title: The Conserved Colletotrichum spp. Effector CEC3 Induces Nuclear Expansion and Cell Death in Plants

abstract: Plant pathogens secrete small proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.


title: Genomics-informed insights into microbial degradation of N,N-dimethylformamide

abstract:
Effective degradation of N,N-Dimethylformamide (DMF), an important industrial waste product, is challenging as only few bacterial isolates are known to be capable of degrading DMF. Aerobic remediation of DMF has typically been used, whereas anoxic remediation attempts are recently made, using nitrate as one electron acceptor, and ideally include methane as a byproduct. Here, we analyzed 20,762 complete genomes and 28 constructed draft genomes for the genes associated with DMF degradation. We identified 952 genomes that harbor genes involved in DMF degradation, expanding the known diversity of prokaryotes with these metabolic capabilities. Our findings suggest acquisition of DMF-degrading gene via plasmids are important in the order Rhizobiales and genus Paracoccus, but not in most other lineages. Degradation pathway analysis reveals that most putative DMF degraders using aerobic Pathway I will accumulate methylamine intermediate, while members of Paracoccus, Rhodococcus, Achromobacter, and Pseudomonas could potentially mineralize DMF completely under aerobic conditions. The aerobic DMF degradation via Pathway II is more common than thought and is primarily present in -and {beta}-Proteobacteria and Actinobacteria. Most putative DMF degraders could grow with nitrate anaerobically (Pathway III), however, genes for the use of methyl-CoM to produce methane were not found. These analyses suggest that microbial consortia could be more advantageous in DMF degradation than pure culture, particularly for methane production under the anaerobic condition. The identified genomes and plasmids form an important foundation for optimizing bioremediation of DMF-containing wastewaters.

ImportanceDMF is extensively used as a solvent in industries, and is classified as a probable carcinogen. DMF is a refractory compound resistant to degradation, and until now, only few bacterial isolates have been reported to degrade DMF. To achieve effective microbial degradation of DMF from wastewater, it is necessary to identify genomic diversity with the potential to degrade DMF and characterize the genes involved in two aerobic degradation pathways and potential anaerobic degradation for methane production. A wide diversity of organisms has the potential to degrade DMF. Plasmid-mediated degradation of DMF is important for Rhizobiales and Paracoccus. Most DMF degraders could grow anaerobically with nitrate as electron acceptor, while co-cultures are required to complete intermediate methanogenesis for methane production. This is the first genomics-based global investigation into DMF degradation pathways. The genomic database generated by this study provides an important foundation for the bioremediation of DMF in industrial waste waters.

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category: microbiology


title: Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

abstract: Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

category: molecular biology


title: Tight nanoscale clustering of Fc gamma-receptors using DNA origami promotes phagocytosis

abstract: Macrophages destroy pathogens and diseased cells through Fc{gamma} receptor (Fc{gamma}R)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple Fc{gamma}Rs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies.

category: cell biology


title: Proteomic Organellar Mapping Reveals Modulation of Peroxisomes by HIV-1

abstract: As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new, unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses we found that classifier performance in this system was organelle-dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine (SVM) learning for mitochondrial and ER proteins, but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, BANDLE, on SVM-classified data. Comparative BANDLE analysis of cells induced to express the wild-type viral genome vs. cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as TCR signaling components and coatomer complex. Lastly, we found that SVM classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.


title: Comparative Analysis of T Cell Spatial Proteomics and the Influence of HIV Expression


title: Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

abstract: Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo-to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P-than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.


title: β-Carotene oxidation products - function and safety

abstract: {beta}-Carotene oxidation products have newly discovered bioactivity in plants and animals. Synthetic fully oxidized {beta}-carotene (OxBC) has application in supporting livestock health, with potential human applications. The safety of synthetic OxBC has been evaluated. An Ames test showed weak-to-moderate mutagenicity in only one cell line at high concentrations. A mouse micronucleus assay established a non-toxic dose of 1800 mg/kg body weight, and no bone marrow micronuclei were induced. Plant sources of {beta}-carotene inevitably contain varying levels of natural OxBC. Vegetable powders and dried forages can be especially rich. Intakes of natural OxBC for humans and livestock alike have been estimated. The exposure range for humans (1-22 mg/serving) is comparable to the safe intake of {beta}-carotene (<15 mg/d). In livestock, OxBC in alfalfa can contribute ~550-850 mg/head/d for dairy cattle but in forage-deficient poultry feed much less (~1 ppm). Livestock intake of supplemental synthetic OxBC is comparable to OxBC potentially available from traditional plant sources. Human intake of synthetic OxBC in meat from livestock fed OxBC is similar to a single serving of food made with carrot powder. It is concluded that consumption of synthetic OxBC at levels comparable to natural OxBC is safe for humans and animals.

category: biochemistry


title: An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease

abstract: Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.


title: Transcription Factor NF-κB Unravels Nucleosomes

abstract: NF-{kappa}B is a transcription factor responsible for activating hundreds of genes in mammalian organisms. To accomplish its function, NF-{kappa}B must interact with DNA occupied by nucleosomes, but how this interaction occurs is unclear. Here we used Atomic Force Microscopy to characterize complexes of NF-{kappa}B with nucleosomes assembled on different DNA templates. The assembly of NF-{kappa}B-nucleosome complexes leads to a substantial decrease of DNA wrapping efficiency. Mapping of the nucleosomes did not reveal displacement of under-wrapped nucleosomes from their original position, suggesting that unravelling involves dissociation of one or both flanks of the nucleosomes. We discovered two binding modes of NF-{kappa}B associated with nucleosome unraveling - NF-{kappa}B bound to the nucleosome core and to the DNA flanks and propose models explaining the interaction of NF-{kappa}B with the nucleosome. We speculate that NF-{kappa}B can function as a pioneer factor enhancing its ability to facilitate rapid transcriptional response to cell stress.

category: biophysics


title: Groundwater bacterial communities evolve over time, exhibiting oscillating similarity patterns in response to recharge

abstract: Time series analyses are a crucial tool for uncovering the patterns and processes shaping microbial communities and their functions, especially in aquatic ecosystems. Subsurface aquatic environments are perceived to be more stable than oceans and lakes, due to the lack of sunlight, the absence of photosysnthetically-driven primary production, low temperature variations, and oligotrophic conditions. However, periodic groundwater recharge should affect the structure and succession of groundwater microbiomes. To disentangle the long-term temporal changes in groundwater bacterial communities of shallow fractured bedrock community, and identify the drivers of the observed patterns, we analysed bacterial 16S rRNA gene sequencing data for samples collected monthly from three groundwater wells over a six-year period (n=230) along a hillslope recharge area. We show that the bacterial communities in the groundwater of limestone-mudstone alternations were not stable over time and showed oscillating dissimilarity patterns which corresponded to periods of groundwater recharge. Further, we observed an increase in dissimilarity over time (generalized additive model P < 0.001) indicating that the successive recharge events result in communities that are increasingly more dissimilar to the initial reference time point. The sampling period was able to explain up to 29.5% of the variability in bacterial community composition and the impact of recharge events on the groundwater microbiome was linked to the strength of the recharge and local environmental selection. Many groundwater bacteria originated from the recharge-related sources (mean = 66.5%, SD = 15.1%) and specific bacterial taxa were identified as being either enriched or repressed during recharge events. Overall, similar to surface aquatic environments, groundwater microbiomes vary through time, though we revealed groundwater recharges as unique driving factors for these patterns. The high temporal resolution employed here highlights the complex dynamics of bacterial communities in groundwater and demonstrated that successive shocks disturb the bacterial communities, leading to decreased similarity to the initial state over time.


