decreased downstream or was not significantly related to position within network, depending on the season. Spatial {beta}-diversity was mostly found to be decreasing in the downstream direction, and this was the case for all seasons for bacteria. Temporal {beta}-diversity was mostly found to be increasing downstream. In general, genus richness values predicted by the model were found to be higher than those obtained by directly analyzing the eDNA data. Overall, stream size (subsumed by drainage area) was generally a poor predictor of patterns of predicted - and {beta}-diversities. Conversely, riverine biodiversity is shaped by a complex interplay of environmental variables, abiotic and biotic factors, which need be taken into account for a correct assessment of its structure.
C_LI

category: ecology


title: Genome-wide analysis of Schistosoma mansoni reveals population structure and praziquantel drug selection pressure within Ugandan hot-spot communities

abstract:
Populations within schistosomiasis control areas, especially those in Africa, are recommended to receive regular mass drug administration (MDA) with praziquantel (PZQ) as the main strategy for controlling the disease. The impact of PZQ treatment on schistosome genetics remains poorly understood, and is limited by a lack of high-resolution genetic data on the population structure of parasites within these control areas. We generated whole-genome sequence data from 174 individual miracidia collected from both children and adults from fishing communities on islands in Lake Victoria in Uganda that had received either annual or quarterly MDA with PZQ over four years, including samples collected immediately before and four weeks after treatment. Genome variation within and between samples was characterised and we investigated genomic signatures of natural selection acting on these populations that could be due to PZQ treatment. The parasite population on these islands was more diverse than found in nearby villages on the lake shore. We saw little or no genetic differentiation between villages, or between the groups of villages with different treatment intensity, but slightly higher genetic diversity within the pre-treatment compared to post-treatment parasite populations. We identified classes of genes significantly enriched within regions of the genome with evidence of recent positive selection among post-treatment and intensively treated parasite populations. The differential selection observed in post-treatment and pre-treatment parasite populations could be linked to any reduced susceptibility of parasites to praziquantel treatment.

Author summarySchistosomiasis is caused by parasitic helminths of the genus Schistosoma. Schistosoma mansoni is the primary cause of intestinal schistosomiasis, a devastating and widespread parasitic infection that causes morbidity, death and socio-economic impact on endemic communities across the world and especially sub-Saharan Africa. Using whole-genome sequencing, we were able to elucidate the parasite population within Lake Victoria island fishing communities in Uganda which are among the major hotspots for schistosomiasis. We further assessed genetic markers that might be linked to recent observations concerning reduced susceptibility to praziquantel, the major drug used in the treatment of this disease. Whole-genome data on the population genetics of S. mansoni in an African setting will provide a strong basis for future functional genomics or transcriptomic studies that will be key to identifying drug targets, improving existing drugs or developing new therapeutic interventions.

category: genomics


title: PIMT suppresses endothelial activation and vascular inflammation through methylation of TRAF6

abstract: As a repair enzyme, protein L-isoaspartyl O-methyltransferase (PIMT) methylates and converts isoaspartyl residues (isoAsp) back to conventional forms to avoid protein damage during aging and stress responses. This study aims to investigate the pathological significance of PIMT in vascular inflammation. Herein, we show that PIMT is highly expressed in lung endothelial cells (ECs), and its reduction significantly exacerbates pulmonary inflammation in a murine model of acute lung injury (ALI). Mechanistically, we identify tumor necrosis factor receptor-associated factor 6 (TRAF6) as a substrate of PIMT. PIMT-mediated methylation of TRAF6 inhibits TRAF6 oligomerization, autoubiquitination, and LPS-induced NF-{kappa}B transactivation in ECs. Importantly, in addition to transcriptionally attenuating expression of adhesion molecules, PIMT post-translationally impedes site specific N-glycosylation of intercellular adhesion molecule-1 (ICAM-1), which leads to an increased protein degradation of ICAM-1 and a subsequent inhibition of EC interaction with immune cells during inflammatory process. Our results for the first time identify PIMT-mediated protein O-methylation as a key post-translational mechanism in controlling vascular inflammation, and suggest that PIMT may represent a novel therapeutic target in inflammatory vascular diseases.


title: Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis

abstract:
Initial TCR engagement of CD8+ T cells results in T cell expansion, and these early events influence the generation of diverse effector and memory populations. During infection, some activated T cells re-encounter cognate antigen, but how these events influence local effector responses or formation of memory populations is unclear. To address this issue, OT-I T cells which express the Nur77-GFP reporter of TCR activation were paired with T. gondii that express OVA to assess the impact of TCR activation on CD8+ T cell responses. During acute infection, TCR stimulation in affected tissues correlated with parasite burden and was associated with markers of effector cells while Nur77-GFP- OT-I showed signs of effector memory potential. However, adoptive transfer of Nur77-GFP negative or positive OT-I from infected mice into naive recipients resulted in formation of similar memory populations. During the chronic stage of infection in the CNS, TCR activation was associated with large scale transcriptional changes and the acquisition of an effector T cell phenotype as well as the generation of a population of CD103+ CD69+ Trm like cells. However, while inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight the contribution of recent TCR activation on the phenotypic heterogeneity of the CD8+ T cell response but suggest that this process has a limited impact on memory populations at acute and chronic stages of infection.

Author SummaryCD8+ T cells are important to control many acute and chronic infections, however the role that recent T cell receptor stimulation plays in the formation of ongoing T cell responses is unclear. Here, we utilize a genetic reporter of TCR stimulation and high parameter flow cytometry to characterize TCR-driven phenotypes of pathogen specific T cell responses to the parasite Toxoplasma gondii during acute and chronic infection in the periphery and central nervous system. This work demonstrates the importance of recent TCR stimulation in driving local effector CD8+ T cell responses in peripheral tissues during infection, as well as the plasticity of the formation of memory T cells. Additionally, we utilize static and live imaging to investigate how Toxoplasma gondii life cycle impacts the ability to present antigen to CD8+ T cells. These studies aid in our understanding of how effector and memory CD8+ T cell responses are generated and maintained during an infection.


title: Essential bromodomain TcBDF2 as a drug target against Chagas disease

abstract: Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein-containing a bromodomain that was recently described. Using the Tet regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing we were able to demonstrate the essentiality of TcBDF2 in T cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results, point to TcBDF2 as a druggable target against T. cruzi.

category: cell biology


title: Selecting an effective amplitude threshold for neural spike detection

abstract: This paper assesses and challenges whether commonly used methods for defining amplitude thresholds for spike detection are optimal. This is achieved through empirical testing of single amplitude thresholds across multiple recordings of varying SNR levels. Our results suggest that the most widely used noise-statistics-driven threshold can suffer from parameter deviation in different noise levels. The spike-noise-driven threshold can be an ideal approach to set the threshold for spike detection, which suffers less from the parameter deviation and is robust to sub-optimal settings.

category: bioengineering


title: TooT-SC: Predicting Eleven Substrate Classes of Transmembrane Transport Proteins

abstract:
BackgroundTransporters form a significant proportion of the proteome and play an important role in mediating the movement of compounds across membranes. Transport proteins are difficult to characterize experimentally, so there is a need for computational tools that predict the substrates transported in order to annotate the large number of genomes being sequenced. Recently we developed a dataset of eleven substrate classes from Swiss-Prot using the ChEBI ontology as the basis for the definition of the classes.

