title: Live-bearing cockroach genome reveals convergent evolutionary mechanisms linked to viviparity in insects and beyond

abstract: Insects provide an unparalleled opportunity to link genomic changes with the rise of novel phenotypes, given tremendous variation in the numerous and complex adaptations displayed across the group. Among these numerous and complex adaptations, live-birth has arisen repeatedly and independently in insects and across the tree of life, suggesting this is one of the most common types of convergent evolution among animals. We sequenced the genome and transcriptome of the Pacific beetle-mimic cockroach, the only truly viviparous cockroach, and performed comparative analyses including two other viviparous insect lineages, the tsetse and aphids, to unravel the genomic basis underlying the transition to viviparity in insects. We identified pathways experiencing adaptive evolution, common in all viviparous insects surveyed, involved in uro-genital remodeling, maternal control of embryo development, tracheal system, and heart development. Our findings suggest the essential role of those pathways for the development of placenta-like structure enabling embryo development and nutrition. Viviparous transition seems also to be accompanied by the duplication of genes involved in eggshell formation. Our findings from the viviparous cockroach and tsetse reveal that genes involved in uterine remodeling are up-regulated and immune genes are down-regulated during the course of pregnancy. These changes may facilitate structural changes to accommodate developing young and protect them from the mothers immune system. Our results denote a convergent evolution of live-bearing in insects and suggest similar adaptive mechanisms occurred in vertebrates, targeting pathways involved in eggshell formation, uro-genital remodeling, enhanced tracheal and heart development, and reduced immunity.


title: Scalable microbial strain inference in metagenomic data using StrainFacts

abstract: While genome databases are nearing a complete catalog of species commonly inhabiting the human gut, their representation of intraspecific diversity is lacking for all but the most abundant and frequently studied taxa. Statistical deconvolution of allele frequencies from shotgun metagenomic data into strain genotypes and relative abundances is a promising approach, but existing methods are limited by computational scalability. Here we introduce StrainFacts, a method for strain deconvolution that enables inference across tens of thousands of metagenomes. We harness a "fuzzy" genotype approximation that makes the underlying graphical model fully differentiable, unlike existing methods. This allows parameter estimates to be optimized with gradient-based methods, speeding up model fitting by two orders of magnitude. A GPU implementation provides additional scalability. Extensive simulations show that StrainFacts can perform strain inference on thousands of metagenomes and has comparable accuracy to more computationally intensive tools. We further validate our strain inferences using single-cell genomic sequencing from a human stool sample. Applying StrainFacts to a collection of more than 10,000 publicly available human stool metagenomes, we quantify patterns of strain diversity, biogeography, and linkage-disequilibrium that agree with and expand on what is known based on existing reference genomes. StrainFacts paves the way for large-scale biogeography and population genetic studies of microbiomes using metagenomic data.

category: bioinformatics


title: Generalized Hypergeometric Distributions Generated by Birth-Death Process in Bioinformatics

abstract: Modern high-throughput biological systems detection methods generate empirical frequency distributions (EFD) which exhibit complex forms and have long right-side tails. Such EFD are often observed in normal and pathological processes, of which the probabilistic properties are essential, but the underlying probability mechanisms are poorly understood. To better understand the probability mechanisms driving biological complexity and the pathological role of extreme values, we propose that the observed skewed discrete distributions are generated by non-linear transition rates of birth and death processes (BDPs). We introduce a (3d+1)-parameter Generalized Gaussian Hypergeometric Probability ((3d+1)-GHP) model with the probabilities defined by a stationary solution of generalized BDP (g-BDP) and represented by generalized hypergeometric series with regularly varying function properties. We study the Regularly Varying 3d-Parameter Generalized Gaussian Hypergeometric Probability (3d-RGHP) functions regular variation properties, asymptotically constant slow varying component, unimodality and upward/ downward convexity which allows us to specify a family of 3d-RGHP models and study their analytical and numerical characteristics. The frequency distribution of unique muta-tions occurring in the human genome of patients with melanoma have been analyzed as an example application of our theory in bioinformatics. The results show that the parameterized model not only fits the  heavy tail well, but also the entire EFD taken on the complete experimental outcome space. Our model provides a rigorous and flexible mathematical framework for analysis and application of skewed distributions generated by BDPs which often occur in bioinformatics and big data science.