title: Establishment and Resilience of Transplanted Gut Microbiota in Aged Mice

abstract: Fecal microbiota transplantation (FMT), a procedure in which fecal material is transferred from a donor to a recipient, has been increasingly used as a treatment to restore healthy gut microbiota. There is a substantial difference in the composition of gut microbiota between young and aged hosts, but little is known about whether age matching between the FMT donor and recipient affects microbiota restoration and long-term maintenance. In the present investigation, we aimed to study the establishment and resilience of transplanted gut microbiota in aged recipients. We treated naturally aged mice (20 months old) with a broad-spectrum antibiotic cocktail and monitored the restoration of gut microbiota over 8 weeks. The diversity of gut microbiota in aged mice failed to reach the baseline level via spontaneous recovery; in contrast, FMT from either (age-)matched or unmatched donors facilitated the recovery of gut microbiota diversity. The microbiota transplanted from different donors successfully established in the aged recipients and had long-term effects on the gene expression profiles of the host colon. Finally, we evaluated the long-term maintenance of transplanted microbiota via intentional disruption of gut homeostasis. We found that lack of age matching between FMT donors and recipients may decrease the resilience of transplanted gut microbiota against colonic inflammation. The results from our study systematically examining the effects of FMT on the gut homeostasis of aged hosts suggest that the compatibility between donors and recipients should be taken into account when implementing FMT.


title: Comprehensive mouse gut metagenomecatalog reveals major difference to thehuman counterpart

abstract: Mouse is the most used model for studying the impact of microbiota on its host, but the repertoire of species from the mouse gut microbiome remains largely unknown. Here, we construct a Comprehensive Mouse Gut Metagenome (CMGM) catalog by assembling all currently available mouse gut metagenomes and combining them with published reference and metagenome-assembled genomes. The 50011 genomes cluster into 1699 species, of which 78.1% are uncultured, and we discovered 226 new genera, 7 new families, and 1 new order. Rarefaction analysis indicates comprehensive sampling of the species from the mouse gut. CMGM enables an unprecedented coverage of the mouse gut microbiome exceeding 90%. Comparing CMGM to the human gut microbiota shows an overlap 64% at the genus, but only 16% at the species level, demonstrating that human and mouse gut microbiota are largely distinct.


title: Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

abstract: Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, its molecular consequences on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. In particular, ribosomes translating on the endoplasmic reticulum are more rapidly dephosphorylated than cytosolic ribosomes. Loss of RPS6 phosphorylation causes a correspondingly larger drop in translation efficiency of mRNAs with short CDSs than long CDSs. Interestingly, mRNAs with 5 TOP motifs are translated well also in the absence of RPS6 phosphorylation despite short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs in different subcellular localizations and the functional consequence on translation.


title: Regulation of transcriptional bursting by core promoter elements in the Drosophila embryo

abstract: Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancers activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters "enhancer responsiveness" by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development.


title: Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryo


title: Myosin turnover controls actomyosin contractile instability

abstract: Actomyosin contractile force is harnessed for diverse functions, from cell division to morphogenesis during development. However, actomyosin contractility is intrinsically unstable to self-reinforcing spatial variations that destroy actomyosin architecture if unopposed. The full instability was rarely observed, and how cells control the instability is not established. Here, we observed the instability run its full course in isolated cytokinetic contractile rings lacking component turnover. Myosin II aggregated hierarchically into aggregates of growing size and separation up to a maximum. Molecularly explicit simulations reproduced hierarchical aggregation that precipitated tension loss and ring fracture, and identified the maximum separation as the length of actin filaments mediating mechanical communication between aggregates. Late stage simulated aggregates had aster-like morphology with polarity sorted actin, similar to late stage actomyosin systems in vitro. Our results suggest myosin II turnover controls actomyosin contractile instability in normal cells, setting myosin aggregate size and intercepting catastrophic hierarchical aggregation and fracture.


title: Glucoregulation and coping behavior after chronic stress: sex differences across the lifespan

abstract: Exposure to prolonged stress during adolescence taxes adaptive and homeostatic processes leading to deleterious behavioral and metabolic outcomes. Although previous pre-clinical studies found effects of early life stress on cognition and stress hormone reactivity, these studies largely focused on males. The purpose of the current study was to determine how biological sex shapes behavioral coping and metabolic health across the lifespan after chronic stress. We hypothesized that examining chronic stress-induced behavioral and endocrine outcomes would reveal sex differences in the biological basis of susceptibility. During the late adolescent period, male and female Sprague-Dawley rats experienced chronic variable stress (CVS). Following completion of CVS, all rats experienced a forced swim test (FST) followed 3 days later by a fasted glucose tolerance test (GTT). The FST was used to determine coping in response to a stressor. Endocrine metabolic function was evaluated in the GTT by measuring glucose and corticosterone, the primary rodent glucocorticoid. Animals then aged to 15 months when the FST and GTT were repeated. In young animals, chronically stressed females exhibited more passive coping and corticosterone release in the FST. Additionally, chronically stressed females had elevated corticosterone and impaired glucose clearance in the GTT. Aging affected all measurements as behavioral and endocrine outcomes were sex specific. Furthermore, regression analysis between hormonal and behavioral responses identified associations depending on sex and stress. Collectively, these data indicate female susceptibility to the effects of chronic stress during adolescence. Further, translational investigation of coping style and glucose homeostasis may identify biomarkers for stress-related disorders.

category: animal behavior and cognition


title: Variability and thermal modulation of locomotor statistics in zebrafish

abstract:
BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way.


title: Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability


title: ENCODING OF LIMB STATE BY SINGLE NEURONS IN THE CUNEATE NUCLEUS OF AWAKE MONKEYS

abstract: The cuneate nucleus (CN) is among the first sites along the neuraxis where proprioceptive signals can be integrated, transformed, and modulated. The objective of the study was to characterize the proprioceptive representations in CN. To this end, we recorded from single CN neurons in three monkeys during active reaching and passive limb perturbation. We found that many neurons exhibited responses that were tuned approximately sinusoidally to limb movement direction, as has been found for other sensorimotor neurons. The distribution of their preferred directions (PDs) was highly non-uniform and resembled that of muscle spindles within individual muscles, suggesting that CN neurons typically receive inputs from only a single muscle. We also found that the responses of proprioceptive CN neurons tended to be modestly amplified during active reaching movements compared to passive limb perturbations, in contrast to cutaneous CN neurons whose responses were not systematically different in the active and passive conditions. Somatosensory signals thus seem to be subject to a "spotlighting" of relevant sensory information rather than uniform suppression as has been suggested previously.

