title: The skeletal muscle circadian clock regulates sarcomere length homeostasis through titin splicing

abstract: Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

category: physiology


title: The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20


title: A comprehensive mass spectral library for human thyroid tissues

abstract: Thyroid nodules occur in about 60% of the population. Current diagnostic strategies, however, often fail at distinguishing malignant nodules before surgery, thus leading to unnecessary, invasive treatments. As proteins are involved in all physio/pathological processes, a proteome investigation of biopsied nodules may help correctly classify and identify malignant nodules and discover therapeutic targets. Quantitative mass spectrometry data-independent acquisition (DIA) enables highly reproducible and rapid throughput investigation of proteomes. An exhaustive spectral library of thyroid nodules is essential for DIA yet still unavailable. This study presents a comprehensive thyroid spectral library covering five types of thyroid tissue: multinodular goiter, follicular adenoma, follicular and papillary thyroid carcinoma, and normal thyroid tissue. Our library includes 925,330 transition groups, 157,548 peptide precursors, 121,960 peptides, 9941 protein groups, and 9826 proteins from proteotypic peptides. This library resource was evaluated using three papillary thyroid carcinoma samples and their corresponding adjacent normal thyroid tissue, leading to effective quantification of up to 7863 proteins from biopsy-level thyroid tissues.

category: systems biology


title: Assessment of Genetic Diversity and Population Genetic Structure of Santalum album L. in India by Genic and Genomic SSR markers

abstract: Sandalwood (Santalum album L.) is highly valued aromatic tropical tree. It is known for its high quality heartwood and oil. In this study, 39 genic and genomic SSR markers were used to analyze the genetic diversity and population structure of 177 S. album accessions from 14 populations of three states in India. High genetic diversity was observed in terms of number of alleles 127, expected heterozygosity (He) ranged from 0.63-0.87 and the average PIC was 0.85. The selected population had relatively high genetic diversity with Shannons information index (I) >1.0. 0.02 mean coefficient of genetic differentiation (FST) and 10.55 gene flow were observed. AMOVA revealed that 92% of the variation observed within individuals. Based on cluster and Structure result, individuals were not clustered as per their geographical origin. Furthermore the clusters were clearly distinguished by principal component analysis analysis and the result revealed that PC1 reflected the moderate contribution in genetic variation (6%) followed by PC2 (5.5%). From this study, high genetic diversity and genetic differentiation was found in S. album populations. The genetic diversity information of S. album populations can be used for selection of superior genotypes and germplasm conservation to promote the tree improvement of S. album populations.

category: molecular biology


title: Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cells in vitro

abstract: Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we describe the 3Cpro effect on human cells in vitro and demonstrate that the enzyme induces ferroptosis which is mediated by iron ions and cause oxidative stress in the cells. This is the first demonstration that proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

category: cell biology


title: Novel Generic Models for Differentiating Stem Cells Reveal Oscillatory Mechanisms

abstract: Understanding cell fate selection remains a central challenge in developmental biology. We present a class of simple yet biologically-motivated mathematical models for cell differentiation that generically generate oscillations and hence suggest alternatives to the standard framework based on Waddingtons epigenetic landscape. The models allow us to suggest two generic dynamical scenarios that describe the differentiation process. In the first scenario gradual variation of a single control parameter is responsible for both entering and exiting the oscillatory regime. In the second scenario two control parameters vary: one responsible for entering, and the other for exiting the oscillatory regime. We analyse the standard repressilator and four variants of it and show the dynamical behaviours associated with each scenario. We present a thorough analysis of the associated bifurcations and argue that gene regulatory networks with these repressilator-like characteristics are promising candidates to describe cell fate selection through an oscillatory process.

category: developmental biology


title: Ecological network analysis of watershed meta-ecosystems: A new perspective on quantifying the integrated watershed ecosystem

abstract: A watershed is an integrated ecosystem. In different disciplines, a watershed has been described as a geomorphic unit, a hydrological unit, an ecological unit, or a socio-economic unit and has been quantitatively described using different indicator systems. Until now, no general indicator system has existed that could quantitatively describe the geomorphic features, hydrologic features, ecological features and socio-economic features of an integrated watershed ecosystem (IWE) simultaneously. Here, we proposed a quantitative descriptive framework for an IWE (QDFIWE). This QDFIWE involved three steps: (1) constructing a watershed meta-ecosystem (WME) based on the hierarchical system of the watershed; (2) constructing flow networks based on the WME; and (3) identifying the holistic properties (such as spatial throughput, spatial organization and spatial resilience) of the WME through analyzing its flow networks based on ecological network analysis (ENA). Then, we applied this method to study the geomorphic topological structure, geomorphic spatial structure, natural water resource system and integrated water resource system of the Yangtze River basin. The results suggested that based on the QDFIWE, (1) one could construct different WMEs and corresponding flow networks for different requirements; (2) one could obtain time series of the holistic properties of an IWE to analyze its evolution; (3) one could compare, classify and cluster any number of IWEs through identifying their holistic properties according to similar strategies; and (4) one could determine or create more indicators, which could provide additional information, based on the holistic properties of an IWE. This study demonstrates that the QDFIWE is a general method of quantifying the holistic properties of all subsystems of an IWE simultaneously. Thus, the method can improve the understanding of the IWE.

category: ecology


title: Individual growth models support the quantification of isotope incorporation rate, trophic discrimination and their interactions

abstract: Two large but independent bodies of literature exist on two essential components of the dynamics of isotopic incorporation: the isotopic incorporation rate ({lambda}) and the trophic discrimination factor ({Delta}). Understanding the magnitude of these two parameters and the factors that shape them is fundamental to interpret the results of ecological studies that rely on stable isotopes. {lambda} scales allometrically with body mass among species and depends on growth within species. Both are often assumed to be constant and independent of each other but evidence accumulates that might be linked and to vary with growth. We built and analyzed a model (IsoDyn) that connects individual growth and isotopic incorporation of nitrogen into whole body and muscle tissues. The model can assume a variety of individual growth patterns including exponential or asymptotic growths. {lambda} depends on the rate of body mass gains which scales allometrically with body mass. {Delta} is a dynamic response variable that depends partly on the ratio between fluxes of gains and losses and covaries negatively with {lambda}. The model can be parameterized either from existing large databases of animal growth models or directly from experimental results. The model was applied to experimental results on three ectotherms and one endotherm and compared to the results of the simpler and widely used time model. IsoDyn model gave a better fit with relatively little calibration. IsoDyn clarifies and expands the interpretation of isotopic incorporation data.


title: Metapopulations with habitat modification

abstract: Across the tree of life, organisms modify their local environment, rendering it more or less hospitable for other species. Despite the ubiquity of these processes, simple models that can be used to develop intuitions about the consequences of widespread habitat modification are lacking. Here we extend the classic Levins metapopulation model to a setting where each of n species can colonize patches connected by dispersal, and when patches are vacated via local extinction, they retain a "memory" of the previous occupant--modeling habitat modification. While this model can exhibit a wide range of dynamics, we draw several overarching conclusions about the effects of modification and memory. In particular, we find that any number of species may potentially coexist, provided that each is at a disadvantage when colonizing patches vacated by a conspecific. This notion is made precise through a quantitative stability condition, which provides a way to unify and formalize existing conceptual models. We also show that when patch memory facilitates coexistence, it generically induces a positive relationship between diversity and robustness (tolerance of disturbance). Our simple model provides a portable, tractable framework for studying systems where species modify and react to a shared landscape.