ResultsWe extend our earlier work TranCEP, which predicted seven substrate classes, to the new dataset with eleven substrate classes. Like TranCEP, TooT-SC combines pairwise amino acid composition (PAAC) of the protein, with evolutionary information captured in a multiple sequence alignment (MSA) using TM-Coffee, and restriction to important positions of the alignment using TCS. Our experimental results show that TooT-SC significantly outperforms the state-of-the-art predictors, including our earlier work, with an overall MCC of 0.82 and the MCC for the eleven classes ranging from 0.66 to 1.00.

ConclusionTooT-SC is a useful tool with high performance covering a broad range of substrate classes. The results quantify the contribution made by each type of information used during the prediction process. We believe the methodology is applicable more generally for protein sequence analysis.

category: bioinformatics


title: A comprehensive evaluation of consensus spectrum generation methods in proteomics

abstract: Spectrum clustering is a powerful strategy to minimize redundant mass spectral data by grouping highly similar mass spectra corresponding to repeatedly measured analytes. Based on spectrum similarity, near-identical spectra are grouped in clusters, after which each cluster can be represented by its so-called consensus spectrum for downstream processing. Although several algorithms for spectrum clustering have been adequately benchmarked and tested, the influence of the consensus spectrum generation step is rarely evaluated. Here, we present an implementation and benchmark of common consensus spectrum algorithms, including spectrum averaging, spectrum binning, the most similar spectrum, and the best-identified spectrum. We have analyzed diverse public datasets using two different clustering algorithms (spectra-cluster and MaRaCluster) to evaluate how the consensus spectrum generation procedure influences downstream peptide identification. The BEST and BIN methods were found the most reliable methods for consensus spectrum generation, including for datasets with post-translational modifications (PTM) such as phosphorylation. All source code and data of the present study are freely available on GitHub at https://github.com/statisticalbiotechnology/representative-spectra-benchmark.


title: Slow-cycling cells in glioblastoma: a specific population in the cellular mosaic of cancer stem cells

abstract: AO_SCPLOWBSTRACTC_SCPLOWGlioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional features including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells 1-7. In this study we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells and cells phenotypically defined based on the expression of markers commonly used to enrich for cancer stem cells (CSCs). Our data support the cancer stem cell mosaicism model with slow-cycling cells representing critical tesserae.

category: cancer biology


title: Toll Like Receptor 9 Pathway Mediates Schlafen+-MDSC Polarization During Helicobacter-Induced Gastric Metaplasias

abstract:
Background and AimsA subset of MDSCs that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia (SPEM). We propose that individuals with a more robust response to damage-activated molecular patterns (DAMPs) and increased Toll-like receptor (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection.

MethodsA mouse or human Transwell co-culture system comprised of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers and Helicobacter were used to dissect the cellular source of interferon (IFN) in the stomach by flow cytometry. Conditioned media from the cocultures polarized primary myeloid cells. Myeloid-derived suppressor cell (MDSC) activity was determined by T cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFN and SLFN12L expression by immunohistochemistry. NF{kappa}B binding to the TLR9 C allele was determined by electrophoretic mobility shift assays.

ResultsHelicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFN. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFN secretion and SLFN+-MDSC polarization. Neutralizing IFN in vivo mitigated Helicobacter-induced SPEM. The TLR9 minor C allele creates an NF{kappa}b binding site associated with higher levels of TLR9, IFN and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer.

ConclusionTLR9 plays an essential role in the production of IFN and polarization of SLFN+-MDSCs upon Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.


title: The vagus nerve mediates the stomach-brain coherence in rats

abstract: Interactions between the brain and the stomach shape both cognitive and digestive functions. Recent human studies report spontaneous synchronization between brain activity and gastric slow waves in the resting state. However, this finding has not been replicated in any animal models. The neural pathways underlying this apparent stomach-brain synchrony is also unclear. Here, we performed functional magnetic resonance imaging while simultaneously recording body-surface gastric slow waves from anesthetized rats in the fasting vs. postprandial conditions and performed a bilateral cervical vagotomy to assess the role of the vagus nerve. The coherence between brain fMRI signals and gastric slow waves was found in a distributed "gastric network", including subcortical and cortical regions in the sensory, motor, and limbic systems. The stomach-brain coherence was largely reduced by the bilateral vagotomy and was different between the fasting and fed states. These findings suggest that the vagus nerve mediates the spontaneous coherence between brain activity and gastric slow waves, which is likely a signature of real-time stomach-brain interactions. However, its functional significance remains to be established.


title: Neural Basis of The Double Drift Illusion

abstract: In the "double-drift" illusion, local motion within a window moving in the periphery alters the windows perceived path. The illusion is strong even when the eyes track a target whose motion matches the window so that the stimulus remains stable on the retina. This implies that the illusion involves the integration of retinal signals with non-retinal eye-movement signals. To identify where in the brain this integration occurs, we measured BOLD fMRI responses in visual cortex while subjects experienced the double drift illusion. We identified a number of cortical areas that responded more strongly during the illusion, but only in area hMT+ was it possible to decode the illusory trajectory. Our results provide evidence for a perceptual representation in human visual cortex that is not linked to retinal position.


title: Using the glial mitochondrial machinery to prevent multilevel dysfunctions observed in a mouse model of Alzheimer s disease

abstract: Alzheimers disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenge as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxydative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.


title: Overexpression of UCP4 in astrocytic mitochondria prevents multilevel dysfunctions in a mouse model of Alzheimer s disease


title: Brain perivascular space imaging across the human lifespan

abstract:
Enlarged perivascular spaces (PVS) are considered a biomarker for vascular pathology and has been observed in several neurological disorders and in normal aging; however, the age-related changes in PVS structure across the normative lifespan are not well understood. We sought to characterize the relationship between PVS anatomical characteristics and age in a large cohort ([~]1400) between 8 and 90 years of age from the Human Connectome Project (HCP) Lifespan datasets. PVS from the white matter regions and the basal ganglia (BG) were automatically segmented and quantified from vesselness maps derived from multimodal structural MRI data. PVS burden increased from childhood to advancing age in both the white matter and BG and was reflected by increasing PVS volume fraction (VF), count and mean cross-sectional diameter and decreasing mean solidity, with elevated PVS burden in males compared to females. Within white matter regions, PVS burden in childhood significantly predicted the rate of PVS expansion across the lifespan (t(66)=-12.15, p<.001, R2=.69), where regions with low PVS VF in childhood undergo rapid growth (e.g., temporal regions) and regions with high PVS VF in childhood undergo slowed growth (e.g., limbic regions). PVS anatomical alterations across the lifespan was not significantly associated with working memory performance. Together, our findings of spatially-varying patterns of PVS enlargement across the lifespan constitutes a feature of normal aging and may reflect alterations to waste clearance functionality. These results provide a normative reference for the spatial distribution of PVS enlargement patterns from which pathological alterations can be compared.