category: neuroscience


title: Accuracy and precision in super-resolution MRI: Enabling spherical tensor diffusion encoding at ultra-high b-values and high resolution

abstract: Diffusion MRI (dMRI) is a useful probe of tissue microstructure but suffers from low signal-to-noise ratio (SNR) whenever high resolution and/or high diffusion encoding strengths are used. Low SNR leads not only to poor precision but also poor accuracy of the diffusion-weighted signal, as the rectified noise floor gives rise to a positive signal bias. Recently, super-resolution techniques have been proposed for signal acquisition at a low spatial resolution but high SNR, whereafter a higher spatial resolution is recovered by image reconstruction. In this work, we describe a super-resolution reconstruction framework for dMRI and investigate its performance with respect to signal accuracy and precision. Using strictly controlled phantom experiments, we show that the super-resolution approach improves accuracy by facilitating a more beneficial trade-off between spatial resolution and diffusion encoding strength before the noise floor affects the signal. Moreover, precision is shown to have a less straightforward dependency on acquisition, reconstruction, and intrinsic tissue parameters. Indeed, we find that a gain in precision from super-resolution reconstruction (SRR) is substantial only when some spatial resolution is sacrificed. We also demonstrated the value of SRR in the challenging combination of high resolution and spherical b-tensor encoding at ultrahigh b-values--a configuration that produces a unique contrast that emphasizes tissue in which diffusion is restricted in all directions. We conclude that SRR is most valuable in low-SNR conditions, where it can suppress rectified noise floor effects and recover signal with high accuracy. The in vivo application showcases a vastly superior image contrast when using SRR compared to conventional imaging, facilitating investigations of brain tissue that would otherwise have prohibitively low SNR, resolution or required non-conventional MRI hardware.


title: Closed-loop auditory stimulation method to modulate sleep slow waves and motor learning performance in rats

abstract: Slow waves and cognitive output have been modulated in humans by phase-targeted auditory stimulation. However, to advance its technical development and further our understanding, implementation of the method in animal models is indispensable. Here, we report the successful employment of slow waves phase-targeted closed-loop auditory stimulation (CLAS) in rats. To validate this new tool both conceptually and functionally, we tested the effects of up- and down-phase CLAS on proportions and spectral characteristics of sleep, and on learning performance in the single pellet-reaching task, respectively. Without affecting 24-h sleep-wake behavior, CLAS specifically altered delta (slow waves) and sigma (sleep spindles) power persistently over chronic periods of stimulation. Down-phase CLAS exerted a detrimental effect on overall engagement and success rate in the behavioral test, and overall CLAS-dependent spectral changes were positively correlated with learning performance. Altogether, our results provide proof-of-principle evidence that phase-targeted CLAS of slow waves in rodents is efficient, safe and stable over chronic experimental periods, enabling the use of this high-specificity tool for basic and preclinical translational sleep research.


title: Behaviour-driven Arc expression is greater in dorsal than ventral CA1 regardless of task or sex differences.

abstract: Evidence from genetic, behavioural, anatomical, and physiological study suggests that the hippocampus functionally differs across its longitudinal (dorsoventral or septotemporal) axis. Although, how to best characterize functional and representational differences in the hippocampus across its long axis remains unclear. While some suggest that the hippocampus can be divided into dorsal and ventral subregions that support distinct cognitive functions, others posit that these regions vary in their granularity of representation, wherein spatial-temporal resolution decreases in the ventral (temporal) direction. Importantly, the cognitive and granular hypotheses make distinct predictions on cellular recruitment dynamics under conditions when animals perform tasks with qualitatively different cognitive-behavioural demands. The cognitive function account implies that dorsal and ventral cellular recruitment differs depending on relevant behavioural demands, while the granularity account suggests similar recruitment dynamics regardless of the nature of the task performed. Here, we quantified cellular recruitment with the immediate early gene (IEG) Arc across the entire longitudinal CA1 axis in female and male rats performing spatial- and fear-guided memory tasks. Our results show that recruitment is greater in dorsal than ventral CA1 regardless of task or sex. This experimentum crucis leads to the strong inference that the granularity hypothesis for functional differences across the longitudinal axis in the rodent hippocampus is correct.


title: Peer presence increases adolescents' prosocial behavior by speeding the evaluation of rewards for others

abstract: Peer presence can elicit maladaptive adolescent decision-making, potentially by increasing sensitivity to the rewards one receives. It remains unknown whether peer presence also increases adolescents sensitivity to others rewards, which could have an adaptive effect in contexts allowing pro-social behaviors. Here, we combine social utility modeling and real-time decision process modeling to characterize how peer presence alters adolescents processing of self and other rewards. We found that adolescents behaved selfishly when privately allocating rewards for themselves and a peer in an incentive-compatible task. In peer presence, however, adolescents became more altruistic. Real-time estimates of reward processing collected using computer mouse tracking showed that altruistic behavior was associated with relatively earlier processing of peer-rewards relative to self-rewards, such that peer presence sped peer-reward processing without altering self-reward processing. Our results indicate a causal mechanism through which peer presence prompts greater prosocial behavior by altering how adolescents process prosocial outcomes.


title: Decreased synthesis and variable gene transcripts of oxytocin in a domesticated avian species

abstract: The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia. Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to white-rumped munias. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We identified intra-strain variability in the untranslated regions of the OT sequence in Bengalese finches in comparison to the munia OT. Several of these changes fall in specific transcription factor binding sites, which show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed significantly lower OT mRNA expression in the diencephalon of the Bengalese finches relative to munias. Our study thus points to a decreased OT synthesis in the domestic strain compared with the wild strain in birds. This is an opposite pattern from that found in some domesticated mammals, suggesting that different processes of OT function might have occurred in mammals and birds under domestication.


title: Structure-function analysis of purified proanthocyanidins reveals a role for polymer size in suppressing inflammatory responses

abstract: Proanthocyanidins (PAC) are dietary compounds that have been extensively studied for beneficial health effects due to their anti-inflammatory properties. However, the structure-function relationships of PAC and their mode-of-action remain obscure. Here, we isolated a wide range of diverse PAC polymer mixtures of unprecedented purity from plant material. Polymer size was a key factor in determining the ability of PAC to regulate inflammatory cytokine responses in murine macrophages. PAC polymers with a medium (9.1) mean degree of polymerization (mDP) induced substantial transcriptomic changes, whereas PAC with either low (2.6) or high (12.3) mDP were significantly less active. Short-term oral treatment of mice with PAC modulated gene pathways connected to nutrient metabolism and inflammation in ileal tissue in a polymerization-dependent manner. Mechanistically, the bioactive PAC polymers modulated autophagic flux and directly inhibited lipopolysaccharide-induced autophagy. Collectively, our results highlight the importance of defined structural features in the health-promoting effects of PAC-rich foods.

category: immunology


title: Type II alveolar cells with constitutive expression of MHCII and limited antigen presentation capacity contribute to improved respiratory viral disease outcomes

abstract: Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in the worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited in comparison to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.


title: Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

abstract:
Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy.