title: Larger is not better: No mate preference by European Common Frog (Rana temporaria) males

abstract: According to classical sexual selection theory, females are the choosy sex in most species. Choosiness is defined as the individual effort to invest energy and time to assess potential mates. In explosive breeding anurans, high intrasexual competition between males leads to a sexual coercion ruled mating system, where males could have evolved preferences for specific female traits. In the current study, we tested male mating preference in the explosive breeding European Common Frog without intrasexual competition. We hypothesized that males show preferences towards larger female body size in the absence of male competition. We conducted mate choice experiments, placing a male and two differently sized females in a box and recorded their mating behavior. Males did not show any preference considering female body size, neither in the attempt to grab a female nor during the formation of pairs. We witnessed a high failure rate of male mating attempts, which might make the evolution of mate choice too costly. However, small males are faster in attempting females, which could be an alternative strategy to get access to females, because their larger competitors have an advantage during scramble competition. Nonetheless, in successfully formed pairs, the females were on average larger than the males, an observation which deviated from our null-model where pairs should be of similar size if mating would be random. This indicates that selection takes place, independent from male mating preference or scramble competition.

category: animal behavior and cognition


title: Pro-regenerative Extracellular Matrix Hydrogel Prevents and Mitigates Pathological Alterations of Pelvic Muscles Following Birth Injury

abstract: Pelvic floor disorders, which include pelvic organ prolapse, and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Even though childbirth, specifically vaginal delivery, has been long recognized as the most important potentially modifiable risk factor for PFM injury, the precise mechanisms of PFM dysfunction following childbirth remain elusive. In this study we demonstrate that PFMs undergo atrophy and severe fibrosis in parous women with symptomatic pelvic organ prolapse compared to age-matched nulliparous cadaveric donors without history of pelvic floor disorders. These pathological alterations are recapitulated in the pre-clinical rat model of simulated birth injury. The transcriptional signature of PFMs post-injury demonstrates a sustained inflammatory response, impairment in muscle anabolism, and persistent expression of extracellular matrix (ECM) remodeling genes. Next, we evaluated the administration of acellular injectable skeletal muscle extracellular matrix hydrogel for the prevention and mitigation of these pathological alterations. Treatment of PFMs with the biomaterial either at the time of birth injury or 4 weeks post-injury reduced muscle atrophy and mitigated fibrotic degeneration. By evaluating gene expression, we demonstrate that these changes are mainly driven by the hydrogel-induced modulation of the immune response and intramuscular fibrosis, as well as enhancement of the endogenous myogenesis. This work furthers our understanding of PFM birth injury and demonstrates proof-of-concept for a new pragmatic pro-regenerative biomaterial approach for treating injured PFMs.

category: bioengineering


title: Noise Reconstruction & Removal Network: A New Architecture to Denoise FIB-SEM Images

abstract:
Recent advances in Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) allows the imaging and analysis of cellular ultrastructure at nanoscale resolution, but the collection of labels and/or noise-free data sets has several challenges, often immutable. Reasons range from time consuming manual annotations, requiring highly trained specialists, to introducing imaging artifacts from the prolonged scanning during acquisition. We propose a fully unsupervised Noise Reconstruction and Removal Network for denoising scanning electron microscopy images.

The architecture, inspired by gated recurrent units, reconstructs and removes the noise by synthesizing the sequential data. At the same time the fully unsupervised training guides the network in distinguishing true signal from noise and gives comparable results to supervised architectures. We demonstrate that this new network specialized on 3D electron microscopy data sets, achieves comparable and even better results than supervised networks.


title: Dysgu: efficient structural variant calling using short or long reads

abstract: Structural variation (SV) plays a fundamental role in genome evolution and can underlie inherited or acquired diseases such as cancer. Long-read sequencing technologies have led to improvements in the characterization of structural variants (SVs), although paired-end sequencing offers better scalability. Here, we present dysgu, which calls SVs or indels using paired-end or long reads. Dysgu detects signals from alignment gaps, discordant and supplementary mappings, and generates consensus contigs, before classifying events using machine learning. Additional SVs are identified by remapping of anomalous sequences. Dysgu outperforms existing state-of-the-art tools using paired-end or long-reads, offering high sensitivity and precision whilst being among the fastest tools to run. We find that combining low coverage paired-end and long-reads is competitive in terms of performance with long-reads at higher coverage values.

category: bioinformatics


title: rPanglaoDB: an R package to download and merge labeled single-cell RNA-seq data from the PanglaoDB database

abstract:
MotivationCharacterizing cells with rare molecular phenotypes is one of the promises of high throughput single-cell RNA sequencing (scRNA-seq) techniques. However, collecting enough cells with the desired molecular phenotype in a single experiment is challenging, requiring several samples preprocessing steps to filter and collect the desired cells experimentally before sequencing. Data integration of multiple public single-cell experiments stands as a solution for this problem, allowing the collection of enough cells exhibiting the desired molecular signatures. By increasing the sample size of the desired cell type, this approach enables a robust cell type transcriptome characterization.

ResultsHere, we introduce rPanglaoDB, an R package to download and merge the uniformly processed and annotated scRNA-seq data provided by the PanglaoDB database. To show the potential of rPanglaoDB for collecting rare cell types by integrating multiple public datasets, we present a biological application collecting and characterizing a set of 157 fibrocytes. Fibrocytes are a rare monocyte-derived cell type, that exhibits both the inflammatory features of macrophages and the tissue remodeling properties of fibroblasts. This constitutes the first fibrocytes unbiased transcriptome profile report. We compared the transcriptomic profile of the fibrocytes against the fibroblasts collected from the same tissue samples and confirm their associated relationship with healing processes in tissue damage and infection through the activation of the prostaglandin biosynthesis and regulation pathway.

Availability and ImplementationrPanglaoDB is implemented as an R package available through the CRAN repositories https://CRAN.R-project.org/package=rPanglaoDB.

Contactdaniecos@uio.no

Supplementary informationCode to replicate the case example and figure 1 is available at https://github.com/dosorio/rPanglaoDB

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/446161v1_fig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b4e67forg.highwire.dtl.DTLVardef@88a46aorg.highwire.dtl.DTLVardef@e25681org.highwire.dtl.DTLVardef@19d5bb7_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO Characterization of the fibrocytes transcriptome. (A) Identification of cells expressing marker genes that differentiate fibrocytes identity from macrophages and fibroblasts (CD34, ACTA2, FN1, Collagen V, FAP and SIRPA). (B) Cross validation of the identified cells expressing CD34+, ACTA2+, COL5A1+, COL5A2+, COL5A3+, FN1+, FAP+, SIRPA+, PTPRC+, MME+, and SEMA7A+ by kernel density estimation through the Nebulosa package. (C) Volcano plot displaying the differential expression between fibrocytes and the fibroblasts collected in the same merged samples. (D) Enrichment of the prostaglandin biosynthesis and regulation pathway using GSEA through the fgsea package. (E) Enrichment of the prostaglandin biosynthesis and regulation pathway using ssGSEA through the GSVA package.