Significance StatementPerivascular spaces (PVS) surround penetrating blood vessels and play an important role in the brain waste clearance system. Enlarged PVS are observed in normal aging and neurological conditions; however, research on the role of PVS in health and disease are hindered by the lack of knowledge regarding the time course of PVS alterations across the lifespan. To address this, we characterized age-related alterations in PVS structure from childhood through advancing age. We found PVS become progressively wider and more numerous with differing regional trajectories. These findings provide an important step towards understanding the evolution of brain clearance mechanisms across the lifespan and can be used as a normative reference to better understand neurological disorders characterized by abnormal waste clearance.


title: Stimulus Whitening Improves the Efficiency of Reverse Correlation

abstract: Human perception depends upon internal representations of the environment that help to organize the raw information available from the senses by acting as reference patterns. Internal representations are widely characterized using reverse correlation, a method capable of producing unconstrained estimates of the representation itself, all on the basis of simple responses to random stimuli. Despite its advantages, reverse correlation is often infeasible to apply because the number of stimulus-response trials needed to provide an accurate estimate is typically very large. Prior approaches have aimed to overcome this sampling inefficiency by incorporating prior knowledge of the representation, which biases the estimate and ultimately limits the essential power of reverse correlation. The present approach, however, improves efficiency via stimulus whitening, a statistical procedure that decorrelates stimuli, making them less redundant, and commensurately more favorable for efficient estimation of an arbitrary target. We provide a mathematical justification for whitening, and demonstrate in simulation that whitening provides greater than 85% improvement in efficiency for a given estimation accuracy, and also a two- to five-fold increase in accuracy for a given sample size. Improving the efficiency of reverse correlation may enable a broader scope of investigations into individual variability and potential universality of perceptual mechanisms.


title: Inhibitory IL-10-producing CD4+ T cells develop in a T-bet-dependent manner and facilitate cytomegalovirus persistence via coexpression of arginase-1

abstract: Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity, but the mechanisms that underpin the development of such regulatory networks in vivo have remained obscure. To address this knowledge gap, we performed a comprehensive functional, phenotypic, and transcriptomic analysis of IL-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed at sites of viral persistence in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enabled better immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the coexpression of Arg1.


title: The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses

abstract: Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid- host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.

category: microbiology


title: The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate

abstract: Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor- gene (IL7R) in MS-risk. Based on evolutionary and functional arguments we postulated that DDX39B enhances immune tolerance decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified ForkheadBox P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized subclass of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

category: molecular biology


title: Zebrafish caudal fin amputation induces a metabolic switch necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration

abstract: Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a shift in the metabolic profile at early stages of regeneration, enhancing glycolysis at the expense of mitochondrial oxidation. This metabolic switch mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.

category: developmental biology


title: A computational method for predicting the most likely evolutionary trajectories in the stepwise accumulation of resistance mutations

abstract:
Pathogen evolution of drug resistance often occurs in a stepwise manner via accumulation of multiple mutations, which in combination have a non-additive impact on fitness, a phenomenon known as epistasis. Epistasis complicates the sequence-structure-function relationship and undermines our ability to predict evolution. We present a computational method to predict evolutionary trajectories that accounts for epistasis, using the Rosetta Flex ddG protocol to estimate drug binding free energy changes upon mutation and an evolutionary model based in thermodynamics and statistical mechanics. We apply this method to predict evolutionary trajectories to known multiple mutations associated with resistant phenotypes in malaria. Resistance to the combination drug sulfadoxine-pyrimethamine (SP) in malaria-causing species Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) has arisen via the accumulation of multiple point mutations in the DHFR and DHPS genes. Four known PfDHFR pyrimethamine resistance mutations are highly prevalent in field-isolates and multiple studies have shown epistatic interactions between these mutations determine the accessible evolutionary trajectories to the highly resistant quadruple mutation N51I,C59R,S108N,I164L. We simulated the possible evolutionary trajectories to this quadruple PfDHFR mutation as well as the homologous PvDHFR mutations. In both cases, our most probable pathways agreed well with those determined experimentally. We also applied this method to predict the most likely evolutionary pathways to observed multiple mutations associated with sulfadoxine resistance in PfDHPS and PvDHPS. This novel method can be applied to any drug-target system where the drug acts by binding to the target.

Significance StatementAntimicrobial resistance (AMR) is a major public health threat, resulting from the overuse and misuse of antimicrobial drugs. Our ability to monitor emerging resistance and direct the most appropriate treatment strategy (stewardship) would be strengthened by the development of methods to accurately predict the mutational pathways that lead to resistance. We present a computational method to predict the most likely resistance pathways that result from mutations that alter drug binding affinity. We utilized the Rosetta Flex ddG protocol and a thermodynamic evolutionary model. This novel approach can accurately capture known resistance trajectories and can be applied to any system where resistance arises via changes in drug binding affinity.

category: evolutionary biology


title: GAIT BIOMECHANICS OF RABBITS WITH EITHER ACHILLES OR TIBIALIS CRANIALIS ARTIFICIAL TENDONS

abstract: Artificial tendons have been developed as a replacement for biological tendons with irreparable pathologies and defects. Previous studies reported the mechanical strength and tissue integration of a polyester suture-based artificial tendon, but not its effect on locomotor function. The objective of this study was to quantify the hindlimb biomechanics during hopping gait of New Zealand White rabbits with surgical replacement of either the Achilles (n=2) or tibialis cranialis (TC, n=2) biological tendons with artificial tendons. Once pre-surgery and for five consecutive weeks post-surgery (starting at about two weeks post-surgery), we measured hindlimb kinematics and ground contact pressures with a video camera and pressure mat, respectively. Promisingly, post-surgical locomotor function was either consistent or improved over time in both tendon replacement groups. However, Achilles rabbits exhibited greater immediate post-surgery functional decline and less post-surgical functional recovery than TC rabbits. Compared to healthy rabbits, at the study endpoint, (1) TC rabbits had a 17.3-degree higher (i.e., more plantarflexed) ankle angle at foot strike; and (2) Achilles rabbits had a 39.2-degree lower (i.e., more dorsiflexed) ankle angle at toe off. These functional deficits suggest that the muscles attached to the artificial tendons had lower force-generating capacity. Future studies of artificial tendons are needed to quantify long-term function, determine the effectiveness of structured rehabilitation exercises, and refine surgical implementation.


title: CavitySpace: A database of potential ligand binding sites in the human proteome

abstract: The ligand binding sites of a protein provide useful information to uncover its functions and to direct the structure-based drug design. However, as binding site detection relies on the three-dimensional (3D) structural data of proteins, functional analysis based on protein ligand binding sites is formidable for proteins without structural information. Recent developments in protein structure prediction and the 3D structures built by AlphaFold provide an unprecedented opportunity for analyzing ligand binding sites in human proteins. We have used the reliable ligand binding site detection program CAVITY to analyse all the proteins in the human proteome and constructed the CavitySpace database, which is the first pocket library for predicted protein structures. CavitySpace can be used to predict protein function based on pocket information, to identify new druggable protein targets for drug design, and to search for new binding sites for known drugs for drug repurposing. CavitySpace is freely available at http://www.pkumdl.cn:8000/cavityspace/.