Graphical abstract

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title: Pro-autoimmune allele of tyrosine phosphatase, PTPN22, enhances tumor immunity

abstract: The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W in encoded protein Lyp) is present in 5-10% of the North American population and is strongly associated with numerous autoimmune diseases. Although much research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its pro-autoimmune allele has in tumor immunity is poorly defined. To interrogate the role this allele may have in the anti-tumor immune response, we used CRISPR/Cas9 to generate mice in which the ortholog of Lyp, PEP, is mutated at position 619 to produce the relevant pro-autoimmune mutation (R619W). Results of this study show that mice homozygous for this alteration (PEP-619WW) resist tumor growth as compared with wildtype mice. Consistent with these results, tumors from PEP-619WW mice have more CD45 infiltrates containing more activated CD8 T cell and CD4 T cells. Additionally, there are more cDC1 cells and less MDSCs in tumors from PEP-619WW mice. Interestingly, the tumor infiltrating PEP-619WW cDC1s have decreased PD-L1 expression compared to cDC1s from PEP-WT mice. Taken together, our data show that the pro-autoimmune allele of Ptpn22 drives a strong anti-tumor response in innate and adaptive immune cells resulting in superior control of tumors.


title: Large-scale computational discovery and analysis of virus-derived microbial nanocompartments

abstract: Protein compartments represent an important strategy for subcellular spatial control and compartmentalization. Encapsulins are a class of microbial protein compartments defined by the viral HK97-fold of their capsid protein, self-assembly into icosahedral shells, and dedicated cargo loading mechanism for sequestering specific enzymes. Encapsulins are often misannotated and traditional sequence-based searches yield many false positive hits in the form of phage capsids. This has hampered progress in understanding the distribution and functional diversity of encapsulins. Here, we develop an integrated search strategy to carry out a large-scale computational analysis of prokaryotic genomes with the goal of discovering an exhaustive and curated set of all HK97-fold encapsulin-like systems. We report the discovery and analysis of over 6,000 encapsulin-like systems in 31 bacterial and 4 archaeal phyla, including two novel encapsulin families as well as many new operon types that fall within the two already known families. We formulate hypotheses about the biological functions and biomedical relevance of newly identified operons which range from natural product biosynthesis and stress resistance to carbon metabolism and anaerobic hydrogen production. We conduct an evolutionary analysis of encapsulins and related HK97-type virus families and show that they share a common ancestor. We conclude that encapsulins likely evolved from HK97-type bacteriophages. Our study sheds new light on the evolutionary interplay of viruses and cellular organisms, the recruitment of protein folds for novel functions, and the functional diversity of microbial protein organelles.


title: The B1.351 and P.1 variants extend SARS-CoV-2 host range to mice

abstract: Receptor recognition is a major determinant of viral host range, infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with novel hosts, and the high mutation rate of RNA viruses may explain their frequent host shifts. SARS-CoV-2 extensive circulation in humans results in the emergence of variants, including variants of concern (VOCs) with diverse mutations notably in the spike, and increased transmissibility or immune escape. Here we show that, unlike the initial and Delta variants, the three VOCs bearing the N501Y mutation can infect common laboratory mice. Contact transmission occurred from infected to naive mice through two passages. This host range expansion likely results from an increased binding of the spike to the mouse ACE2. Together with the observed contact transmission, it raises the possibility of wild rodent secondary reservoirs enabling the emergence of new variants.


title: Variants with the N501Y mutation extend SARS-CoV-2 host range to mice, with contact transmission


title: Angiotensin II disrupts the cytoskeletal architecture of human urine-derived podocytes and results in activation of the renin-angiotensin system

abstract:
High blood pressure is one of the major public health problems which causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin-angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory and -hypertrophic milieu that causes remodelling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CDK).

In the past, disease modeling of kidney-associated malignancies was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of SIX2-positive urine derived renal progenitor cells (UdRPCs) into mature podocytes bearing typical foot processes. The UdRPCs-derived podocytes show the ability to execute Albumin endocytosis and the activation of the renin-angiotensin system by being responsive to ANGII stimulation. Our data reveals the ANGII dependent downregulation of NPHS1 and SYNPO, resulting in the disruption of the complex podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of -ACTININ. In the present manuscript we confirm and propose UdRPCs as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening.


title: Potential Role of Red Palm Oil Supplemented Diet on Oxidative Stress Enzymes in Plasmodium berghei Induced Malaria

abstract:
BackgroundMalaria parasites are very vulnerable to oxidative stress during the part of their life cycle when they inhabit the erythrocytes. Studies have shown that dietary intake of antioxidant plays a role in stabilizing oxidative stress.

MethodsThe objective of this research work was to examine the antioxidative effect of red palm oil on Plasmodium berghei malaria induced oxidative stress. Sixty (60) mice were distributed into five groups. Group A served as the negative control (healthy mice with normal feed); group B as positive control (healthy mice fed with red palm oil without malaria parasite.while the other groups (C to E) served as the test groups. Group C served as group of healthy mice fed with red palm oil (pelletized), infected with malaria parasite without antimalaria drug. Group D served as group of healthy mice fed with red palm oil (pelletized), infected with malaria parasite and treated with amodiaquine. Group E served as group of healthy mice fed with normal feed, infected with malaria parasite and treated with amodiaquine. The parasitemia levels were estimated on days 1,4 and 5. The activity of oxidative stress enzymes biomarkers were determined spectrophotometrically.

ResultGroup A showed a statistically significant increase in the activity of SOD (1.90 {+/-} 0.16 units/mg protein), GST (1.68 {+/-} 0.086 units/L) compared to group C, SOD (3.54 {+/-} 0.83 units/mg protein), GST (2.12 {+/-} 0.20 units/L). Group B showed a statistical significant decrease in the activities of SOD (3.22 {+/-} 0.33 units/mg protein), Catalase (49.11 {+/-} 2.35 {micro}mol/min), GSH-R (31.50 {+/-} 2.48 units/L) compared to group E, SOD (2.18 {+/-} 0.39 units/mg protein), Catalase (44.07 {+/-} 3.88 {micro}mol/min), GSH-R (27.75 {+/-} 1.64 units/L).

ConclusionThe dietary intake of red palm oil helps to reduce free radical mediated injury to the tissue thus preventing oxidative stress induced by malaria or any other factors.


title: Phospholipids stabilize binding of pituitary adenylate cyclase-activating peptide to vasoactive intestinal polypeptide receptor

abstract: Vasoactive intestinal polypeptide receptor (VIP1R) is a class B G-protein coupled receptor (GPCR) that is widely distributed throughout the central nervous system, T-lymphocytes, and peripheral tissues of organs like lungs and liver. Critical functions of these receptors render them potential pharmacological targets for the treatment of a broad spectrum of inflammatory and neurodegenerative diseases. Here we use atomistic studies to show that phospholipids can act as potent regulators of peptide binding on to the receptor. We simulated the binding of neuropeptide pituitary adenylate cyclase-activating peptide (PACAP27) into the transmembrane bundle of the receptor. The simulations reveal two lipid binding sites on the peptidic ligand for the negatively charged phosphodiester of phospholipids in the extracellular leaflet which lower the peptide-receptor binding free energy by ~8kBT. We further simulated the effect of anionic lipids phosphatidylinositol-4,5-bisphosphate (PIP2). These lipids show much stronger interaction, lowering the peptide-receptor binding energy by an additional ~7kBT compared to POPC lipids. These findings suggest that lipids can play an active role in catalyzing peptide-receptor binding and activating vasoactive intestinal polypeptide receptors.


title: Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport

abstract: Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse EPR measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm towards the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, where TonB binding alone is sufficient to produce allosteric rearrangements in the transporter. They also demonstrate the importance of studying outer membrane proteins in their native environment.


title: Momelotinib is a highly potent inhibitor of FLT3-mutant AML

abstract:
Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogenic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues, D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL) and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