C_FIG


title: Structure Learning for Hierarchical Regulatory Networks

abstract: Network analysis offers a powerful technique to model the relationships between genes within biological regulatory networks. Inference of biological network structures is often performed on high-dimensional data, yet is hindered by the limited sample size of high throughput "omics" data typically available. To overcome this challenge, we exploit known organizing principles of biological networks that are sparse, modular, and likely share a large portion of their underlying architecture. We present SHINE - Structure Learning for Hierarchical Networks - a framework for defining data-driven structural constraints and incorporating a shared learning paradigm for efficiently learning multiple networks from high-dimensional data. We show through simulations SHINE improves performance when relatively few samples are available and multiple networks are desired, by reducing the complexity of the graphical search space and by taking advantage of shared structural information. We evaluated SHINE on TCGA Pan-Cancer data and found learned tumor-specific networks exhibit expected graph properties of real biological networks, recapture previously validated interactions, and recapitulate findings in literature. Application of SHINE to the analysis of subtype-specific breast cancer networks identified key genes and biological processes for tumor maintenance and survival as well as potential therapeutic targets for modulating known breast cancer disease genes.


title: Circulatory systems and mortality rates

abstract: Aging is a complex process involving multiple factors and subcellular processes, ultimately leading to the death of an organism. The microscopic processes that cause aging are relatively well understood and effective macroscopic theories help explain the universality of aging in complex systems. However, these theories fail to explain the diversity of aging observed for various lifeforms. As such, more complete "mesoscopic" theories of aging are needed, combining the biophysical details of microscopic failure and the macroscopic structure of complex systems. Here we explore two models: (1) a network theoretic model, and (2) a convection diffusion model emphasizing the biophysical details of communicated signals. The first model allows us to explore the effects of connectivity structures on aging. In our second model, cells interact through cooperative and antagonistic factors. We find by varying the ratio at which these factors affect cell death, as well as the reaction kinetics, diffusive and flow parameters, we obtain a wide diversity of mortality curves. As such, the connectivity structures as well as the biophysical details of how various factors are transported in an organism may explain the diversity of aging observed across different lifeforms.

category: biophysics


title: An Efficient Method to Quantify Structural Distributions in Heterogeneous cryo-EM Datasets

abstract: Cryogenic Electron Microscopy (cryo-EM) preserves the ensemble of protein conformations in solution and thus provide a promising way to characterize conformational changes underlying protein functions. However, it remains challenging for existing software to elucidate distributions of multiple conformations from a heterogeneous cryo-EM dataset. We developed a new algorithm: Linear Combinations of Template Conformations (LCTC) to obtain distributions of multiple conformations from cryo-EM datasets. LCTC assigns 2D images to the template 3D structures obtained by Multi-body Re-finement of RELION via a novel two-stage matching algorithm. Specifically, an initial rapid assignment of experimental 2D images to template 2D images was applied based on auto-correlation functions of image contours that can efficiently remove the majority of irrelevant 2D images. This is followed by pixel-pixel matching of images with fewer number of 2D images, which can accurately assign the 2D images to the template images. We validate the LCTC method by demonstrating that it can accurately reproduce the distributions of 3 Thermus aquaticus (Taq) RNA polymerase (RNAP) structures with different degrees of clamp opening from a simulated cryo-EM dataset, in which the correct distributions are known. For this dataset, we also show that LCTC greatly outperforms clustering-based Manifold Embedding and Maximum Likelihood-based Multi-body Re-finement algorithms in terms of reproducing the structural distributions. Lastly, we also successfully applied LCTC to reveal the populations of various clamp-opening conformations from an experimental Escherichia coli RNAP cryo-EM dataset. Source code is available at https://github.com/ghl1995/LCTC.


title: Robust Segmentation of Cellular Ultrastructure on Sparsely Labeled 3D Electron Microscopy Images using Deep Learning

abstract: A deeper understanding of the cellular and subcellular organization of tumor cells and their interactions with the tumor microenvironment will shed light on how cancer evolves and guide effective therapy choices. Electron microscopy (EM) images can provide detailed view of the cellular ultrastructure and are being generated at an ever-increasing rate. However, the bottleneck in their analysis is the delineation of the cellular structures to enable interpretable rendering. We have mitigated this limitation by using deep learning, specifically, the ResUNet architecture, to segment cells and subcellular ultrastructure. Our initial prototype focuses on segmenting nuclei and nucleoli in 3D FIB-SEM images of tumor biopsies obtained from patients with metastatic breast and pancreatic cancers. Trained with sparse manual labels, our method results in accurate segmentation of nuclei and nucleoli with best Dice score of 0.99 and 0.98 respectively. This method can be extended to other cellular structures, enabling deeper analysis of inter- and intracellular state and interactions.

category: cancer biology


title: Multi-dimensional analyses identify genes of high priority for pancreatic cancer research

abstract:
Genomic profiling has unveiled the molecular subtypes and mutational landscape of pancreatic ductal adenocarcinoma (PDAC). However, there is a knowledge gap on the consistency of gene expression across PDAC tumors profiled in independent studies and this limits follow up research. To facilitate novel drug target prioritization and biomarker discovery, we investigated the most consistently expressed genes in human PDAC. We identified ~4,000 genes highly or lowly expressed in at least 4 of 5 microarrays (adjusted P<0.05) and validated their expression pattern in additional datasets, bulk tumor and single-cell RNA sequencing samples. Over 50% of the genes were previously uncharacterized in PDAC; many correlated with proliferation, metastasis, mutation, tumor grade, and ~41% predicted overall survival. We identified 185 high-priority targets (notably in cell cycle and glycolysis) whose inhibition suppressed PDAC cell viability in multiple RNA interference datasets and these genes predicted treatment in mouse models. Our results represent important milestone in the quest for mechanisms, drug targets and biomarkers in PDAC, and originate from an adaptable analytical concept that can aid discovery in other cancers.

HighlightsO_LIIdentifies ~4,000 consistent genes across PDAC microarrays, >50% of which have not been studied
C_LIO_LIGlycolysis and cell cycle are the most consistent processes in PDAC
C_LIO_LIHeterogeneous pathways underlie or correlate with clinicopathological variables
C_LIO_LIIdentifies 205 genes with similar expression pattern in PDAC tissues and peripheral blood
C_LIO_LIHighlights 185 upregulated genes that are high priority therapeutic targets in PDAC
C_LI


title: Activating P2Y1 receptors improves function in arteries with repressed autophagy

abstract:
ObjectiveThe importance of endothelial cell (EC) autophagy to vascular homeostasis is evolving. Earlier we reported that purinergic 2Y1 receptor (P2Y1-R) activation rejuvenates shearstress induced nitric oxide (NO) generation in bovine aortic endothelial cells that is otherwise compromised after pharmacological and genetic autophagy repression. Here we determined the translational and functional relevance of these findings.