title: IDSL.CCDB: a database for exploring inter-chemical correlations in metabolomics and exposomics datasets

abstract: Inter-chemical correlations in metabolomics and exposomics datasets provide valuable information for studying relationships among reported chemicals measured in human specimens. With an increase in the size of these datasets, a network graph analysis and visualization of the correlation structure is difficult to interpret. While co-regulatory genes databases have been developed, a similar database for metabolites and chemicals have not been developed yet. We have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics - Chemical Correlation Database (IDSL.CCDB), as a systematic catalogue of inter-chemical correlation in publicly available metabolomics and exposomics studies. The database has been provided via an online interface to create single compound-centric views that are clear, readable and meaningful. We have demonstrated various applications of the database to explore: 1) the chemicals from a chemical class such as Per- and Polyfluoroalkyl Substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalates and tobacco smoke related metabolites; 2) xenobiotic metabolites such as caffeine and acetaminophen; 3) endogenous metabolites (acyl-carnitines); and 4) unannotated peaks for PFAS. The database has a rich collection of 36 human studies, including the National Health and Nutrition Examination Survey (NHANES) and high-quality untargeted metabolomics datasets. IDSL.CCDB is supported by a simple, interactive and user-friendly web-interface to retrieve and visualize the inter-chemical correlation data. The IDSL.CCDB has the potential to be a key computational resource in metabolomics and exposomics facilitating the expansion of our understanding about biological and chemical relationships among metabolites and chemical exposures in the human body. The database is available at www.ccdb.idsl.me site.


title: An approachable, flexible, and practical machine learning workshop for biologists

abstract: The increasing prevalence and importance of machine learning in biological research has created a need for machine learning training resources tailored towards biological researchers. However, existing resources are often inaccessible, infeasible, or inappropriate for biologists because they require significant computational and mathematical knowledge, demand an unrealistic time-investment, or teach skills primarily for computational researchers. We created the Machine Learning for Biologists (ML4Bio) workshop, a short, intensive workshop that empowers biological researchers to comprehend machine learning applications and pursue machine learning collaborations in their own research. The ML4Bio workshop focuses on classification and was designed around 3 principles: (a) focusing on preparedness over fluency or expertise, (b) necessitating minimal coding and mathematical background, and (c) requiring low time investment. It incorporates active learning methods and custom open source software that allows participants to explore machine learning workflows. After multiple sessions to improve workshop design, we performed a study on 3 workshop sessions. Despite some confusion around identifying subtle methodological flaws in machine learning workflows, participants generally reported that the workshop met their goals, provided them with valuable skills and knowledge, and greatly increased their beliefs that they could engage in research that uses machine learning. ML4Bio is an educational tool for biological researchers, and its creation and evaluation provides valuable insight into tailoring educational resources for active researchers in different domains. The workshop materials are available from https://carpentries-incubator.github.io/ml4bio-workshop/ and the ml4bio software is available from https://github.com/gitter-lab/ml4bio.

category: scientific communication and education


title: The metric backbone preserves community structure and is a primary transmission subgraph in contact networks

abstract: The structure of social networks strongly affects how different phenomena spread in human society, from the transmission of information to the propagation of contagious diseases. It is well-known that heterogeneous connectivity strongly favors spread, but a precise characterization of the redundancy present in social networks and its effect on the robustness of transmission is still lacking. This gap is addressed by the recently introduced metric backbone, a subgraph that is sufficient to compute all shortest paths of weighted graphs. We show that the metric backbones of nine contact networks obtained from proximity sensors in a variety of social contexts are generally very small, 49% of the original graph for one and ranging from about 6% to 20% for the others. This reflects a surprising amount of redundancy and reveals that shortest paths on these networks are very robust to random attacks and failures. We show that the metric backbone preserves the community structures of the full contact networks and is a primary subgraph in epidemic transmission. The metric backbone, thus, is a principled graph reduction technique that does not affect shortest path connectivity for any nodes in a network. It is an important subgraph to characterize epidemic spread, the robustness of social networks, and any communication dynamics that depend on complex network shortest paths.

category: systems biology


title: Test-retest reproducibility of human brain multi-slice 1H FID-MRSI data at 9.4 T after optimization of lipid regularization, macromolecular model and spline baseline stiffness

abstract:
* PurposeThis study analyzes the effects of retrospective lipid suppression, a simulated macromolecular prior knowledge and different spline baseline stiffness values on 9.4 T multi-slice proton FID-MRSI data spanning the whole cerebrum of human brain and its reproducibility of metabolite ratio (/tCr) maps for 10 brain metabolites.