Key pointsO_LIMomelotinib shows high efficacy against FLT3 mutated AML cells including quizartinib-resistant activation loop variants.
C_LIO_LIMomelotinib effectively suppresses intrinsic resistance conferred by FLT3 ligand and hematopoietic cytokines (GM-CSF and IL3).
C_LI

category: cancer biology


title: PI3Kβ inhibition restores ALK inhibitor sensitivity in ALK-rearranged lung cancer

abstract: For non-small cell lung cancer (NSCLC) patients with ALK-rearranged tumors, treatment with ALK inhibitors can improve outcomes. However, clinical resistance typically develops over time, and in the majority of cases resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen, and deployed functional drug screens to identify modulators of resistance to ALK inhibitors. This identified a role for PI3K{beta} and EGFR in regulating resistance to ALK inhibition. Furthermore, inhibition of ALK elicited activation of EGFR, and inhibition of PI3K{beta} rescued EGFR-mediated ALK inhibitor resistance. In ALK-rearranged primary cultures, cell lines and in vivo xenograft models, combined inhibition of ALK and PI3K{beta} prevented compensatory MAPK and PI3K-AKT pathway reactivation and selectively targeted the cancer cells. The combinatorial effect was seen even in the background of TP53 mutations and in epithelial-mesenchymal transformed cells. In conclusion, combinatorial ALK and PI3K{beta} inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.


title: Comprehensive patient-level classification and quantification of driver events in TCGA PanCanAtlas cohorts

abstract:
There is a growing need to develop novel therapeutics for targeted treatment of cancer. The prerequisite to success is the knowledge about which types of molecular alterations are predominantly driving tumorigenesis. To shed light onto this subject, we have utilized the largest database of human cancer mutations - TCGA PanCanAtlas, multiple established algorithms for cancer driver prediction (2020plus, CHASMplus, CompositeDriver, dNdScv, DriverNet, HotMAPS, OncodriveCLUSTL, OncodriveFML) and developed four novel computational pipelines: SNADRIF (Single Nucleotide Alteration DRIver Finder), GECNAV (Gene Expression-based Copy Number Alteration Validator), ANDRIF (ANeuploidy DRIver Finder) and PALDRIC (PAtient-Level DRIver Classifier). A unified workflow integrating all these pipelines, algorithms and datasets at cohort and patient levels was created.

We have found that there are on average 12 driver events per tumour, of which 0.6 are single nucleotide alterations (SNAs) in oncogenes, 1.5 are amplifications of oncogenes, 1.2 are SNAs in tumour suppressors, 2.1 are deletions of tumour suppressors, 1.5 are driver chromosome losses, 1 is a driver chromosome gain, 2 are driver chromosome arm losses, and 1.5 are driver chromosome arm gains. The average number of driver events per tumour increases with age (from 7 to 15) and cancer stage (from 10 to 15) and varies strongly between cancer types (from 1 to 24). Patients with 1 and 7 driver events per tumour are the most frequent, and there are very few patients with more than 40 events. In tumours having only one driver event, this event is most often an SNA in an oncogene. However, with increasing number of driver events per tumour, the contribution of SNAs decreases, whereas the contribution of copy-number alterations and aneuploidy events increases.

Author SummaryBy analysing genomic and transcriptomic data from 10000 cancer patients through our custom-built computational pipelines and previously established third-party algorithms, we have found that half of all driver events in a patients tumour appear to be gains and losses of chromosomal arms and whole chromosomes. We therefore suggest that future therapeutics development efforts should be focused on targeting aneuploidy. We have also found that approximately a third of driver events in a patient are whole gene amplifications and deletions. Thus, therapies aimed at copy-number alterations also appear very promising. On the other hand, drugs aiming at point mutations are predicted to be less successful, as these alterations are responsible for just a couple of drivers per tumour. One notable exception are patients having only one driver event in their tumours, as this event is almost always a single nucleotide alteration in an oncogene.


title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

abstract: The E3 ligase RNF168 has been suggested to have roles at DNA replication forks in addition to its canonical functions in DNA double-strand break (DSB) signaling. However, the precise role of RNF168 in DNA replication remains unclear. Here we demonstrate that RNF168 is recruited to DNA replication factories independent of the canonical DSB response pathway regulators and identify a degenerate PCNA-Interacting Peptide (DPIP) motif in the C-terminus of RNF168 which mediates its binding to PCNA. An RNF168 mutant harboring substitutions in the DPIP box fails to interact with PCNA and is not recruited to sites of DNA synthesis, yet fully retains its ability to promote DSB-induced 53BP1 foci. Surprisingly, the RNF168 DPIP mutant also retains the ability to support ongoing DNA replication fork movement, demonstrating that PCNA-binding is dispensable for normal S-phase functions. However, replisome-associated RNF168 functions to suppress the DSB-induced 53BP1 DNA damage response during S-phase. Moreover, we show that WT RNF168 can perform PCNA ubiquitylation independently of RAD18 and also synergizes with RAD18 to amplify PCNA ubiquitylation. Taken together, our results identify non-canonical functions of RNF168 at the replication fork and demonstrate new mechanisms of cross talk between the DNA damage and replication stress response pathways.


title: The Vaginal Microbiome in Women of Reproductive Age with Healthy Weight versus Overweight/Obesity

abstract:
ObjectiveEvaluate differences between the vaginal microbiome of reproductive-aged women with overweight and obesity (Ow/Ob) compared with healthy weight (HW).

MethodsA cohort of 367 non-pregnant women (18-40 years) with Ow/Ob (body mass index [BMI] [≥]25 kg/m2) was case-matched with 367 women with HW (BMI 18.0-24.9 kg/m2). The study was a secondary analysis of 16S rRNA vaginal microbiome surveys through the Vaginal Human Microbiome Study (VaHMP). Groups were matched on age, race/ethnicity, income, and nulliparity status.

ResultsMean age and BMI of Ow/Ob and HW groups were 26.8 versus 26.7 years and 37.0 versus 22.1 kg/m2, respectively. The overall vaginal microbiome composition differed between groups (PERMANOVA, p=0.035). Women with Ow/Ob had higher alpha-diversity compared with women with HW (Wilcoxon test, Shannon index p=0.025; Inverse Simpson index p=0.026). Lactobacillus dominance ([≥]30% proportional abundance) was observed in a greater proportion of women with HW (48.7%) compared with Ow/Ob (40.1%; p=0.026).