title: hudson: A User-Friendly R Package to Extend Manhattan Plots

abstract: The Manhattan plot is one of the most widely used visualization techniques when plotting summary statistics from genome-wide or phenome-wide association studies. While there are a number of existing tools to create these plots, there is room for extending their utility to satisfy increasingly complex and comprehensive analyses as well as the need for comparisons between different sets of results or between discovery and replication datasets. The R package presented here, hudson, provides user-friendly plotting functions intended for use with genome, phenome, and exposome-wide association studies, but its flexible framework can be utilized for a wide variety genome-wide analyses. Further, we have extended these figures to allow for interactive elements to facilitate results exploration for the ever-increasing large-scale dimensionality of these data. hudson can be obtained from https://github.com/RitchieLab/hudson.


title: Needler: An Algorithm to Develop a Comprehensive Targeted MS Method Capable of Monitoring the Human Proteome

abstract: Targeted proteomics for addressing biological hypotheses through quantitative mass spectrometry (MS) is increasingly popular because of its ability to deliver sensitive and reproducible results to inform protein concentration and dynamics. While the technique is powerful, the associated tooling to develop unique targeted data acquisition methods per experiment is limited, often focused on producing a minimally viable analytical approach for a limited set of analytes, rather than one which can maximally quantitate all targets of interest. We developed the Needler algorithm to produce comprehensive targeted MS methods capable of quantitating the maximum number of proteins given available instrument constraints. Using this algorithm, we demonstrate that the minimum instrument parameters required to produced targeted MS quantitation of all tryptic peptides from the human proteome are well in excess of current capabilities. Given these constraints, we provide a visualized scope of the boundaries for practical MS monitoring to maximize proteome and subproteome coverage dependent upon instrument duty cycle capabilities.


title: Soft topographical patterns trigger a stiffness-dependent cellular response to contact guidance

abstract:
Directional migration is involved in multiple physiological and pathological processes. Among other external signals, the architecture of the extracellular matrix can trigger directed cell migration through a phenomenon known as contact guidance: cells elongate, align, and migrate along the direction set by aligned extracellular matrix fibers. This process involves the orientation of focal adhesions, actin, and tubulin cytoskeleton along the direction of those fibers. Contact guidance has been extensively studied on stiff materials with topographical grooved patterns. However, how it translates to softer physiologically relevant compliances is not known. Here we show that substrate stiffness modulates the cellular response to topographical contact guidance. We found that for fibroblasts, while focal adhesions and actin responded to topography independently of the stiffness, microtubules showed a stiffness-dependent response that regulates contact guidance. On the other hand, both clusters and single breast carcinoma epithelial cells displayed stiffness-dependent contact guidance migration, leading to more directional and efficient migration when increasing substrate stiffness. These results suggest that both matrix stiffening and alignment of extracellular matrix fibers cooperate during directional cell migration, and both should be accounted when studying processes such as cancer cell invasion.

TeaserChanges in the stiffness of topographical patterns modify how mesenchymal and epithelial cells perform contact guidance.

category: biophysics


title: Bistable nerve conduction

abstract: The essence of the nerve system is to transmit information from and to different parts of the body in response to environmental changes. Here we show that a bistable conduction behavior can occur in the nerve system, which exhibits stimulus-dependent fast and slow conduction waves. The bistable behavior is caused by a positive feedback loop of the wavefront upstroke speed, mediated by the sodium channel inactivation properties, which is further potentiated by the calcium current. This provides a mechanism for the slow and fast conduction in the same nerve system observed experimentally. We show that the bistable conduction behavior is robust with respect to the experimentally determined activation thresholds of the known sodium and calcium channel families. The theoretical insights provide a generic mechanism for stimulus-dependent fast and slow conduction in the nerve system, which is applicable to conduction in other electrically excitable tissue, such as cardiac muscles.


title: Reversible adhesion by type IV pili leads to formation of irreversible localized clusters

abstract: Despite the fact a fundamental first step in the physiopathology of many disease-causing bacteria is the formation of long-lived, localized, multicellular clusters, the spatio-temporal dynamics of the cluster formation process, particularly on host tissues, remains poorly understood. Experiments on abiotic surfaces suggest that the colonization of a surface by swimming bacteria requires i) irreversible adhesion to the surface, ii) cell proliferation, and iii) a phenotypic transition from an initial planktonic state. Here, we investigate how Pseudomonas aeruginosa (PA) infects a polarized MDCK epithelium and show that contrary to what has been reported on the colonization of abiotic surfaces, PA forms irreversible bacterial clusters on apoptotic epithelial cell without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is regulated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion on the biotic surface, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible adhesion with a characteristic attachment time. Using a simple kinetic model, we explain how such reversible adhesion process leads to the formation of irreversible bacterial clusters and quantify the cluster growth dynamics.


title: A cellular hierarchy framework for understanding heterogeneity and predicting drug response in AML

abstract: The treatment landscape of AML is evolving with promising therapies entering clinical translation, yet patient responses remain heterogeneous and biomarkers for tailoring treatment are lacking. To understand how disease heterogeneity links with therapy response, we determined the leukemia cell hierarchy make-up from bulk transcriptomes of over 1000 patients through deconvolution using single-cell reference profiles of leukemia stem, progenitor, and mature cell types. Leukemia hierarchy composition was associated with functional, genomic, and clinical properties and converged into four overall classes, spanning Primitive, Mature, GMP, and Intermediate. Critically, variation in hierarchy composition along the Primitive vs GMP or Primitive vs Mature axes were associated with response to chemotherapy or drug sensitivity profiles of targeted therapies, respectively. A 7-gene biomarker derived from the Primitive vs Mature axis was predictive of patient response to 105 investigational drugs. Thus, hierarchy composition constitutes a novel framework for understanding disease biology and advancing precision medicine in AML.


title: Establishment and characterization of novel autologous pair primary cultures from two Indian non-habitual tongue carcinoma patients

abstract: Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India owing to very high percentages of patients with smoking and chewing habits. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To better understand disease progression, knowledge of cross talk between tumor stroma and the tumor cells becomes indispensable. Patient-derived in vitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, especially the cancer associated fibroblasts (CAFs). In the present study, two novel autologous pairs have been immortalized spontaneously from non-habitual, HPV-positive patients, presented with tongue squamous cell carcinoma. The epithelial and fibroblast primary lines had typical polygonal and spindle shaped morphology, respectively. Positive staining with Pan-cytokeratin (PanCK) and Fibroblast Specific Protein (FSP-1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. Increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF-conditioned medium proved the "CAF-ness" of the established fibroblast cells. The established cultures are the first of their kind which would serve as an useful platform in understanding the cross talk between tumor-stroma and tumor, along with studying tongue cancer progression.


title: Physiological Responses to Caprine Coccidiosis During a 100-year Rain Event in Oklahoma: An Opportunistic Case Study with a implications for climate change and clinical diagnosis.