* MethodsMeasurements were performed twice on five volunteers using a non-accelerated FID MRSI 2D sequence at 9.4 T. The effects of retrospective lipid L2-regularization, macromolecular spectrum and different LCModel baseline flexibilities on SNR, FWHM, fitting residual, CRLB and the concentration ratio maps were investigated. Intra-subject, inter-session coefficient of variation of the mean metabolite ratios (/tCr) of each slice was calculated.

* ResultsL2-regularization provided effective suppression of lipid-artifacts, but should be avoided if no artifacts are detected. Transversal, sagittal and coronal of many metabolite ratio maps correspond to anatomically expected concentration relations in gray and white matter for the majority of the cerebrum when using a flexible baseline in LCModel fit. Additionally, results from the second measurements of the same subjects show that slice positioning and data quality correlate significantly to the first measurement.

* ConclusionConcentration ratio maps (/tCr) for 4 metabolites (tCho, NAA, Glu, mI) spanning the majority and six metabolites (NAAG, GABA, GSH, Tau, Gln, Asp) covering 32 mm in the upper part of the brain were acquired at 9.4 T using multi-slice FID MRSI with retrospective lipid suppression, a macromolecular spectrum and a flexible LCModel baseline.

category: neuroscience


title: Top-down control of the left visual field bias in cued visual spatial attention

abstract: A left visual field (LVF) bias in perceptual judgements, response speed and discrimination accuracy are well documented in humans. However, LVF bias can be modulated by perceptual and task demands. For example, cuing spatial attention can reduce or eliminate the LVF bias, suggesting that attentional control can compensate for the LVF bias. We investigated this possibility directly by recording pupillometry together with fMRI in a cued visual spatial attention task. Prior to the onset of a task-relevant target stimulus, we observed that the pupil was significantly more dilated following attend-right than attend-left cues even though task performance did not differ. This difference in pupil dilation was inversely related to the corresponding difference in later target-evoked pupil dilation and in the reaction times to those targets, suggesting that an increased attentional effort was triggered by the attend-right cues, and this offset the LVF bias, equating behavioral performance. The differences in pupil dilation to the right versus left hemifield were correlated with corresponding fMRI differences primarily in the right hemisphere, supporting the idea that the increased attentional effort for rightward attention is mediated by activity in right hemisphere networks, which illuminates how attentional control mediates attentional biases in vision.


title: Taurine or N-acetylcysteine treatments prevent memory impairment and metabolite profile alterations in the hippocampus of high-fat diet-fed female mice

abstract: Obesity impacts brain metabolism and function, and constitutes a risk factor for dementia development. Long-term exposure to obesogenic diets leads to taurine accumulation in the hippocampus of rodents. Taurine has putative beneficial effects on cell homeostasis, and is reported to improve cognition. Therefore, hippocampal taurine accumulation in obese and diabetic models might constitute a counteracting response to metabolic stress. This study tested the hypothesis that treatment with taurine or with N-acetylcysteine (NAC), which provides cysteine for the synthesis of taurine and the antioxidant glutathione, prevent obesity-associated hippocampal alterations and memory impairment. Female mice were fed either a regular diet or a high-fat diet (HFD). For the same period, some mice had access to 3%(w/v) taurine or 3%(w/v) NAC in the drinking water. After 2 months, magnetic resonance spectroscopy was used to measure metabolite profiles. Then, memory was assessed in novel object and novel location recognition tests. HFD feeding caused memory impairment in both tests, and reduced concentration of lactate, phosphocreatine-to-creatine ratio, and the neuronal marker N-acetylaspartate in the hippocampus. Both taurine and NAC prevented HFD-induced memory impairment and N-acetylaspartate reduction. NAC, but not taurine, prevented the HFD-induced reduction of lactate and phosphocreatine-to-creatine ratio. Analysis of the metabolite profile further revealed prominent NAC/taurine-induced increase of glutamate and GABA levels in the hippocampus. We conclude that NAC and taurine prevent of neuronal dysfunction, while only NAC prevents energy metabolism dysregulation in HFD-induced obesity in female mice.


title: Why Are Alzheimer's Disease Patients Unaware of their Memory Deficits?