ConclusionsThe vaginal microbiome differs in reproductive-aged women with Ow/Ob compared with women with HW, with increased alpha-diversity and decreased predominance of Lactobacillus. Observed differences in vaginal microbiome may partially explain differences in preterm birth and bacterial vaginosis risk between these populations.


title: Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

abstract: Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.


title: Dynamic changes of genomic and transcriptomic tumor diversity during melanoma progression

abstract: Metastatic melanoma is an aggressive disease that is notoriously difficult to treat. Here, we used a BRAFV600E-driven mouse model to study melanoma progression and metastasis in the absence of extrinsic mutagenic factors such as UV radiation. The tumors were grown for different time periods in seven different mice such that disease progression was tracked over time. We sequenced the whole exome and whole transcriptome of 16 melanoma samples from these mice. The timed samples revealed that many genes are increasingly upregulated during progression, such as genes involved in extracellular matrix organization, and Vegfc, a factor that induces lymphatic vessel growth and metastases to lymph nodes, which was identified as an important signaling hub in our analysis. The Vegfc gene interaction network highlighted multiple genes from the MAPK pathway that are increasingly deregulated during tumor evolution. The genomic diversity of primary and metastatic tumors showed an increase over time. Additionally, our integrative multi-omics analysis adds further evidence that Rock1 is a crucial player driving metastasis formation, and may be upregulated due to mutations in the PI3K/AKT pathway. In summary, our study contributes towards an improved understanding of melanoma which may eventually lead to improved treatment strategies.


title: Novel epigenetic-metabolic inhibitor combination treatment blocks platinum-induced ovarian cancer stem cell enrichment

abstract: High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer. Platinum resistance is a common occurrence in HGSOC and a main cause of tumor relapse resulting in high patient mortality rates. Recurrent OC is enriched in aldehyde dehydrogenase (ALDH)+ ovarian cancer stem cells (OCSCs), which are resistant to platinum agents. We demonstrated that acute platinum treatment induced a DNA damage-dependent decrease in BRCA1 levels. In a parallel response associated with G2/M arrest, platinum treatment also induced an increase in expression of NAMPT, the rate limiting regulator of NAD+ production from the salvage pathway, and levels of NAD+, the cofactor required for ALDH activity. Concurrent inhibition of DNA methyltransferases (DNMTs) and NAMPT synergistically abrogated the platinum-induced increase in OCSCs. Combining pharmacological inhibitors of DNMT and NAMPT with carboplatin reduced tumorigenesis and OCSC percentage in vivo. We conclude that both epigenetic and metabolic alterations lead to platinum induced OCSC enrichment, providing preclinical evidence that in the neoadjuvant setting, combining DNMT and NAMPT inhibitors with platinum has the potential to reduce OC recurrence and avert the development of platinum resistance.


title: Combined epigenetic and metabolic inhibition blocks platinum-induced ovarian cancer stem cell enrichment


title: Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

abstract: Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC-specific targeted therapies exist. In this study we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC versus non-IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non-IBC cell lines. High expression was associated with poor-prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced proliferation, colony formation, migration, and cancer stem cell populations in vitro, and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2-silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.


title: Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background

abstract: The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between proximal (cis) or distal (trans) elements affect the behaviour of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5 UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.

category: synthetic biology


title: Curtobacterium glycinis sp. nov. from Glycine max, Curtobacterium gossypii sp. nov. from Gossypium hirsutum and Curtobacterium oryzae sp. nov. from Oryza sativa, three new Curtobacterium species and endophytes from agricultural crops

abstract: Three new Curtobacterium species from healthy tissues of agricultural crop plants in the United States are reported. They are Curtobacterium glycinis sp. nov. from soybean in Missouri, Curtobacterium gossypii from cotton in Puerto Rico and corn in Missouri, and Curtobacterium oryzae sp. nov. from rice in Texas.


title: Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammation in vivo and more rapid induction of pro-inflammatory responses in primary bursal cells stimulated ex vivo.

abstract: In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.


title: The SAGA and NuA4 component Tra1 regulates Candida albicans drug resistance and pathogenesis

abstract:
Candida albicans is the most common cause of death from fungal infections. Emergence of resistant strains reducing the efficacy of first line therapy with echinocandins such as caspofungin calls for the identification of alternative therapeutic strategies. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase domain. In Saccharomyces cerevisiae, the assembly and function of SAGA and NuA4 is compromised by a version of Tra1 (Tra1Q3) with three arginine residues in the putative ATP-binding cleft changed to glutamine, Whole transcriptome analysis of the S. cerevisiae tra1Q3 strain highlights Tra1s role in global transcription, stress response and cell wall integrity. As a result, tra1Q3 increases susceptibility to multiple stressors, including caspofungin. Moreover, the same tra1Q3 allele in the pathogenic yeast Candida albicans causes similar phenotypes, suggesting that Tra1 broadly mediates the antifungal response across yeast species. Transcriptional profiling in C. albicans identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion and filamentous growth. Indeed, the tra1Q3 allele reduces filamentation and other pathogenesis traits in C. albicans. We identified EVP1, which encodes a putative plasma membrane protein, amongst the Tra1-regulated genes, Disrupting EVP1 results in reduced filamentation and infection capacity in C. albicans. Thus,Tra1 emerges as a promising therapeutic target for fungal infections.

ImportanceFungal pathogens such as Candida albicans are important agents of infectious disease, with increasing rates of drug resistance, and limited available antifungal therapeutics. In this study, we characterize the role of C. albicans Tra1, a critical component of acetyltransferase complexes, involved in transcriptional regulation and responses to environmental stress. We find C. albicans genetic mutants with impaired Tra1 function have reduced tolerance to cell-wall targeting stressors, including the clinically-important antifungal caspofungin. We further use RNA-sequencing to profile the global fungal response to the tra1 mutation, and identify a previously uncharacterized C. albicans gene, EVP1. We find that both TRA1 and EVP1 play an important role in phenotypes associated with fungal pathogenesis, including cellular morphogenesis, biofilm formation, and toxicity towards host immune cells. Together, this work describes the key role for Tra1 in regulating fungal drug tolerance and pathogenesis, and positions this protein as a promising therapeutic target for fungal infections.


title: Binding affinity prediction for protein-ligand complex using deep attention mechanism based on intermolecular interactions

abstract:
Accurate prediction of protein-ligand binding affinity is important in that it can lower the overall cost of drug discovery in structure-based drug design. For more accurate prediction, many classical scoring functions and machine learning-based methods have been developed. However, these techniques tend to have limitations, mainly resulting from a lack of sufficient interactions energy terms to describe complex interactions between proteins and ligands. Recent deep-learning techniques show strong potential to solve this problem, but the search for more efficient and appropriate deep-learning architectures and methods to represent protein-ligand complexes continues. In this study, we proposed a deep-neural network for more accurate prediction of protein-ligand complex binding affinity. The proposed model has two important features, descriptor embeddings that contains embedded information about the local structures of a protein-ligand complex and an attention mechanism for highlighting important descriptors to binding affinity prediction. The proposed model showed better performance on most benchmark datasets than existing binding affinity prediction models. Moreover, we confirmed that an attention mechanism was able to capture binding sites in a protein-ligand complex and that it contributed to improvement in predictive performance. Our code is available at https://github.com/Blue1993/BAPA.

Author summaryThe initial step in drug discovery is to identify drug candidates for a target protein using a scoring function. Existing scoring functions, however, lack the ability to accurately predict the binding affinity of protein-ligand complexes. In this study, we proposed a deep learning-based approach to extract patterns from the local structures of protein-ligand complexes and to highlight the important local structures via an attention mechanism. The proposed model showed good performance for various benchmark datasets compared to existing models.

category: bioinformatics


title: DGCyTOF:  deep learning with graphic cluster visualization to predict cell types of single cell mass cytometry data

abstract:
BackgroundSingle-cell mass cytometry, also known as cytometry by time of flight (CyTOF) is a powerful high-throughput technology that allows analysis of up to 50 protein markers per cell for the quantification and classification of single cells. Traditional manual gating utilized to identify new cell populations has been inadequate, inefficient, unreliable, and difficult to use, and no algorithms to identify both calibration and new cell populations has been well established.