Approach and ResultsFirst we assessed translational relevance using older humans and mice that exhibit blunted EC autophagy at rest together with impaired arterial function vs. appropriate controls. Rhythmic handgrip exercise elevated radial artery shear rate similarly in adult and older males for 60-min. Compared to baseline, autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs from adult but not older subjects. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older mice in response to 60-min treadmill-running. Next we questioned whether an inability to initiate EC autophagy precipitates arterial dysfunction. Age-associated reductions in intraluminal flow-mediated vasodilation observed in older vs. adult mice were recapitulated in arteries from adult mice by : (i) NO synthase inhibition; (ii) autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) P2Y1-R blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy.

ConclusionsArterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.


title: Late-in-life treadmill-training rejuvenates autophagy, protein aggregate clearance, and function in mouse hearts.

abstract: ABSTRACT AND KEYWORDSThere is evidence for a progressive decline of protein quality control mechanisms during the process of cardiac aging. This enables the accumulation of protein aggregates and damaged organelles that contribute to age-associated cardiac dysfunction. Macroautophagy (referred to as autophagy) is the process by which post-mitotic cells such as cardiomyocytes clear defective proteins and organelles. We hypothesized that late-in-life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs adult mice. As expected, 24-month old male C57BL/6J mice (old) exhibited : (i) repressed autophagosome formation and protein aggregate accumulation in the heart; (ii) systolic and diastolic dysfunction; and (iii) reduced exercise capacity, vs. 8-month old (adult) mice (all p< .05). Separate cohorts of 21 month old mice completed a 3-month progressive resistance treadmill-running program (old-ETR) that improved (all < .05) : (i) body composition; (ii) exercise capacity; and (iii) soleus muscle citrate synthase activity, vs. age-matched mice that did not train (old-SED). Importantly, (iv) protein expression of autophagy markers indicated trafficking of the autophagosome to the lysosome increased, (v) protein aggregate clearance improved, and (vi) overall function was enhanced (all p<0.05), in hearts from old-ETR vs. old- SED mice. Dietary maneuvers and pharmacological interventions shown to elevate basal autophagy are reported to mitigate / reverse age-associated cardiac dysfunction. Here we show the first evidence that a physiological intervention initiated late-in-life improves autophagic flux, protein aggregate clearance, and overall function in mouse hearts.


title: Implicit adaptation to mirror-reversal is in the correct coordinate system but the wrong direction

abstract:
Learning in visuomotor adaptation tasks is the result of both explicit and implicit processes. Explicit processes, operationalized as re-aiming an intended movement to a new goal, account for the lions share of learning while implicit processes, operationalized as error-dependent learning that gives rise to aftereffects, appear to be highly constrained. The limitations of implicit learning are highlighted in the mirror-reversal task, where implicit corrections act in opposition to performance. This is surprising given the mirror-reversal task has been viewed as emblematic of implicit learning. One potential confound of these studies is that both explicit and implicit processes were allowed to operate concurrently, which may interact, potentially in opposition. Therefore, we sought to further characterized implicit learning in a mirror-reversal task with a clamp design to isolate implicit learning from explicit strategies. We confirmed that implicit adaptation is in the wrong direction for mirror-reversal and operates as if the perturbation were a rotation, and only showed a moderate attenuation after three days of training. This result raised the question of whether implicit adaptation blindly operates as though perturbations were a rotation. In a separate experiment, which directly compared a mirror-reversal and a rotation, we found that implicit adaptation operates in a proper coordinate system for different perturbations: adaptation to a mirror-reversal and rotational perturbation is more consistent with Cartesian and polar coordinate systems, respectively. It remains an open question why implicit process would be flexible to the coordinate system of a perturbation but continue to be directed inappropriately.

Public Significance StatementPatients with severe amnesia can improve their performance from day to day in mirror-reversal tasks. These findings led, in part, to the codification of explicit and implicit processes in classic theories regarding the taxonomy of memory systems, with motor learning resting firmly in the branch of implicit memory. However, recent evidence has shown that explicit processes also play an important role in motor learning. Whats more, these studies have found that implicit learning doesnt operate in a useful way in the mirror-reversal task. In the present study, we further examine this puzzling behavior of implicit learning in a mirror-reversal task using a design that can isolate implicit processes from explicit strategies. We clearly showed that the implicit system adapts in the wrong direction for a mirror-reversal, acting as if the perturbation were a rotation. Surprisingly, however, we found that although adaptation is in the wrong direction, the implicit system is sensitive to a particular coordinate system. These findings further challenge the flexibility of this implicit adaptation process in motor learning.


title: The outward Shaker channel OsK5.2 is beneficial to the plant salt tolerance through its role in K+ translocation and its control of leaf transpiration

abstract: High soil salinity constitutes a major environmental constraint to crop production worldwide, and the identification of genetic determinants of plant salt tolerance is awaited by breeders. While the leaf K+ to Na+ homeostasis is considered as key parameter of plant salt tolerance, the underlying mechanisms are not fully identified. Especially, the contribution of K+ channels to this homeostasis has been scarcely examined. Here, we show, using a reverse genetics approach, that the outwardly-rectifying K+ channel OsK5.2, involved in K+ translocation to the shoot and K+ release by guard cells for stomatal closure, is a strong determinant of rice salt tolerance. Upon saline treatment, OsK5.2 function in xylem sap K+ load was maintained, and even transiently increased, in roots. OsK5.2 selectively handled K+ in roots and was not involved in xylem sap Na+ load. In shoots, OsK5.2 expression was up-regulated from the onset of the saline treatment, enabling fast reduction of stomatal aperture, decreased transpirational water flow and therefore decreased trans-plant Na+ flux and reduced leaf Na+ accumulation. Thus, the OsK5.2 functions allowed shoot K+ nutrition while minimizing arrival of Na+, and appeared highly beneficial to the leaf K+ to Na+ homeostasis, the avoidance of salt toxicity and plant growth maintaining.

category: plant biology


title: Structure-based Classification of Tauopathies

abstract: Ordered assembly of the tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimers disease (1,2), chronic traumatic encephalopathy (CTE) (3), Picks disease (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from typical and atypical progressive supranuclear palsy (PSP), the most common tauopathy after Alzheimers disease, define a previously unknown, three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT, Types I and II) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The majority of tau filaments from aging-related tau astrogliopathy (ARTAG) also have the AGD fold. Surprisingly, tau protofilament structures from inherited cases with mutations +3/+16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are identical to those from AGD, suggesting that a relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimers disease and primary age-related tauopathy (PART). These structures provide the basis for a classification of tauopathies that also allows identification of new entities, as we show here for a case diagnosed as PSP, but with abundant spherical 4R tau inclusions in limbic and other brain areas. The structures of the tau fold of this new disease (Limbic-predominant Neuronal inclusion body 4R Tauopathy, LNT) were intermediate between those of GGT and PSP.

category: neuroscience


title: Retrosplenial and hippocampal synchrony during retrieval of old memories in macaques

abstract: To understand the neural activity behind recollections of the distant past, we recorded from the hippocampus and retrosplenial cortex of macaques as they retrieved year-old and newly-acquired object-scene associations. Year-old memoranda preferentially evoked retrosplenial sigma oscillations (10-15 Hz), and phase-locking between RSC and eye movements during retrieval. In contrast, gamma-band retrosplenial-hippocampal synchrony was stronger during retrieval of new items. Primate retrosplenial oscillations may therefore guide retrieval of visuospatial events long past.