abstract: Anosognosia, or the lack of awareness of ones own impairment, is frequent for memory deficits in patients with Alzheimers disease (AD). Although often related to frontal dysfunctions, the neural mechanisms of anosognosia remain largely unknown. We hypothesized that anosognosia in AD may result from a failure in the error-monitoring system, thus preventing patients from being aware of and learning from their own errors. We therefore investigated the event-related potentials evoked by erroneous responses during a memory task in two groups of amyloid positive individuals who had only subjective memory complaints at study entry: 1) those who progressed to AD; and 2) those who remained cognitively normal after five years of follow-up. Our findings revealed direct evidence of a failure in the error-monitoring system at early stages of AD, suggesting that it may be the critical neural substrate of anosognosia in this neurodegenerative disorder.


title: Reinstating olfactory bulb derived limbic gamma oscillations alleviates depression

abstract:
Although the etiology of major depressive disorder remains poorly understood, impairment of gamma oscillations recently emerged as a potential biomarker for major depression. The olfactory bulb (OB) is a major source of brain wide gamma oscillations and bulbectomy is an animal model for depression. Here we demonstrate that chemogenetic suppression of OB neuronal activity or temporally suppressing the OB to pyriform cortex synaptic transmission decreased gamma oscillation power in multiple brain areas associated with depression-like behaviors. To assess the hypothesized link between depression and diffuse depression of gamma oscillations, we employed gamma phase-dependent closed loop neuromodulation of cortical areas, paced by the native OB output. This procedure alleviated depressive-like behaviors in animals and suggests that restoring gamma oscillations may improve depression in humans.

One Sentence SummaryRole of limbic gamma oscillations in depression


title: Information theory rules out the reflex-chain model of C. elegans locomotion

abstract: Despite decades of research, whether the C. elegans traveling-wave sinusoidal body pattern during locomotion is produced (a) by the undulations of the head followed by wave propagation down the body, or (b) via centrally coordinated posture control along the body, is still under debate. By studying relationships between the time series of postural angles along the body extracted from videos of moving worms, we find that the reflex-chain model can be refuted, in both forward and backward locomotion as well as during swimming and crawling behaviors. We show that information theory applied to animal behavior can yield insights into the neural control of behavior.


title: Tandem Walk in Simulated Martian Gravity and Visual Environment

abstract: Astronauts returning from long-duration spaceflights experience visual-vestibular conflicts that causes motion sickness, perceptions that the environment is moving when it is not, problems with walking, and other functional tasks. To evaluate whether astronauts will have similar decrements associated with visual-vestibular conflicts after they land on Mars following exposure to weightlessness, participants were held by a device that offloads their weight; first entirely (0 G) for 10 minutes, and then partially (0.38 G) or not at all (1 G) for 15 minutes. Tandem (heel-to-toe) walk was used to assess the subjects walking performance. Ten subjects performed 2 trials of 10 steps on a medium-density foam surface. Four conditions were investigated: (a) 1 G in virtual reality (VR); (b) 1 G in VR with a superimposed disorienting optokinetic simulation (VR+DOS); (c) 0.38 G in VR; and (d) 0.38 G in VR+DOS. Tandem walk performance decreased in VR+DOS compared to VR in both 1 G and simulated 0.38 G. Tandem walking performance in VR+DOS was better in 0.38 G compared to 1 G. Tandem walking performance in VR+DOS in 1 G was not significantly different from tandem walking performance after spaceflight or bed rest. The increased tandem walking performance in 0.38 G compared to 1 G was presumably due to an increased cone of stability, allowing larger amplitude of body sway without resulting in a fall. Tandem walking on a compliant foam surface in VR+DOS is a potential analog for simulating postflight dynamic balance deficits in astronauts.


title: Joint coding of visual input and eye/head position in V1 of freely moving mice

abstract:
Visual input to the brain during natural behavior is highly dependent on movements of the eyes, head, and body. Neurons in mouse primary visual cortex (V1) respond to eye and head movements, but how information about eye and head position is integrated with visual processing during free movement is unknown, since visual physiology is generally performed under head-fixation. To address this, we performed single-unit electrophysiology in V1 of freely moving mice while simultaneously measuring the mouses eye position, head orientation, and the visual scene from the mouses perspective. We mapped spatiotemporal receptive fields using a generalized linear model (GLM) that predicted the activity of V1 neurons based on gaze-corrected visual input. Critically, a large fraction of visually-responsive neurons showed tuning for eye position and head orientation. Incorporating these variables into the GLM revealed that visual and positional signals in most neurons are multiplicatively integrated, consistent with computation via gain fields and nonlinear mixed selectivity. These results provide new insight into coding in mouse V1, and more generally provide a paradigm for performing visual physiology under natural conditions, including active sensing and ethological behavior.