MethodsA deep learning with graphic cluster (DGCyTOF) visualization is developed as a new integrated embedding visualization approach in identifying canonical and new cell types. The DGCyTOF combines deep-learning classification and hierarchical stable-clustering methods to sequentially build a tri-layer construct for known cell types and the identification of new cell types. First, deep classification learning is constructed to distinguish calibration cell populations from all cells by softmax classification assignment under a probability threshold, and graph embedding clustering is then used to identify new cell populations sequentially. In the middle of two-layer, cell labels are automatically adjusted between new and unknown cell populations via a feedback loop using an iteration calibration system to reduce the rate of error in the identification of cell types, and a 3-dimensional (3D) visualization platform is finally developed to display the cell clusters with all cell-population types annotated.

ResultsUtilizing two benchmark CyTOF databases comprising up to 43 million cells, we compared accuracy and speed in the identification of cell types among DGCyTOF, DeepCyTOF, and other technologies including dimension reduction with clustering, including Principal Component Analysis (PCA), Factor Analysis (FA), Independent Component Analysis (ICA), Isometric Feature Mapping (Isomap), t-distributed Stochastic Neighbor Embedding (t-SNE), and Uniform Manifold Approximation and Projection (UMAP) with k-means clustering and Gaussian mixture clustering. We observed the DGCyTOF represents a robust complete learning system with high accuracy, speed and visualization by eight measurement criteria. The DGCyTOF displayed F-scores of 0.9921 for CyTOF1 and 0.9992 for CyTOF2 datasets, whereas those scores were only 0.507 and 0.529 for the t-SNE+k-means; 0.565 and 0.59, for UMAP+ k-means. Comparison of DGCyTOF with t-SNE and UMAP visualization in accuracy demonstrated its approximately 35% superiority in predicting cell types. In addition, observation of cell-population distribution was more intuitive in the 3D visualization in DGCyTOF than t-SNE and UMAP visualization.

ConclusionsThe DGCyTOF model can automatically assign known labels to single cells with high accuracy using deep-learning classification assembling with traditional graph-clustering and dimension-reduction strategies. Guided by a calibration system, the model seeks optimal accuracy balance among calibration cell populations and unknown cell types, yielding a complete and robust learning system that is highly accurate in the identification of cell populations compared to results using other methods in the analysis of single-cell CyTOF data. Application of the DGCyTOF method to identify cell populations could be extended to the analysis of single-cell RNASeq data and other omics data.

Availability and implementationA Python package (Python 3) and analysis scripts for reproducing the results are available at https://lijcheng12.github.io/DGCyTOF/.

DGCyTOF python package contactLijun.cheng@osu.edu.

Supplementary informationSupplementary data are available at journal **** online.

Key pointsO_LIDGCyTOF has been developed for the automatic identification of known and new cell populations.
C_LIO_LIOptimal dimension reduction that integrates graphic-clustering technology facilitates highly accurate identification of new cell populations.
C_LIO_LIUse of a feedback-calibration system can enhance the homology of cell types for the identification of cell populations.
C_LIO_LIThree-dimensional visualization technology allows vivid depiction of cell types to improve their classification.
C_LI


title: The N-terminal domain of RfaH plays an active role in protein fold-switching

abstract:
The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a {beta}-barrel while RfaH CTD is forming an -hairpin blocking such site. Upon recognition of the ops exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a {beta}-barrel structurally identical to NusG CTD.

Although RfaH transformation has been extensively characterized computationally, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards -folded CTD, whereas the NTD interferes with {beta}CTD folding, becoming trapped in a {beta}-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the -folded RfaH by specifically orienting the CTD upon interdomain binding and also by favoring {beta}-barrel rupture into an intermediate from which fold-switching proceeds.


title: Testing in vitro toxicity of nanoparticles in 3D cell culture with various extracellular matrix scaffold

abstract: Nanomaterials are used in a variety of fields and toxicity assessment is paramount for their development and application. Although most toxicity assessments have been performed in 2D (2-Dimensional) cell culture, the inability to adequately replicate the in vivo environment and toxicity is a limitation. To overcome the limitation, a 3D (3-Dimensional) cell culture method has been developed to make an environment closer to an in vivo system. In this study, 20 nm SiO2 nanoparticles were dispersed in serum-containing (SC) and serum-free (SF) media to compare 2D cell culture and 3D cell culture toxicity. The cells were subjected to a 3D cell culture method in which HepG2, a human-derived liver cancer cell line, was mixed on a scaffold. We found that nanoparticles induced toxicity in 2D cell culture, but toxicity was not observed in 3D cell culture similar to in vivo environment. However, differences in toxicity were observed between the three types of scaffolds in the absence of serum as the number of cells decreased.

category: pharmacology and toxicology


title: Brain Water as a Function of Age and Weight in Normal Rats

abstract: Rats are frequently used to study water content of normal and injured brain, as well as changes in response to various osmotherapeutic regimens. Magnetic resonance imaging in humans has shown that brain water content declines with age as a result of progressive myelination. The purpose of this study was to quantify changes in brain water content during rat development and aging. Brain water content was measured by standard techniques in 129 normal male Sprague-Dawley rats that ranged in age (weight) from 13 to 149 days (18 to 759 g). Overall, the results demonstrated a decrease from 85.59% to 76.56% water content with increasing age (weight). Nonlinear allometric functions relating brain water to age and weight were determined. These findings provide age-related context for prior rat studies of brain water, emphasize the importance of using similarly aged controls in studies of brain water, and indicate that age-related changes in brain water content are not specific to humans.

category: physiology


title: Personalized Closed-Loop Brain Stimulation for Effective Neurointervention Across Participants

abstract: Accumulating evidence from human-based research has highlighted that the prevalent one-size-fits-all approach for neural and behavioral interventions is inefficient. This approach can benefit one individual, but be ineffective or even detrimental for another. Studying the efficacy of the large range of different parameters for different individuals is costly, time-consuming and requires a large sample size that makes such research impractical and hinders effective interventions. Here an active machine learning technique is presented across participants--personalized Bayesian optimization (pBO)--that searches available parameter combinations to optimize an intervention as a function of an individuals ability. This novel technique was utilized to identify transcranial alternating current stimulation frequency and current strength combinations most likely to improve arithmetic performance, based on a subjects baseline arithmetic abilities. The pBO was performed across all subjects tested, building a model of subject performance, capable of recommending parameters for future subjects based on their baseline arithmetic ability. pBO successfully searches, learns, and recommends parameters for an effective neurointervention as supported by behavioral, stimulation, and neural data. The application of pBO in human-based research opens up new avenues for personalized and more effective interventions, as well as discoveries of protocols for treatment and translation to other clinical and non-clinical domains.


title: Critical behaviour of the stochastic Wilson-Cowan model

abstract:
Spontaneous brain activity is characterized by bursts and avalanche-like dynamics, with scale-free features typical of critical behaviour. The stochastic version of the celebrated Wilson-Cowan model has been widely studied as a system of spiking neurons reproducing non-trivial features of the neural activity, from avalanche dynamics to oscillatory behaviours. However, to what extent such phenomena are related to the presence of a genuine critical point remains elusive. Here we address this central issue, providing analytical results in the linear approximation and extensive numerical analysis. In particular, we present results supporting the existence of a bona fide critical point, where a second-order-like phase transition occurs, characterized by scale-free avalanche dynamics, scaling with the system size and a diverging relaxation time-scale. Moreover, our study shows that the observed critical behaviour falls within the universality class of the mean-field branching process, where the exponents of the avalanche size and duration distributions are, respectively, -3/2 and -2. We also provide an accurate analysis of the system behaviour as a function of the total number of neurons, focusing on the time correlation functions of the firing rate in a wide range of the parameter space.