abstract: The interaction between inclement weather and disease acquisition is a long-recognized relationship. In the case of coccidiosis, a parasitic infestation of the intestines, wet weather is known to promote incidences in livestock. Our opportunistic investigation tracks blood chemistries of goats that were exposed to coccidian in a 100 year rain event in Oklahoma. Our results suggest a potentially patterned response of blood chemistries that may be developed into a clinical tool in the future and provide warnings for producers of the risks associated with growing incidences of excessively wet weather related to climate change.


title: Weighting of celestial and terrestrial cues in the monarch butterfly central complex

abstract: Monarch butterflies rely on external cues for orientation during their annual long-distance migration from Northern US and Canada to Central Mexico. These external cues can be celestial cues, such as the sun or polarized light, which are processed in the internal compass of the brain, termed the central complex (CX). Previous research typically focused on how individual simulated celestial cues are encoded in the butterflys CX. However, in nature, the butterflies perceive several celestial cues at the same time and need to integrate them to effectively use the compound of all cues for orientation. In addition, a recent behavioral study revealed that monarch butterflies can rely on terrestrial cues, such as the panoramic skyline, for orientation and use them in combination with the sun to maintain a directed flight course. How the CX encodes a combination of celestial and terrestrial cues and how they are weighted in the butterflys CX is still unknown. Here, we examined how input neurons of the CX, termed TL neurons, combine celestial and terrestrial information. While recording intracellularly from the neurons, we presented a sun stimulus and polarized light to the butterflies as well as a simulated sun and a panoramic scene simultaneously. Our results show that celestial cues are integrated linearly in these cells, while the combination of the sun and a panoramic skyline did not always follow a linear integration of action potential rates. Interestingly, while the weighting between the sun and polarized light was invariant between individual input neurons, it varied strongly when the sun stimulus and the panoramic skyline were presented simultaneously. Taken together, this dynamic weighting between celestial and terrestrial cues may allow the butterflies to flexibly set their cue preference during navigation.


title: Temporal learning modulates post-interval ERPs in a categorization task with hidden reference durations.

abstract: The investigation of time-related activity in human electrophysiological activity has recently expanded from signals during the estimated interval to post-interval activity. Previous findings show timing-associated event-related potentials (ERPs) in both early ([~]150ms) and late positive components (LPC; [~]300ms) post-interval signals. However, it is still unclear whether and what aspects of temporal information these different patterns of EEG activity are capturing, especially, if these signals are associated with interval duration or other task parameters. In the present work, we designed a modified temporal categorization task to investigate if participants adaptation to a changing decisional context led to changes in post-interval ERPs. Participants had to learn a hidden reference duration on each block to categorize the presented target intervals correctly. We found an early posterior N200 potential correlated to target durations and decisional context. A central LPC, which interacted with duration and learning of the hidden reference, predicted behavioral responses. Our findings add to the current evidence that interval duration modulates post-interval activity in timing categorization tasks and shows that late signals are associated with interval duration and decisional context.


title: Preparing for the unknown: How working memory provides a link between perception and anticipated action

abstract: What mechanisms underlie the transfer of a working memory representation into a higher-level code for guiding future actions? Electrophysiological correlates of attentional selection and motor preparation processes within working memory were investigated in two retrospective cuing tasks. In the first experiment, participants stored the orientation and location of a grating. Subsequent feature cues (selective vs. neutral) indicated which feature would be the target for later report. The oscillatory response in the mu and beta frequency range with an estimated source in the sensorimotor cortex contralateral to the responding hand was used as correlate of motor preparation. Mu/beta suppression was stronger following the selective feature cues compared to the neutral cue, demonstrating that purely feature-based selection is sufficient to form a prospective motor plan. In the second experiment, another retrospective cue was included to study whether knowledge of the task at hand is necessary to initiate motor preparation. Following the feature cue, participants were cued to either compare the stored feature(s) to a probe stimulus (recognition task) or to adjust the memory probe to match the target feature (continuous report task). An analogous suppression in the mu frequency range was observed following a selective feature cue, even though the task at hand was not specified yet. Further, a subsequent selective task cue again elicited a mu/beta suppression, which was stronger after a continuous report task cue. This indicates that working memory is able to flexibly store different types of information in higher-level mental codes to provide optimal prerequisites for all required action possibilities.


title: Machine Learning for the Identification of Viral Attachment Machinery from Respiratory Virus Sequences

abstract: At the outset of an emergent viral respiratory pandemic, sequence data is among the first molecular information available. As viral attachment machinery is a key target for therapeutic and prophylactic interventions, rapid identification of viral "spike" proteins from sequence can significantly accelerate the development of medical countermeasures. For five families of respiratory viruses, covering the vast majority of airborne and droplet-transmitted diseases, host cell entry is mediated by the binding of viral surface glycoproteins that interact with a host cell receptor. In this report it is shown that sequence data for an unknown virus belonging to one of the five families above provides sufficient information to identify the protein(s) responsible for viral attachment and to permit an assignment of viral family. Random forest models that take as input a set of respiratory viral sequences can classify the protein as "spike" vs. non-spike based on predicted secondary structure elements alone (with 97.8 % correctly classified) or in combination with N-glycosylation related features (with 98.1 % correctly classified). In addition, a Random Forest model developed using the same dataset and only secondary structural elements was able to predict the respiratory virus family of each protein sequence correctly 89.0 % of the time. Models were validated through 10-fold cross-validation as well as bootstrapping. Surprisingly, we showed that secondary structural element and N-glycosylation features were sufficient for model generation. The ability to rapidly identify viral attachment machinery directly from sequence data holds the potential to accelerate the design of medical countermeasures for future pandemics.


title: On the flexibility of the cellular amination network in E. coli

abstract: Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine source and found that several amino acids complement the auxotrophy, either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids {beta}-alanine, alanine, glycine and serine as new amine sources and identified D-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

category: systems biology


title: Impact of various vaccine boosters on neutralization against Omicron following prime vaccinations with inactivated or adenovirus-vectored vaccine

abstract: Since the first report on November 24, 2021, the Omicron SARS-CoV-2 variant is now overwhelmingly spreading across the world. Two SARS-CoV-2 inactivated vaccines (IAVs), one recombinant protein subunit vaccine (PRV), and one adenovirus-vectored vaccine (AdV) have been widely administrated in many countries including China to pursue herd immunity. Here we investigated cross-neutralizing activities in 341 human serum specimens elicited by full-course vaccinations with IAV, PRV and AdV, and by various vaccine boosters following prime IAV and AdV vaccinations. We found that all types of vaccines induced significantly lower neutralizing antibody titers against the Omicron variant than against the prototype strain. For prime vaccinations with IAV and AdV, heterologous boosters with AdV and PRV, respectively, elevated serum Omicron-neutralizing activities to the highest degrees. In a mouse model, we further demonstrated that among a series of variant-derived RBD-encoding mRNA vaccine boosters, it is only the Omicron booster that significantly enhanced Omicron neutralizing antibody titers compared with the prototype booster following a prime immunization with a prototype S-encoding mRNA vaccine candidate. In summary, our systematical investigations of various vaccine boosters inform potential booster administrations in the future to combat the Omicron variant.