Highlights* Neurons in mouse V1 respond to both vision and self-motion, but it is unclear how these are combined.
* We record neural activity in V1 concurrent with measurement of the visual input during free movement.
* These data provide the first measurement of visual receptive fields in freely moving animals.
* Eye position and head orientation tuning primarily contributes a multiplicative gain on visual responses, consistent with gain fields and nonlinear mixed selectivity.


title: Corticospinal excitability is influenced by the recent history of electrical digital stimulation: implications for the relative magnitude of short-latency afferent inhibition

abstract:
BackgroundElectrical stimulation of the hand can suppress the motor evoked potential (MEP) elicited by transcranial magnetic stimulation (TMS) of the primary motor hand area (M1-HAND) when the afferent stimulus arrives in M1-HAND at the time of TMS. The magnitude of short-latency afferent inhibition (SAI) is expressed as the ratio between the conditioned and unconditioned MEP and is widely used to probe sensorimotor interactions in human physiological studies.

Objective/HypothesisWe hypothesized that corticospinal excitability and SAI are influenced by the recent history of peripheral electrical stimulation.

MethodsIn twenty healthy participants, we recorded MEPs from the right first dorsal interosseus muscle. MEPs were evoked by single-pulse TMS of the left M1-HAND alone (unconditioned TMS) or by TMS preceded by electrical stimulation of the right index finger ("homotopic" conditioning) or little finger ("heterotopic" conditioning). The three conditions were pseudo-randomly intermixed or delivered in blocks in which a single condition was repeated five or ten times. MEP amplitudes and SAI magnitudes were compared using linear mixed effect models.

ResultsAll stimulation protocols consistently produced SAI, which was stronger after homotopic stimulation. Randomly intermingling the three stimulation conditions reduced the relative magnitude of homotopic and heterotopic SAI as opposed to blocked stimulation. The apparent attenuation of SAI was caused by a suppression of the unconditioned but not the conditioned MEP amplitude during the randomly intermixed condition.

Conclusion(s)The recent history of afferent stimulation modulates corticospinal excitability. This "history effect" impacts on the relative magnitude of SAI depending on how conditioned and unconditioned responses are intermixed and needs to be taken into consideration when probing afferent inhibition and corticospinal excitability.


title: Emergence of time persistence in an interpretable data-driven neural network model

abstract: Establishing accurate as well as interpretable models of networks activity is an open challenge in systems neuroscience. Here we infer an energy-based model of the ARTR, a circuit that controls zebrafish swimming statistics, using functional recordings of the spontaneous activity of hundreds of neurons. Although our model is trained to reproduce the low-order statistics of the network activity at short time-scales, its simulated dynamics quantitatively captures the slowly alternating activity of the ARTR. It further reproduces the modulation of this persistent dynamics by the bath temperature and visual stimulation. Mathematical analysis of the model unveils a low-dimensional landscape-based representation of the ARTR activity, where the slow network dynamics reflects Arrhenius-like barriers crossings between metastable states. Our work thus shows how data-driven models built from large neural populations recordings can be reduced to low-dimensional functional models in order to reveal the fundamental mechanisms controlling the collective neuronal dynamics.


title: How exposure to land use impacts and climate change may prune the tetrapod tree of life

abstract: Human domination of landscapes is a key driver of biodiversity loss, with the fingerprint of climate change becoming increasingly pronounced. Frameworks and tools for identifying threats to biodiversity are required to meet Post-2020 Global Biodiversity Framework targets for 2030 that call for, among other things, reducing or halting species extinction rates (1). Hence, we compiled a phylogenetic tree for terrestrial tetrapods, mapped hotspots of geographically restricted and evolutionarily distinct lineages, and identified which hotspots may simultaneously face the highest magnitudes of land use impacts and climate change. Across a quarter of Earths surface, hotspots contain the entire ranges of 45% of tetrapods, representing 39% of terrestrial tetrapod evolutionary heritage. By 2070, we estimate 8-13% of this heritage to occur entirely within hotspots highly exposed to climate change, with 13-29% of hotspots projected to experience high exposure to both stressors simultaneously. Most hotspots at highest risk occur in countries least able to take action. Our analysis highlights the need for global ambition and coordination to avoid catastrophic loss of tetrapod evolutionary heritage.