Author summaryNetworks of spiking neurons are introduced to describe some features of the brain activity, which are characterized by burst events (avalanches) with power-law distributions of size and duration. The observation of this kind of noisy behaviour in a wide variety of real systems led to the hypothesis that neuronal networks work in the proximity of a critical point. This hypothesis is at the core of an intense debate. At variance with previous claims, here we show that a stochastic version of the Wilson-Cowan model presents a phenomenology in agreement with the existence of a bona fide critical point for a particular choice of the relative synaptic weight between excitatory and inhibitory neurons. The system behaviour at this point shows all features typical of criticality, such as diverging timescales, scaling with the system size and scale-free distributions of avalanche sizes and durations, with exponents corresponding to the mean-field branching process. Our analysis unveils the critical nature of the observed behaviours.


title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

abstract:
The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. This actin remodeling event exhibits relatively rapid dynamics that, through quantitative live-cell imaging, was revealed to consist of three phases - a fast recruitment phase which abruptly transitions to a fast turnover phase before resolving into a slow turnover of actin that indicates the end of actin remodeling. Here, we investigate Chlamydia invasion in the context of actin dynamics. Efficient invasion is associated with robust actin remodeling kinetics that results from a collaborative functional interaction between two different classes of actin nucleators - formins, including formin 1 and the diaphanous-related formins mDia1 and mDia2, and the Arp2/3 complex. Recruitment of these nucleators requires the presence of the chlamydial type III effector TarP, which enables the respective nucleating activities of formin and Arp2/3 to collaboratively generate a robust actin network. A collaborative model is supported by the observation that co-inhibition of Fmm1 and Arp2/3 further reduced both actin dynamics and invasion efficiency than either treatment alone. Furthermore, inhibition of recruitment of Fmn1 and/or Arp2/3 by deleting TarP was sufficient to similarly attenuated actin kinetics and invasion efficiency, supporting a model wherein TarP is the major contributor to robust actin remodeling via its recruitment of the two classes of actin nucleators. At the population level, the kinetics of recruitment and turnover of actin and its nucleators were linked. However, a more detailed analysis of the data at the level of individual elementary bodies showed significant variation and a lack of correlation between the kinetics of recruitment and turnover, suggesting that accessory factors variably modify actin kinetics at individual entry sites. In summary, efficient chlamydial invasion requires a specific profile of actin dynamics which are coordinated by TarP-dependent recruitment of two classes of actin nucleators.

Author SummaryThe obligate intracellular pathogen Chlamydia trachomatis relies upon manipulation of the host actin cytoskeleton to drive its entry into host cells, such that impairment of actin dynamics attenuates Chlamydia invasion. Collaboration between two classes of actin nucleators, formin and Arp2/3, are known to enhance actin recruitment and turnover; we found that recruitment of both proteins to the signaling complex established by the type III secreted effector, TarP, was important for pathogen internalization. Furthermore, Formin 1 and Arp2/3 are co-recruited to sites of entry, and pharmacological inhibition of either actin nucleator impaired recruitment of the other, indicating a functional cooperation between branched and filamentous actin nucleation within pathogen entry sites. Disruption of this cooperation negatively impacted both actin dynamics and Chlamydia internalization, indicating that TarP-dependent entry of Chlamydia into non-phagocytic cells operates through the recruitment and activation of Arp2/3 and Formin 1. Finally, kinetic analysis of actin recruitment and turnover revealed that these processes were independently regulated, in addition to implicating the presence of local factors that fine-tune actin dynamics and subsequent invasion.


title: Systematic review of Plasmodium falciparum and Plasmodium vivax polyclonal infections: Impact of prevalence, study population characteristics, and laboratory procedures

abstract: Multiple infections of genetically distinct clones of the same Plasmodium species are common in many malaria endemic settings. Mean multiplicity of infection (MOI) and the proportion of polyclonal infections are often reported as surrogate marker of transmission intensity, yet the relationship with traditional measures such as parasite prevalence is not well understood. We have searched Pubmed for articles on P. falciparum and P. vivax multiplicity, and compared the proportion of polyclonal infections and mean MOI to population prevalence. The impact of the genotyping method, number of genotyping markers, method for diagnosis (microscopy/RDT vs. PCR), presence of clinical symptoms, age, geographic region, and year of sample collection on multiplicity indices were assessed. For P. falciparum, 153 studies met inclusion criteria, yielding 275 individual data points and 33526 genotyped individuals. The proportion of polyclonal infections ranged from 0-96%, and mean MOI from 1-6.1. For P. vivax, 54 studies met inclusion criteria, yielding 115 data points and 13325 genotyped individuals. The proportion of polyclonal infections ranged from 0-100%, and mean MOI from 1-3.8. For both species, the proportion of polyclonal infections ranged from very low to close to 100% at low prevalence, while at high prevalence it was always high. Each percentage point increase in prevalence resulted in a 0.34% increase in the proportion of polyclonal P. falciparum infections (P<0.001), and a 0.78% increase in the proportion of polyclonal P. vivax infections (P<0.001). In multivariable analysis, higher prevalence, typing multiple markers, diagnosis of infections by PCR, and sampling in Africa were found to result in a higher proportion of P. falciparum polyclonal infections. For P. vivax, prevalence, year of study, typing multiple markers, and geographic region were significant predictors. In conclusion, polyclonal infections are frequently present in all settings, but the association between multiplicity and prevalence is weak.


title: Infection of immune competent macrophages expressing functional Slc11a1 alters global gene expression, regulation of metal ions, and infection outcomes

abstract:
Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron availability, increasing exposure to bactericidal copper, and manipulating zinc to restrict the growth of pathogens. Manipulation of zinc at the host-pathogen interface depends on both the pathogens identity and the nature of the host cell. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing our results to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response, with 129S6 macrophages transitioning from M1 to M2 polarization over the course of infection and showing signs of recovery. Our results reveal that functional Slc11a1 profoundly affects the transcriptional landscape upon infection. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogens access to zinc while simultaneously ensuring sufficient zinc to support the immune response.

Author summaryMetal ions play an important role in influencing how immune cells such as macrophages respond to infection by pathogens. Because metal ions are both essential to survival, as well toxic when present is excessive amounts, the host and the pathogen have evolved diverse strategies to regulate metal acquisition and availability. Here, we show that the metal transporter slc11a1 plays a critical role in defining the host response to Salmonella infection. Infection causes widespread changes in expression of metal regulatory genes to limit the pathogens access to iron, increase its exposure to copper, and remodel zinc to ensure increased zinc in the cytosol and limited zinc for the pathogen. Macrophages expressing functional slc11a1 have a different profile of metal regulation and vastly different outcomes compared to immune compromised macrophage, demonstrating significantly different nutritional immune responses in immune competent versus immune compromised macrophages.