title: Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis

abstract: Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidisation is unknown. Here we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its biologically unprecedented protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence and the mechanical stiffness of the cell surface, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from a model oxidised protein substrate, bovine serum albumin, and prevented its oxidative aggregation in vitro. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

category: microbiology


title: Rapid evolution of bacterial AB5 toxin B subunits independent of A subunits: sialic acid binding preferences correlate with host range and toxicity

abstract: Cytotoxic A subunits of bacterial AB5 toxins enter the cytosol following B subunit binding to host cell glycans. We report that A subunit phylogeny evolves independently of B subunits and suggest a future B subunit nomenclature based on species name. Phylogenetic analysis of B subunits that bind sialic acids (Sias) with homologous molecules in databases also show poor correlation with phylogeny. These data indicate ongoing lateral gene transfers between species, with mixing of A and B subunits. Some B subunits are not even associated with A subunits e.g., YpeB of Yersinia pestis, the etiologic agent of plague epidemics. Plague cannot be eradicated because of Y. pestis adaptability to numerous hosts. YpeB shares 58% identity/79% similarity with the homo-pentameric B subunit of E. coli Subtilase cytotoxin, and 48% identity/68% similarity with the B subunit of S. Typhi typhoid toxin. We previously showed selective binding of B5 pentamers to a sialoglycan microarray, with Sia preferences corresponding to hosts e.g., N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Consistent with much broader host range of Y. pestis, YpeB binds all mammalian sialic acid types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, abolished by a mutation (Y77F) eliminating Sia recognition, suggesting cell proliferation and death via lectin-like cross-linking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.


title: Development and applications of sialoglycan-recognizing probes (SGRPs) with defined specificities: exploring the dynamic mammalian sialoglycome

abstract: Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems i.e., in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally-evolved microbial molecules (bacterial adhesins, toxin subunits and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of Sialoglycan Recognizing Probes (SGRPs: SGRP1-SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding non-binding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, Western Blot, flow cytometry and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild type mice and human-like Cmah-/- mice.


title: Sustained ability of a natural microbial community to remove nitrate from groundwater

abstract:
Microbial-mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon-bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located up-gradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by concomitant ethanol and nitrate removal and subsequent sulfate removal upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by concomitant ethanol and nitrate removal whereas the control did not. Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic- and/or genetic-levels. The results of this study demonstrated that the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor. Moreover, these results implied that the electron-donor exposure history of nitrate-contaminated groundwater can play an important role nitrate attenuation.

Article Impact StatementGroundwater microbes sustain ability to remove nitrate in absence of carbon and energy source.


title: Pandemic Vibrio cholerae Acquired Competitive Traits from an Environmental Vibrio Species

abstract:
BackgroundVibrio cholerae, the causative agent of cholera, is a human pathogen that thrives in estuarine environments. V. cholerae competes with neighboring microbes by the contact-dependent translocation of toxic effectors with the type VI secretion system (T6SS). Effector types are highly variable across V. cholerae strains, but all pandemic isolates encode the same set of distinct effectors. It is possible that acquisition of these effectors via horizontal gene transfer played a role in the development of pandemic V. cholerae.

ResultsWe assessed the distribution of V. cholerae T6SS loci across multiple Vibrio species. We showed that the fish-pathogen V. anguillarum encodes all three V. cholerae core loci as well as two of the four additional auxiliary clusters. We further demonstrated that V. anguillarum shares T6SS effectors with V. cholerae, including every pandemic-associated V. cholerae effector. We identified a novel T6SS cluster (Accessory Aux1) that is widespread in V. anguillarum and encodes the pandemic V. cholerae effector TseL. We highlighted potential gene transfer events of Accessory Aux1 from V. anguillarum to V. cholerae. Finally, we showed that TseL from V. cholerae can be neutralized by the V. anguillarum Accessory Aux1 immunity protein and vice versa, indicating V. anguillarum as the donor of tseL to the V. cholerae species.

ConclusionsV. anguillarum constitutes an environmental reservoir of pandemic-associated V. cholerae T6SS effectors. V. anguillarum and V. cholerae likely share an environmental niche, compete, and exchange T6SS effectors. Further, our findings highlight the fish as a potential reservoir of pandemic V. cholerae.

category: genomics


title: Detecting effective starting point of genomic selection by divergent trends from BLUP and ssGBLUP in pigs, beef cattle, and broilers

abstract: Genomic selection has been adopted nationally and internationally in different livestock and plant species. However, understanding whether genomic selection has been effective or not is an essential question for both industry and academia. Once genomic evaluation started being used, estimation of breeding values with pedigree BLUP became biased because this method does not consider selection using genomic information. Hence, the effective start point of genomic selection can be detected in two possible ways including the divergence of genetic trends and Realized Mendelian sampling (RMS) trends obtained with BLUP and Single-step genomic BLUP (ssGBLUP). This study aimed to find the start date of genomic selection for a set of economically important traits in three livestock species by comparing trends obtained using BLUP and ssGBLUP. For this purpose, three datasets comprised a pig dataset with 117k genotypes and 1.3M animals in pedigree, Angus cattle dataset consisted of ~842k genotypes and 11.5M animals in pedigree, and a purebred broiler chicken dataset included ~154k genotypes and 1.3M birds in pedigree were used. The genetic trends for pigs diverged for the genotyped animals born in 2014 for average daily gain and backfat. In beef cattle, the trends started diverging in 2009 for weaning weight and in 2016 for postweaning gain, with little diverging for birth weight. In broiler chickens, the genetic trends estimated by ssGBLUP and BLUP diverged at breeding cycle 6 for two out of three production traits. The RMS trends for the genotyped pigs diverged for animals born in 2014, more for average daily gain than for backfat. In beef cattle, the RMS trends started diverging in 2009 for weaning weight and in 2016 for postweaning gain, with a trivial trend for birth weight. In broiler chickens, the RMS trends from ssGBLUP and BLUP diverged strongly for two production traits at breeding cycle 6, with a slight divergence for another trait. Divergence of the genetic trends from ssGBLUP and BLUP indicates onset of the genomic selection. Presence of trends for RMS indicates selective genotyping, with or without the genomic selection. The onset of genomic selection and genotyping strategies agree with industry practices across the 3 species. In summary, the effective start of genomic selection can be detected by the divergence between genetic and RMS trends from BLUP and ssGBLUP.


title: Regulation of osteoblast autophagy based on PI3K/AKT/mTOR signaling pathway study on the effect of β-ecdysterone on fracture healing

abstract:
PurposeTo investigate the effects of {beta}-ecdysterone on fracture healing and the underlying mechanism.

MethodsMTT assay was used to detect the cell viability and alkaline phosphatase (ALP) activity was measured using a commercial kit. AO/PI and flow cytometry assays were used to determine the state of apoptosis of osteoblasts. The expression level of RunX2, ATG7 and LC3 was evaluated by qRT-PCR and Western blot assays. X-ray and HE staining were conducted on the fractured femur to evaluate the pathological state. Immunohistochemical assay was used to detect the expression level of Beclin-1 and immunofluorescence assay was used to measure the expression level of LC3 in the fractured femurs. Western blot was utilized to determine the expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K.