category: ecology


title: Trait divergence and trade-offs among Brassicaceae species differing in elevational distribution

abstract: Species have restricted geographic distributions and the causes are still largely unknown. Temperature has long been associated with distribution limits, suggesting that there are ubiquitous constraints to the evolution of the climate niche. Here we investigated the traits involved in such constraints by macroevolutionary comparisons involving around 100 Brassicaceae species differing in elevational distribution. Plants were grown under three temperature treatments (regular frost, mild, regular heat) and phenotyped for phenological, morphological and thermal resistance traits. Trait values were analysed by assessing the effect of temperature and elevational distribution, by comparing models of evolutionary trajectories, and by correlative approaches to identify trade-offs. Analyses pointed to size, leaf morphology and growth under heat as among the most discriminating traits between low- and high-elevation species, with high-elevation species growing faster under the occurrence of regular heat bouts, at the cost of much reduced size. Mixed models and evolutionary models supported adaptive divergence for these traits, and correlation analysis indicated their involvement in moderate trade-offs. Finally, we found asymmetry in trait evolution, with evolvability across traits being 50% less constrained under regular frost. Overall, results suggest that trade-offs between traits under adaptive divergence contribute to the disparate distribution of species along the elevational gradient.


title: Deep Learning-based Microbubble Localization for Ultrasound Localization Microscopy

abstract: Ultrasound localization microscopy (ULM) is an emerging vascular imaging technique that overcomes the resolution-penetration compromise of ultrasound imaging. Accurate and robust microbubble (MB) localization is essential for successful ULM. In this study, we present a deep learning (DL)- based localization technique that uses both Field-II simulation and in vivo chicken embryo chorioallantoic membrane (CAM) data for training. Both radiofrequency (RF) and in-phase quadrature (IQ) data were tested in this study. The simulation experiment shows that the proposed DL-based localization was able to reduce both missing MB localization rate and MB localization error. In general, RF data showed better performance than IQ. For the in vivo CAM study with high MB concentration, DL-based localization was able to reduce the vessel MB saturation time by more than 50% as compared to conventional localization. Additionally, we propose a DL-based framework for real-time visualization of the high-resolution microvasculature. The findings of the paper support the use of DL for more robust and faster MB localization, especially under high MB concentrations. The results indicate that further improvement could be achieved by incorporating temporal information of the MB data.

category: bioengineering


title: IDSL.UFA assigns high confidence molecular formula annotations for untargeted LC/HRMS datasets in metabolomics and exposomics

abstract: Untargeted LC/HRMS assays in metabolomics and exposomics aims to characterize the small molecule chemical space in a biospecimen. To gain maximum biological insights from these datasets, LC/HRMS peaks should be annotated with chemical and functional information including molecular formula, structure, sub-structure, chemical class and metabolic pathways. Among these, a molecular formula can be assigned to a majority of LC/HRMS peaks using the theoretical and observed isotopic profiles (MS1) of the underlying ionized compound. For this, we have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics - Ultimate Formula Annotation (IDSL.UFA) R package. In the untargeted metabolomics validation tests, IDSL.UFA assigned 54.31%-85.51% molecular formula for true positive annotations as the top hit, and 90.58%-100% within the top five hits. Molecular formula annotations were further validated by MS/MS data. We have implemented novel strategies to 1) generate formula sources and their theoretical isotopic profiles 2) optimize the formula hits ranking on the individual and the aligned peak lists and 3) scale IDSL.UFA based workflows for studies with larger sample sizes. Annotating the raw data for a publicly available pregnancy metabolome study using IDSL.UFA highlighted hundreds of new pregnancy related compounds, and also suggested presence of chlorinated perfluorotriether alcohols (Cl-PFTrEAs) in human specimens. IDSL.UFA is useful for human metabolomics and exposomics studies where we need to minimize the loss of biological insights in untargeted LC/HRMS datasets. The IDSL.UFA package is available in the R CRAN repository https://cran.r-project.org/package=IDSL.UFA. Detailed documentation and tutorials are also provided at www.ufa.idsl.me.