title: Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance

abstract: Coronaviruses, as exemplified by SARS-CoV-2, can evolve and spread rapidly to cause severe disease morbidity and mortality. Direct acting antivirals (DAAs) are highly effective in decreasing disease burden especially when they target essential viral enzymes, such as proteases and polymerases, as demonstrated in HIV-1 and HCV and most recently SARS-CoV-2. Optimization of these DAAs through iterative structure-based drug design has been shown to be critical. Particularly, the evolutionarily conserved molecular mechanisms underlying viral replication can be leveraged to develop robust antivirals against rapidly evolving viral targets. The main protease (Mpro) of SARS-CoV-2, which is evolutionarily constrained to recognize and cleave 11 specific sites to promote viral maturation, exemplifies one such target. In this study we define the substrate envelope of Mpro by determining the molecular basis of substrate recognition, through nine high-resolution cocrystal structures of SARS-CoV-2 Mpro with the viral cleavage sites. These structures enable identification of evolutionarily vulnerable sites beyond the substrate envelope that may be susceptible to drug resistance and compromise binding of the newly developed Mpro inhibitors.

category: biochemistry


title: Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19

abstract: While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.


title: JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum

abstract:
Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma japonicum. Because the parasites eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg- induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis.

Author summaryAmong neglected tropical diseases, schistosomiasis is a serious disease caused by infection with the parasite Schistosomiasis japonicum. Treatment of schistosomiasis is currently almost exclusively with praziquantel, which kills mainly adult parasites, with minimal effectiveness against immature schistosomes and eggs. However, the parasites eggs are primarily responsible for schistosomiasis dissemination and pathology. In addition, overuse of praziquantel in epidemic areas has led to drug resistance and a reduced cure rate. Thus, new parasite targets for the development of novel therapeutics are crucial. Here, we evaluated the potential of JQ-1, a bromodomain and extra-terminal protein inhibitor, to suppress the production of S. japonicum eggs. Application of JQ-1 to S. japonicum in vitro decreased the number of mature germ cells, the rates of oviposition, and the number of eggs produced in each male-female pairing. JQ-1 treatment of mice infected with S. japonicum ameliorated hepatic granuloma and decreased serum liver enzymes, suggesting improved liver function. These results indicate that JQ-1 inhibits reproductive development and egg production in S. japonicum, providing supporting evidence that JQ-1 warrants additional study for use as a novel approach in the prevention or treatment of schistosomiasis.


title: Evaluation of heat inactivation of human norovirus in freshwater clams using human intestinal enteroids

abstract: Foodborne disease attributed to consumption of shellfish contaminated with human norovirus (HuNoV) is one of many global health concerns. Our study aimed to determine conditions of heat-inactivation of HuNoV in freshwater clams (Corbicula japonica) using a recently developed HuNoV cultivation system employing stem-cell derived human intestinal enteroids (HIEs). We first measured the internal temperature of clam tissue in a water bath during boiling at 90 {degrees}C and found that approximately 2 minutes are required for the tissue to reach 90 {degrees}C. Next, GII.4 HuNoV was spiked into the center of the clam tissue followed by boiling at 90 {degrees}C for 1, 2, 3, or 4 minutes. The infectivity of the HuNoV in clam tissue homogenates was evaluated using HIEs. We demonstrated that HuNoV in unboiled clam tissue homogenates replicated in HIEs, whereas infectivity was lost in all boiled samples, indicating that heat treatment at 90 {degrees}C for 1 minute is sufficient to inactivate HuNoV in freshwater clams. To our knowledge, this is the first study to determine the infectivity of HuNoV tolerability in shellfish using HIEs and our results will be informative to develop strategies to inactivate HuNoV in foods.


title: Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13

abstract:
The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2 motif which, during virus entry, requires cleavage by a cell surface protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2 motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the S2 motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689) and also the reverse effect was seen. This swap had no impact on TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by TMPRSS2. Our findings are the first to demonstrate which S2 residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2 motif changes as they may appear in new SARS-CoV-2 variants.

IMPORTANCESince the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2 cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2 site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2 site variations as soon as they are detected during variant surveillance.


title: The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition

abstract: The ongoing SARS-CoV-2 pandemic continues to be a significant threat to global health. First reported in November 2021, the Omicron variant (B.1.1.529) is more transmissible and can evade immunity better than previous SARS-CoV-2 variants, fueling an unprecedented surge in cases. To produce functional proteins from this polyprotein, SARS-CoV-2 relies on the cysteine proteases Nsp3/papain-like protease (PLpro) and Nsp5/Main Protease (Mpro)/3C-like protease to cleave at three and more than 11 sites, respectively.1 Therefore, Mpro and PLpro inhibitors are considered to be some of the most promising SARS-CoV-2 antivirals. On December 22, 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for PAXLOVID, a ritonavir-boosted formulation of nirmatrelvir. Nirmatrelvir is a first-in-class orally bioavailable SARS-CoV-2 Mpro inhibitor.2 Thus, the scientific community must vigilantly monitor potential mechanisms of drug resistance, especially because SARS-CoV-2 is naive to Mpro inhibitors. Mutations have been well identified in variants to this point.3 Notably, Omicron Mpro (OMpro) harbors a single mutation- P132H. In this study we characterize the enzymatic activity, drug inhibition, and structure of OMpro while evaluating the past and future implications of Mpro mutations.


title: Ultraviolet Dosage and Decontamination Efficacy was Widely Variable Across 14 UV Devices after Testing a Dried Enveloped Ribonucleic Acid Virus Surrogate for SARS-CoV-2

abstract:
AimsThe dosages and efficacy of 14 ultraviolet (UV) decontamination technologies were measured against a SARS-CoV-2 surrogate virus that was dried on to different materials for lab and field testing.

Methods and ResultsA live enveloped, ribonucleic acid virus surrogate for SARS-CoV-2 was dried on stainless steel 304 (SS304), Navy Top Coat-painted SS304 (NTC), cardboard, polyurethane, polymethyl methacrylate (PMMA), and acrylonitrile butadiene styrene (ABS) at > 8.0 log10 plaque-forming units (PFU) per test coupon. The coupons were then exposed to UV light during both lab and field testing. Commercial and prototype UV-emitting devices were measured for efficacy; 4 handheld devices, 3 room/surface-disinfecting machines, 5 air-disinfection devices, and 2 larger custom-made machines. UV device dosages ranged from 0.01-729 mJ cm-2. Anti-viral efficacy among the different UV devices ranged from no decontamination up to nearly achieving sterilization. Importantly, cardboard required far more dosage than SS304.

ConclusionsEnormous variability in dosage and efficacy was measured among the different UV devices. Porous materials limit the utility of UV decontamination.