ResultsThe ALP activity and expression of RunX2 in fractured osteoblasts were significantly suppressed by 3-methyladenine and elevated by rapamycin, 60, and 80 M {beta}-ecdysterone. The apoptotic state of fractured osteoblasts was enhanced by 3-methyladenine and alleviated by rapamycin, 60, and 80 M {beta}-ecdysterone. The state of autophagy both in fractured osteoblasts and femurs was inhibited by 3-methyladenine and facilitated by rapamycin and {beta}-ecdysterone. Compared to control, Garrett score in 3-methyladenine group was significantly decreased and promoted in rapamycin and {beta}-ecdysterone groups, accompanied by ameliorated pathological state. Lastly, the PI3K/AKT/mTOR pathway both in fractured osteoblasts and femurs was activated by 3-methyladenine and inhibited by rapamycin and {beta}-ecdysterone.

Conclusion{beta}-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.


title: A universal, open-source, high-performance tool for automated sleep staging

abstract: The creation of a completely automated sleep-scoring system that is highly accurate, flexible, well validated, free and simple to use by anyone has yet to be accomplished. In part, this is due to the difficulty of use of existing algorithms, algorithms having been trained on too small samples, and paywall demotivation. Here we describe a novel algorithm trained and validated on +27,000 hours of polysomnographic sleep recordings across heterogeneous populations around the world. This tool offers high sleep-staging accuracy matching or exceeding human accuracy and interscorer agreement no matter the population kind. The software is easy to use, computationally low-demanding, open source, and free. Such software has the potential to facilitate broad adoption of automated sleep staging with the hope of becoming an industry standard.


title: Cellular-resolution monitoring of ischemic stroke pathologies in the rat cortex

abstract: Stroke is a leading cause of disability in the Western world. Current post-stroke rehabilitation treatments are only effective in approximately half of the patients. Therefore, there is a pressing clinical need for developing new rehabilitation approaches for enhancing the recovery process, which requires the use of appropriate animal models. Here we study the activity patterns of multiple cortical regions in the rat brain using two-photon microscopy. We longitudinally recorded the fluorescence signal from thousands of neurons labeled with a genetically-encoded calcium indicator before and after an ischemic stroke injury, and found substantial functional changes across motor, somatosensory, and visual cortical regions during the post-stroke cortical reorganization period. We show that a stroke injury in the primary motor cortex has an effect on the activity patterns of neurons not only in the motor and somatosensory cortices, but also in the more distant visual cortex, and that these changes include modified firing rates and kinetics of neuronal activity patterns in response to a sensory stimulus. Changes in neuronal population activity provided animal-specific, circuit-level information on the poststroke cortical reorganization process, which may be essential for evaluating the efficacy of new approaches for enhancing the recovery process.


title: Learning Rates Are Not All the Same: The Interpretation of Computational Model Parameters Depends on the Context

abstract:
Reinforcement Learning (RL) models have revolutionized the cognitive and brain sciences, promising to explain behavior from simple conditioning to complex problem solving, to shed light on developmental and individual differences, and to anchor cognitive processes in specific brain mechanisms. However, the growing RL literature shows increasingly contradictory results, potentially casting doubt on the usefulness of the method. We hypothesized that the observed contradictions instead arise from two fundamental assumptions about computational model parameters that are overly strong: That parameters generalize between contexts (e.g., tasks, models) and that they capture interpretable neurocognitive processes. If these assumptions are corrected, many contradictions dissolve. To test this idea, we asked 291 participants aged 8-30 years to complete three learning tasks during a single experimental session, and fitted their behavior using state-of-the-art RL models. As predicted, we found less-than-perfect parameter generalization between tasks (with better results for RL decision noise/exploration than learning rates) and a fundamental lack of interpretability (especially for learning rates), with a potential role of task similarity for generalization. We conclude that the systematic study of context (including task features and model parameterization) will enhance the generalizability of computational cognitive models.

HighlightsO_LIMany efforts in computational cognitive modeling are based on two implicit assumptions: 1) Model parameters generalize between studies and models. 2) Parameters are interpretable, i.e., neurally, cognitively, and/or mechanistically specific.
C_LIO_LIWe empirically investigate these assumptions in Reinforcement Learning (RL), using a large developmental dataset featuring three different learning tasks in a within-participant design.
C_LIO_LIWe find that RL decision noise/exploration parameters generalize between tasks in terms of captured between-participant variation, but not in terms of absolute parameter values. RL learning rates and Forgetting parameters fail to generalize. All parameters interpretability is low.
C_LIO_LIThese findings are in accordance with previous claims that decision noise/exploration decreases during development, but suggest that claims about the development of learning rates or Forgetting should not be generalized between tasks: parameters are unlikely to capture the same neuro-cognitive processes.
C_LIO_LIWe suggest ways forward to improve the generalizability and interpretability of model parameters, in particular by focusing more on task context.
C_LI


title: Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

abstract: Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

category: pharmacology and toxicology


title: Mild traumatic brain injury/ concussion initiates an atypical astrocyte response caused by blood-brain barrier dysfunction

abstract: Mild traumatic brain injury/ concussion (mTBI) account for 70-90% of all reported TBI cases and cause long lasting neurological consequences in 10 to 40% of patients. Recent clinical studies revealed increased blood-brain barrier (BBB) permeability in mTBI patients, which correlated with secondary damage after mTBI. However, the cascade of cellular events initiated by exposure to blood-borne factors resulting in sustained damage are not fully resolved. We previously reported that astrocytes respond atypically to mTBI rapidly downregulating many proteins essential to their homeostatic function while classic scar formation does not occur. Here, we tested the hypothesis that mTBI -induced BBB damage causes atypical astrocytes through exposure to blood-borne factors. Using a mTBI mouse model, 2-photon imaging, an endothelial cell-specific genetic ablation approach, and serum-free primary astrocyte cultures, we demonstrated that areas with atypical astrocytes coincide with BBB damage and that exposure of astrocytes to plasma proteins is sufficient to initiate downregulation of astrocyte homeostatic proteins. While mTBI resulted in frequent impairment of both physical and metabolic BBB properties and leakage of small-sized blood-borne factors, deposition of the coagulation factor fibrinogen or vessel rupture were rare. Surprisingly, even months after mTBI BBB repair did not occur in areas with atypical astrocytes. Together, these findings implicate that even relatively small BBB disturbances are sustained long-term and render nearby astrocytes dysfunctional, likely at the cost of neuronal health and function.


title: Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity

abstract: The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the worlds population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (OX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus OX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus OX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

category: immunology


title: Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 and Spike Protein Subunit 1

abstract: A hallmark of COVID-19 is a hyperinflammatory state that is associated with severity. Various anti-inflammatory therapeutics have shown mixed efficacy in treating COVID-19, and the mechanisms by which hyperinflammation occurs are not well understood. Previous research indicated that monocytes, a key innate immune cell, undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response in monocytes. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism that was associated with production of pro-inflammatory cytokines including interleukin-6 and tumor necrosis factor-. This response was dependent on hypoxia-inducible factor-1, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production in monocytes, and abrogated glycolytic and mitochondrial metabolism. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments in monocytes. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.


title: SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia

abstract:
The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases fatality is linked to age.