title: Federated analysis in COINSTAC reveals functional network connectivity and spectral links to smoking and alcohol consumption in nearly 2,000 adolescent brains

abstract: With the growth of decentralized/federated analysis approaches in neuroimaging, the opportunities to study brain disorders using data from multiple sites has grown multi-fold. One such initiative is the Neuromark, a fully automated spatially constrained independent component analysis (ICA) that is used to link brain network abnormalities among different datasets, studies, and disorders while leveraging subject-specific networks. In this study, we implement the neuromark pipeline in COINSTAC, an open-source neuroimaging framework for collaborative/decentralized analysis. Decentralized analysis of nearly 2000 resting-state functional magnetic resonance imaging datasets collected at different sites across two cohorts and co-located in different countries was performed to study the resting brain functional network connectivity changes in adolescents who smoke and consume alcohol. Results showed hypoconnectivity across the majority of networks including sensory, default mode, and subcortical domains, more for alcohol than smoking, and decreased low frequency power. These findings suggest that global reduced synchronization is associated with both tobacco and alcohol use. This work demonstrates the utility and incentives associated with large-scale decentralized collaborations spanning multiple sites.


title: patcHwork: A user-friendly pH sensitivity analysis web server for protein sequences and structures

abstract:
pH regulates protein function and interactions by altering the charge of individual residues causing the loss or gain of intra-molecular non-covalent bonds, which may additionally lead to structural rearrangements. While tools to analyze residue-specific charge distribution of protein sequences and structures at a given pH exist, currently no tool is available to investigate non-covalent bond changes at two different pH values. In an effort to make protein pH sensitivity analysis more accessible to researchers without computational structural biology background, we developed patcHwork, a web server that combines the identification of amino acids undergoing a charge shift with the determination of affected non-covalent bonds at two user-defined pH values. At the sequence-only level, patcHwork applies the Henderson-Hasselbalch equation to determine pH-sensitive residues. When the 3D protein structure is available, patcHwork can be employed to gain a deeper mechanistic understanding of the effect of pH on a protein of interest. This is achieved using the PDB2PQR and PROPKA tools and non-covalent bond determination algorithms. A user-friendly interface allows visualizing pH-sensitive residues as well as affected salt bridges, hydrogen bonds and aromatic (pi-pi and cation-pi) interactions. Importantly, patcHwork can be used to identify patches, a new concept we propose of pH-sensitive residues in close proximity on the protein structure, which may have a major impact on function. We demonstrate the attractiveness of patcHwork studying experimentally investigated pH-sensitive proteins. (Access:https://patchwork.biologie.uni-freiburg.de/)

Graphical abstract

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title: New Functions Identified for HIV gp120 with the New ProtSub Sequence Matching

abstract: The reduced cost of sequencing is leading to an explosive growth in the number of available sequences across diverse genomes, and for individual patients. Inferring meaningful functions of individual genes/proteins is lagging, which hinders the deeper understanding of biological function and evolution. Traditionally, protein function has been determined by time consuming experimental methods or by sequence matching that often does not agree with the experimental findings. We have significantly improved protein sequence matching, by accounting for inter-dependent amino acid substitutions observed within densely packed protein structures, which yields additional substitutions beyond those usually seen, with good matches to additional proteins, some having new functions, not identified by conventional sequence matching. In the current study, we have applied this approach to predict novel functions for the proteins from HIV. These newly found functional annotations are then manually reviewed and many are validated from the literature, here for the HIV envelope protein gp120. These new functions are both more specific as well as some being entirely novel functions. We also show statistically that on average our new functional annotations are more informative than those given by conventional substitution matrices such as BLOSUM62. These results suggest that the new ProtSub protein sequence matching that incorporates structural information generally yields better identifications of related proteins, which can have broader and often gains in identifying more specific functions


title: In vitro effect of a non-immunosuppressive FKBP ligand, FK1706, on SARS-CoV-2 replication in combination with antivirals

abstract: FKBP, a naturally occurring ubiquitous intracellular protein, has been proposed as a potential target for coronavirus replication. A non-immunosuppressive FKBP ligand, FK1706, was studied in vitro in a Vero cell model to assess potential activity alone and in combination with antivirals against SARS-CoV-2 replication. When combined with remdesivir, synergistic activity was seen (summary synergy score 24.7+9.56). FK1706 warrants in vivo testing as a potential new combination therapeutic for the treatment of COVID-19 infections.