Significance and Impact of the StudyUV devices have wide variability in dosages, efficacy, hazards, and UV output over time indicating that each UV device needs independent technical measurement and assessment for product development, and prior to use.


title: Serum proteomics identifies immune pathways and candidate biomarkers of coronavirus infection in wild vampire bats

abstract: The apparent ability of bats to harbor many virulent viruses without showing disease is likely driven by distinct immune responses that coevolved with mammalian flight and the exceptional longevity of this order. Yet our understanding of the immune mechanisms of viral tolerance is restricted to a small number of bat-virus relationships and remains poor for coronaviruses (CoVs), despite their relevance to human health. Proteomics holds particular promise for illuminating the immune factors involved in bat responses to infection, because it can accommodate especially low sample volumes (e.g., sera) and thus can be applied to both large and small bat species as well as in longitudinal studies where lethal sampling is necessarily limited. Further, as the serum proteome includes proteins secreted from not only blood cells but also proximal organs, it provides a more general characterization of immune proteins. Here, we expand our recent work on the serum proteome of wild vampire bats (Desmodus rotundus) to better understand CoV pathogenesis. Across 19 bats sampled in 2019 in northern Belize with available sera, we detected CoVs in oral or rectal swabs from four individuals (21.1% positivity). Phylogenetic analyses identified all vampire bat sequences as novel -CoVs most closely related to known human CoVs. Across 586 identified serum proteins, we found no strong differences in protein composition nor abundance between uninfected and infected bats. However, receiver operating characteristic curve analyses identified seven to 32 candidate biomarkers of CoV infection, including AHSG, C4A, F12, GPI, DSG2, GSTO1, and RNH1. Enrichment analyses using these protein classifiers identified downregulation of complement, regulation of proteolysis, immune effector processes, and humoral immunity in CoV-infected bats alongside upregulation of neutrophil immunity, overall granulocyte activation, myeloid cell responses, and glutathione processes. Such results denote a mostly cellular immune response of vampire bats to CoV infection and identify putative biomarkers that could provide new insights into CoV pathogenesis in wild and experimental populations. More broadly, applying a similar proteomic approach across diverse bat species and to distinct life history stages in target species could improve our understanding of the immune mechanisms by which wild bats tolerate viruses.


title: Multivalent interactions essential for lentiviral integrase function

abstract: A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits (1). Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 [A] resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the -helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching revealed that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 resulted in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.


title: Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes

abstract: Robust translation elongation of any given amino acid sequence is a prerequisite to shape proteomes. Nevertheless, nascent peptides could destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains stochastically can induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in the N-terminal regions could prematurely terminate translation, producing premature products as peptidyl-tRNA species. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we found many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk. Indeed, a bioinformatics analysis revealed that the N-terminal regions of ORFs avoid D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.


title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

abstract: Alpha-synuclein (-syn), a major component of Lewy bodies found in Parkinsons disease (PD) patients, has been found exported outside of cells and may mediate its toxicity via cell-to-cell transmission. Here, we reconstituted soluble, monomeric -syn secretion by the expression of DnaJ homolog subfamily C member 5 (DNAJC5) in HEK293T cells. DNAJC5 undergoes palmitoylation and anchors on the membrane. Palmitoylation is essential for DNAJC5-induced -syn secretion, and the secretion is not limited by substrate size or unfolding. Cytosolic -syn is actively translocated and sequestered in an endosomal membrane compartment in a DNAJC5-dependent manner. Reduction of -syn secretion caused by a palmitoylation-deficient mutation in DNAJC5 can be reversed by a membrane-targeting peptide fusion-induced oligomerization of DNAJC5. We propose that DNAJC5 forms a palmitoylated oligomer to accommodate and export -syn.


title: Molecular insights into the MCM8/9 helicase complex in DNA unwinding and translocation

abstract: The minichromosome maintenance protein 8 and 9 (MCM8 and MCM9) form a functional helicase complex (MCM8/9) that plays an essential role in DNAhomologous recombination (HR) repair for DNA double-strand break. However, the assembly mechanism and the structural characterization of MCM8/9 remain unclear. Here, we report structures of the MCM8/9 complex combining X-ray crystallography and cryo-electron microscopy reconstruction analysis. The structures reveal that MCM8/9 is arranged by a 3-fold symmetry axis to form a heterohexamer with a central channel to accommodate DNA. Multiple characteristic hairpins from the N-terminal oligosaccharide/oligonucleotide (OB) domains of MCM8/9 hexameric ring protrude to the central channel and serve to unwind the duplex DNA. Importantly, the MCM8/9 hexamer is consisted of an N-tier ring and a C-tier ring that are connected by the flexible linkers (N-C linkers) which are much longer than those of MCM2-7. Our structural dynamic analyses based on the cryo-EM data reveal that the flexible C-tier ring showed rotating motions relative to the N-tier ring, which maybe mediated by the N-C linkers as shorten the length of the N-C linkers will greatly decrease the helicase activity and chemoresistance of MCM8/9. Our structural and biochemical results implicate an unusual DNA unwinding and translocation mechanism of MCM8/9 helicase complex in HR.


title: The IMD and JNK pathways drive the functional integration of the immune and circulatory systems of mosquitoes

abstract: The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial hemocytes, destroy pathogens in the areas of the body that experience the swiftest hemolymph (blood) flow. An infection recruits additional periostial hemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial hemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNAseq in the African malaria mosquito, Anopheles gambiae, and discovered that an infection upregulates multiple components of the IMD and JNK pathways in the heart with periostial hemocytes. This upregulation is greater in the heart with periostial hemocytes than in the circulating hemocytes or the entire abdomen. RNAi-based knockdown then showed that the IMD and JNK pathways drive periostial hemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating how these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones.


title: The antimicrobial peptide cathelicidin is critical for the development of Th17 responses in severe inflammatory disease

abstract: Multiple Sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-{gamma} production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system.


title: Germinal center-derived broadly neutralizing antibodies adapt to SARS-CoV-2 antigenic drift

abstract: The outbreak of SARS-CoV-2 variant Omicron which harbors a striking number of mutations in the spike protein has been raising concerns about the effectiveness of vaccines and antibody treatment1. Here, we confirmed a substantial reduction in neutralizing potency against Omicron in all convalescent and vaccinated sera. However, we found that some people infected by the early strain show relatively higher neutralization to Omicron. From those B cells, we developed neutralizing antibodies inhibiting broad variants including Delta and Omicron. Unlike reported antibodies, one had an extremely large interface and widely covered receptor binding motif of spike, thereby interfering with diversified variants. Somatic mutations introduced by long-term germinal center reaction contributed to the key structure of antibodies and the universal interaction with spike variants. Recalling such rare B cells may confer sustainable protection against SARS-CoV-2 variants emerging one after another.


title: Direct type I Interferon signaling in hepatocytes control malaria

abstract: Malaria is a devastating disease impacting over half of the worlds population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, preventing the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a novel transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary and sufficient to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire new anti-malarial drugs and vaccine strategies.