Significance StatementBronchial epithelia are the primary target for SARS-CoV-2 infections. Our work uses reconstituted bronchial epithelia from adults and children. We show that infection of adult epithelia with SARS-CoV-2 is rapid and results in the synchronized release of large clusters of infected cells and syncytia into the apical lumen contributing to the released infectious virus dose. Infection of children derived bronchial epithelia revealed an intrinsic resistance to infection and virus spread, probably as a result of a faster onset of interferon secretion. Thus, our data provide direct evidence for the epidemiological observation that children are less susceptible to SARS-CoV-2.


title: Evaluating the risk of SARS-CoV-2 transmission to bats using a decision analytical framework

abstract:
Preventing wildlife disease outbreaks is a priority issue for natural resource agencies, and management decisions can be urgent, especially in epidemic circumstances. With the emergence of SARS-CoV-2, wildlife agencies were concerned whether the activities they authorize might increase the risk of viral transmission from humans to North American bats but had a limited amount of time in which to make decisions. We provide a description of how decision analysis provides a powerful framework to analyze and re-analyze complex natural resource management problems as knowledge evolves. Coupled with expert judgment and avenues for the rapid release of information, risk assessment can provide timely scientific information for evolving decisions. In April 2020, the first rapid risk assessment was conducted to evaluate the risk of transmission of SARS-CoV-2 from humans to North American bats.

Based on the best available information, and relying heavily on formal expert judgment, the risk assessment found a small possibility of transmission during summer work activities. Following that assessment, additional knowledge and data emerged, such as bat viral challenge studies, that further elucidated the risks of human-to-bat transmission and culminated in a second risk assessment in the fall of 2020. We update the first SARS-CoV-2 risk assessment with new estimates of little brown bat (Myotis lucifugus) susceptibility and new management alternatives, using findings from the prior two risk assessments and other empirical studies. We highlight the strengths of decision analysis and expert judgment not only to frame decisions and produce useful science in a timely manner, but also to serve as a framework to reassess risk as understanding improves. For SARS-CoV-2 risk, new knowledge led to an 88% decrease in the median number of bats estimated to be infected per 1000 encountered when compared to earlier results. The use of facemasks during, or a negative COVID-19 test prior to, bat encounters further reduced those risks. Using a combination of decision analysis, expert judgment, rapid risk assessment, and efficient modes of information distribution, we provide timely science support to decision makers for summer bat work in North America.


title: Biophysical characterization of the SARS-CoV-2 E protein

abstract: SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.


title: Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium


title: The N-terminal and central domains of CoV-2 nsp1 play key functional roles in suppression of cellular gene expression and preservation of viral gene expression

abstract: Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.


title: Loss of N1-methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression

abstract: N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the A site. Enzyme and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the starvation-induced stringent response, consistent with the notion that loss of tRNA aminoacylation activates the response. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.


title: Contribution of metal transporters of the ABC, ZIP, and NRAMP families to manganese uptake and infective endocarditis virulence in Streptococcus sanguinis

abstract: Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a {Delta}ssaACB {Delta}tmpA mutant was far more attenuated for serum growth and somewhat more attenuated for virulence in a rabbit model than its {Delta}ssaACB parent. The growth of both mutants was restored by supplemental manganese, but the {Delta}ssaACB {Delta}tmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. In contrast to ssaACB and tmpA, which appear ubiquitous in S. sanguinis, a mntH gene encoding an NRAMP-family transporter has been identified in relatively few strains, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its {Delta}ssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions.


title: Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo

abstract:
Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a {Delta}ssaACB mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and {Delta}ssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its {Delta}ssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis and point to strategies for enhancing the safety of oral probiotics.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/446192v1_ufig1.gif" ALT="Figure 1">
View larger version (44K):
org.highwire.dtl.DTLVardef@a5afa7org.highwire.dtl.DTLVardef@1af3cdforg.highwire.dtl.DTLVardef@1c58693org.highwire.dtl.DTLVardef@1063980_HPS_FORMAT_FIGEXP  M_FIG Depiction of methods. Streptococcal strains used are depicted at the top. The four methods illustrated are: 1. S. sanguinis cells were grown in media at pH 7.3 and pH 6.2 and quantified by plating. 2. S. sanguinis cells were grown in a fermentor vessel for RNA-sequencing and metal analysis. 3. S. sanguinis and S. mutans cells were grown in 12-well plates singly or in competition, then plated and the pH of the media measured. 4. S. sanguinis and S. mutans cells were inoculated into the mouths of mice. Oral swabs and dental biofilms from molars were assayed for bacterial composition by qPCR. (Biorender)

C_FIG


title: Digital PCR to quantify ChAdOx1 nCoV-19 copies in blood and tissues

abstract: Vaccination with the adenoviral-vector based Astra Zeneca ChAdOx1 nCov-19 vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR is an excellent tool to quantify transgene copies in vivo. Here we present a highly sensitive digital PCR for in-situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human serum 24, 72 and 168 hours post vaccination, and in a variety of murine tissues in an experimental vaccination model 30 minutes post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.


title: The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin

abstract: The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence 681PRRAR/S686. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.


title: A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

abstract: Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.

category: genetics


title: Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

abstract: Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5 or 3 junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5 or 3 junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.


title: Mutational hotspot in the SARS-CoV-2 Spike protein N-terminal domain conferring immune escape potential

abstract: Global efforts are being taken to monitor the evolution of SARS-CoV-2, aiming at early identification of mutations with the potential of increasing viral infectivity or virulence. We report a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. We investigate the impact these mutations might have on the evasion of neutralizing antibodies. Finally, we uncover that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path on which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape.


title: Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan

abstract: Hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between megasynthases. In the nematode Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides in animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional stand-alone enzymes that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes in the biosynthetic pathway and discover a novel mechanism of trafficking intermediates between a PKS and an NRPS. Specifically, we show that the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

category: biochemistry


title: Decomposition of the SARS-CoV-2-ACE2 interface reveals a common trend among emerging viral variants

abstract:
New viral variants of the SARS-CoV-2 virus show enhanced infectivity compared to wild type, resulting in an altered pandemic situation in affected areas. These variants are the B.1.1.7 (United Kingdom), B.1.1.7 with the additional E484K mutation, the B.1.351 variant (South Africa) and the P.1 variant (Brazil). Understanding the binding modalities between these viral variants and the host cell receptor ACE2 allows depicting changes, but also common motifs of virus-host cell interaction. The trimeric spike protein expressed at the viral surface contains the receptor-binding domain (RBD) that forms the molecular interface with ACE2. All the above-mentioned variants carry between one and three amino acid exchanges within the interface-forming region of the RBD, thereby altering the binding interface with ACE2. Using molecular dynamics simulations and decomposition of the interaction energies between the RBD and ACE2, we identified phenylalanine 486, glutamine 498, threonine 500 and tyrosine 505 as important interface-forming residues across viral variants. We also suggest a reduced binding energy between RBD and ACE2 in viral variants with higher infectivity, attributed to residue-specific differences in electrostatic interaction energy. Importantly, individual amino acid exchanges not only influence the affected position, but also alter the conformation of surrounding residues and affect their interaction potential as well. We demonstrate how computational methods can help to identify changed as well as common motifs across viral variants. These identified motifs might play a crucial role, in the strategical development of therapeutic interventions against the fast mutating SARS-CoV-2 virus.

Significance StatementThe COVID-19 pandemic caused by the SARS-CoV-2 virus has significantly changed our lives. To date, there is a lack of neutralizing drugs that specifically target SARS-CoV-2. Hope lies in newly developed vaccines that effectively prevent severe cases of acute respiratory syndrome. However, emerging viral variants escape vaccine-induced immune-protection. Therefore, identification of appropriate molecular targets across viral variants is important for the development of second- and third-generation vaccines and inhibitory antibodies. In this study, we identify residues across viral variants that are important for viral binding to the host cell. As such residues cannot be replaced without diminishing infectivity of the virus, these residues represent primary targets for intervention, for example by neutralizing antibodies.


title: Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate

abstract:
Xylem sap is the major transport route for nutrients from roots to shoots. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, while nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased whereas salicylic acid and jasmonoyl-isoleucine decreased with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Significance statementThis study reports the largest, currently available plant xylem sap proteome and metabolome databases and highlights novel discoveries of specialized metabolites and phytohormones in the xylem sap. This is the first multi-omics study linking differences in nitrogen supply with changes xylem sap composition, endophyte growth and transcriptional defenses in leaves.


title: Mitochondrial cyclophilin D promotes disease tolerance by licensing NK cell development and IL-22 production against influenza virus

abstract: Immunity to infectious disease involves a combination of host resistance, which eliminates the pathogen, and disease tolerance, which limits tissue damage. While the severity of most pulmonary viral infections, including influenza A virus (IAV), is linked to excessive inflammation, our mechanistic understanding of this observation remains largely unknown. Here we show that mitochondrial cyclophilin D (CypD) protects against IAV infection via disease tolerance. Mice deficient in CypD (CypD-/- mice) are significantly more susceptible to IAV infection despite comparable antiviral immunity. Instead, this susceptibility resulted from damage to the lung epithelial barrier caused by a significant reduction of IL-22 production by conventional NK cells in IAV-infected CypD-/- mice. Transcriptomic and functional data revealed that the compromised IL-22 production by NK cells resulted from dysregulated lymphopoiesis, stemming from increased cell death in NK cell progenitors, as well as the generation of immature NK cells that exhibited altered mitochondrial metabolism. Importantly, following IAV infection, administration of recombinant IL-22 abrogated pulmonary damage and enhanced survival of CypD-/- mice. Collectively, these results demonstrate a key role for CypD in NK cell-mediated disease tolerance.


title: Diverse Genetic Determinants of Nitrofurantoin Resistance in UK Escherichia coli

abstract:
Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2018. Nitrofurantoin resistance is reported in <6% urinary E. coli isolates in the UK. However, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify genetic determinants of nitrofurantoin resistance in a local E. coli collection and assess their prevalence in a larger dataset of E. coli genomes. Deleterious point mutations and gene-inactivating insertion sequences in both chromosomal nitroreductase genes nfsA and nfsB were identified in genomes of nine nitrofurantoin-resistant urinary E. coli isolates collected from north west London. Eight types of genetic alterations were identified when comparing sequences of nfsA, nfsB, and the associated gene ribE in 12,412 E. coli genomes collected from across the UK. Evolutionary analysis revealed homoplasic mutations and explained the order of stepwise mutations. An algorithm was developed to predict nitrofurantoin susceptibility and predictions for 20 accessible isolates were experimentally validated. Only one genome carrying oqxAB, a mobile gene complex associated with reduced nitrofurantoin susceptibility, was identified. In conclusion, mutations and insertion sequences in nfsA and nfsB are leading causes of nitrofurantoin resistance in UK E. coli. As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored.

ImportanceThis study expands knowledge about the genetic basis of nitrofurantoin resistance in E. coli isolates using whole-genome sequencing and genomic analysis. We report novel and previously known deleterious mutations of chromosomal genes nfsA, nfsB, and ribE as well as the interruption of nfsA and nfsB by insertion sequences, recapitulating the roles of oxygen-insensitive nitroreductases in the development of nitrofurantoin resistance in E. coli. We revealed and categorised the genotypic diversity in these three genes in a large collection of UK E. coli genomes. A scoring algorithm is provided to predict nitrofurantoin susceptibility from genotypes. Our predictions suggest that acquired nitrofurantoin resistance is not of immediate concern in the UK. However, experimental validation of predictions suggested the involvement of mechanisms other than alterations in nfsA, nfsB, or ribE in determining nitrofurantoin susceptibility, emphasising the need for monitoring nitrofurantoin resistance amongst E. coli.


title: Differential transcriptomic response of Anopheles arabiensis to Plasmodium vivax and Plasmodium falciparum infection

abstract:
Plasmodium vivax malaria is now recognized as the second most dangerous parasitic threat to human health with the regular decrease of Plasmodium falciparum worldwide over recent decades. A very limited numbers of studies address the interaction of P. vivax with its Anopheles mosquito vectors. Those studies were conducted in P. vivax endemic countries with P.vivax local major vectors for which limited genomic and genetic tools are available. Despite the presence of P. vivax in several African countries and increasing reports on its occurrence in many others, there is virtually no data on the molecular responses of Anopheles arabiensis, a major African mosquito vector, to P. vivax, which limits the development of further "mosquito-targeted" interventions aimed at reducing P. vivax transmission. Taking advantage of the situation of Madagascar where P. falciparum, P. vivax and An. arabiensis are present, we explore the molecular responses of An. arabiensis towards these two human malaria parasites. RNA sequencing on RNAs isolated from mosquito midguts dissected at the early stage of infection (24 hours) was performed using mosquitoes fed on the blood of P. vivax and P. falciparum gametocyte carriers in a field station. From a de novo assembly of An. arabiensis midgut total RNA transcriptome, the comparative analysis revealed that a greater number of genes were differentially expressed in the mosquito midgut in response to P. vivax (209) than to P. falciparum (81). Among these, 15 common genes were identified to be significantly expressed in mosquito midgut 24 hours after ingesting P. vivax and P. falciparum gametocytes, including immune responsive genes and genes involved in amino-acid detoxification pathways. Importantly, working with both wild mosquitoes and field circulating parasites, our analysis revealed a strong mosquito genotype by parasite genotype interaction. Our study also identified 51 putative long non-coding RNAs differentially expressed in An. arabiensis mosquito infected midgut. Among these, several mapped to the published An. arabiensis genome at genes coding immune responsive genes such as gambicin 1, leucine-rich repeat containing genes, either on sense or antisense strands.

This study constitutes the first comparison of An. arabiensis molecular interaction with P. vivax and P. falciparum, investigating both coding and long non-coding RNAs for the identification of potential transcripts, that could lead to the development of novel approaches to simultaneously block the transmission of vivax and falciparum malaria.