title: KaiC-like proteins improve stress resistance in environmental Pseudomonas species

abstract: KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria that are not known to have or need a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC-like proteins found in environmental Pseudomonas species. Using a bioinformatic approach, we describe the diversity and distribution of members of this protein family in the Pseudomonas genus and sketch, through comparative genomics, a conserved minimal interaction network comprising a histidine kinase and a response regulator. We then characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in sulfur uptake and alternative carbon source utilization in P. protegens. As expected, it physically interacts with its cognate histidine kinase and response regulator. Moreover, KaiC homologs are phosphorylated at one (P. putida) or two (P. protegens) sites and KaiC phosphorylation patterns change over time; however, in Pseudomonas species, changes in KaiC phosphorylation are driven by the age of the culture rather than by circadian cues as is the case in Cyanobacteria. In this study, through thorough bioinformatic and experimental analyses, we shed light onto the functional diversification and evolution of a unique protein family, diversely involved in bacterial rhythmic interactions with their environment. By so doing, we present a striking example of a protein whose general purpose is conserved in evolution, but whose molecular mechanics and participation in bacterial physiology can change dramatically across species.


title: Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

abstract:
Vector competence influences the ability of Anopheles mosquitoes to transmit Plasmodium parasites. The aim of this study was to determine the competence of male and female Anopheles mosquitoes to support the development of Plasmodium parasites. Male and female A. stephensi (STE2 strain) were infected with in vitro-cultured P. berghei (ANKA strain) ookinetes. We found that the number of oocysts produced was higher in males than in females. RNA-seq analysis of male and female mosquitoes injected with P. berghei ookinetes showed that predominantly genes of unknown function changed in expression levels in response to ookinete infection; however, further studies are required to elucidate their functions. Moreover, male mosquitoes were injected with in vitro-cultured P. falciparum (3D7 strain) gametocytes or zygotes. The development of P. falciparum in males was detected using nested polymerase chain reaction. We found the DNA content was higher on day 15 than on day 0, indicating that P. falciparum developed in the mosquito hemocoel. This study revealed promising new mechanisms underlying the interactions between Plasmodium and mosquitoes.

Author SummaryAnopheles mosquitoes transmit Plasmodium parasites that cause malaria disease. Vector competence is the ability of vectors to support pathogen development. The aim of this study was to determine the competence of male and female mosquitoes for Plasmodium parasites. Oocysts were formed in both male and female mosquitoes injected with ookinetes from in vitro-cultured Plasmodium berghei, which causes malaria in mice. The number of oocysts was higher in males, indicating that the competence in males was higher than that in females. RNA-seq analysis showed that the expression of genes of unknown function was highly variable in males and females. The genes defining the competence factors need further study, but the results indicate novel genes may be discovered. Furthermore, the development of Plasmodium falciparum, which causes the most severe malaria in humans, in male mosquitoes injected with in vitro-cultured gametocytes or zygotes was detected. As male mosquitoes do not suck blood, this method may allow us to experiment with Plasmodium more safely.


title: A novel bacteriocin produced by Bifidobacterium longum subsp. infantis has dual antimicrobial and immunomodulatory activity

abstract:
Bacteriocins are ribosomally-synthesized antimicrobial peptides produced by bacteria with either narrow or broad spectrum activity. Many genome mining studies have indicated that bacteriocin gene clusters are widespread within certain gut microbiota members. In early life, Bifidobacterium comprise the dominant microbiota genus in vaginally delivered and breast-fed infants, with high levels associated with improved health. However, in many cases the mechanisms underlying these beneficial effects are unknown, although a limited number of studies have suggested that bacteriocin production by Bifidobacterium may represent a key mechanism for preventing pathogen over-growth. Here, we used BAGEL4 and antiSMASH to identify putative bacteriocin sequences in the whole genome sequences of 33 Bifidobacterium strains isolated from infants participating in two clinical studies. We identified a novel non-lantibiotic bacteriocin from Bifidobacterium longum subsp. infantis LH_664, with 40% sequence homology to Lactococcin 972 from Lactococcus lactis subsp. lactis. The putative bacteriocin (Bifidococcin_664) was chemically synthesized and studied for antimicrobial and immune-modulatory activities. We determined it has discrete activity against Clostridium perfringens and it appears to have novel immune stimulatory activities, promoting macrophage phagocytosis and specific cytokine release. These data highlight strain-specific beneficial properties in the early life genus Bifidobacterium, and suggest avenues for development of novel and highly specific dual action antimicrobials, and possible probiotic strains, that are active against clinically important bacterial pathogens.

Data summarySamples LH_9 to LH_666 were previously sequenced and deposited to ENA under accession numbers ERS2658025-ERS2658043. Samples LH_986 to LH_1052 are newly sequenced and deposited to NCBI under accession numbers SAMN24838598-SAMN24838611. Additionally, previously assembled publicly available sequences (n=7) were retrieved online from NCBI Genomes database. See Supplementary Table S1 for further details.


title: Ultramicrobacteria in various fermented cabbages

abstract: Little is known about the ultramicrobial communities of foods. Several bacteria, including pathogenic species, can form ultramicrobial communities when exposed to harsh conditions, making their detection via conventional culture techniques difficult. This study aimed to explore ultramicrobial communities within the microbial communities of fermented cabbage products, such as kimchi, sauerkraut, and suancai, which are produced through specific manufacturing methods based on the laws and culture of respective regions. We used single-molecule real-time sequencing with tangential flow filtration for fermented cabbages after pre-filtration and transmission electron microscopy to confirm the identity of ultramicrobacteria (UMB).To the best of our knowledge, this is the first study to identify the differences between ultramicrobial communities and microbial communities of fermented cabbages. Although the size of the ultramicrobial communities was smaller than that of the latter, their diversity was not lower. In addition, some UMB underwent cell shrinkage due to unfavorable environments, while others maintained their small size regardless. Major pathogens were not detected in the ultramicrobial communities of fermented cabbages. Nevertheless, several suspicious strains were detected. Our method can be used to screen food materials for the presence of UMB undetectable via conventional methods. Ultramicrobial and microbial communities were efficiently separated using tangential flow filtration and analyzed via single-molecule real-time sequencing. The ultramicrobial communities of fermented vegetables were different from conventional microbial communities. This study provides new insights into the ecology of UMB in foods.


title: An Optimal Regulation of Fluxes Dictates Microbial Growth In and Out of Steady-State

abstract: Effective coordination of cellular processes is critical to ensure the competitive growth of microbial organisms. Pivotal to this coordination is the appropriate partitioning of cellular resources between protein synthesis via translation and the metabolism needed to sustain it. Here, we present a coarse-grained and organism-agnostic theory of microbial growth centered on the dynamic regulation of this resource partitioning. At the core of this regulation is an optimization of metabolic and translational fluxes, mechanistically achieved via the perception of charged- and uncharged-tRNA turnover. An extensive comparison with {approx} 50 data sets from Escherichia coli establishes the theorys veracity and shows that it can predict a remarkably wide range of growth phenomena in and out of steady-state with quantitative accuracy. This predictive power, achieved with only a few biological parameters, cements the preeminent importance of protein synthesis in microbial growth and establishes the theory as an ideal physiological framework to interrogate the dynamics of growth, competition, and adaptation in complex and ever-changing environments.