category: pharmacology and toxicology


title: Circulatory exosomes from COVID-19 patients trigger NLRP3 inflammasome in endothelial cells

abstract: SARS-CoV-2 infection induces inflammatory response, cytokine storm, venous thromboembolism, coagulopathy, and multiple organ damage. Resting endothelial cells prevent coagulation, control blood flow and inhibit inflammation. However, it remains unknown how SARS-CoV-2 induces strong molecular signals in distant cells for immunopathogenesis. In this study, we examined the consequence of human endothelial cells (microvascular endothelial cells (HMEC-1) and liver endothelial cells (TMNK-1)) to exosomes from plasma of severe COVID-19 patients. We observed a significant induction of NLRP3, caspase-1 and IL-1{beta} mRNA expression in the endothelial cells following exposure to exosomes from plasma of COVID-19 patients as compared to that of healthy donors. Activation of caspase-1 was noted in the endothelial cell culture medium following exposure to the COVID-19 exosomes. Further, COVID-19 exosomes significantly induced mature IL-1{beta} secretion in the endothelial cell culture medium. Thus, our results demonstrated for the first time that exosomes from COVID-19 plasma trigger NLRP3 inflammasome in endothelial cells of distant organs.

category: immunology


title: In vitro phenotypic and transcriptomic variation in Neisseria musculi morphotypes correlate with colonization variability and persistence in vivo

abstract:
Asymptomatic colonization of the upper respiratory tract is a common trait of the two human restricted pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. In vivo models of pathogenic neisserial infections are heterologous systems that permit short-term colonization but do not fully recapitulate infections in humans. Studying Neisseria musculi (Nmus), an oral commensal, in laboratory mice allows investigation of Neisseria-host interactions that avoids host restriction barriers. Nmus produces smooth and rough morphotypes on solid media. We compared the in vitro phenotypes, biofilm transcriptomes, in vivo colonization patterns and burdens of the two Nmus morphotypes. We observed that the two morphotypes differ in biofilm formation, pilin production, transformation frequency, and aggregation in vitro. These phenotypes strongly correlated with differential expression of a set of genes in the Nmus biofilms including those that encoded factors for bacterial attachment. In vivo, the smooth morphotype stably colonized the oral cavities of all inoculated A/J and C57BL/6J mice at higher burdens relative to the rough. Interestingly, both morphotypes colonized the oral cavities of A/Js at higher magnitudes than in C57BL/6Js. Gut colonization by the smooth morphotype was qualitatively higher than the rough. Nasal colonization in the A/Js were transient following nasal inoculations. Collectively, our results demonstrate that colonization by Nmus can be affected by various factors including Nmus morphotypes, inoculation routes, anatomical niches, and host backgrounds. The Nmus-mouse model can use variable morphotype-host combinations to study the dynamics of neisserial asymptomatic colonization and persistence in multiple extragenital niches.

IMPORTANCEAnimal models for human adapted pathogenic Neisseria spp. do not fully mimic human infections and are complicated by host restriction barriers that can hinder long-term persistence. Such barriers can be avoided by studying Neisseria spp. native to the animal host used for disease models. Neisseria musculi (Nmus) isolated from wild mice colonizes the oral cavity and gut of laboratory mice for extended periods. Nmus shares host interaction factors with species pathogenic to humans and thus provides a native system to study orthologs of factors that may facilitate asymptomatic colonization and persistence in the human upper respiratory tract. We investigated the Nmus-mouse system to compare in vitro and in vivo phenotypes of two Nmus morphotypes. Our results support the hypothesis that the two morphotypes vary in different aspects of Neisseria-host interactions. Future use of the Nmus-host system will help identify molecular mechanisms required for neisserial asymptomatic colonization, dissemination, and persistence.

category: microbiology


title: Re-sensitising XDR biofilms using a combination of bacteriophage cocktails and colistin: a natural approach

abstract: Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control MDR/XDR bacterial pathogens. 81 samples were screened for phages against selected clinical pathogens. 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae and 16 phages obtained against P. aeruginosa. The novel phages were observed to be strain-specific with a complete growth inhibition of up to 6 hrs. Phage plus colistin combinations further reduced the MBEC of colistin up to 16 folds. Notably, cocktail of phages exhibited supreme efficacy with complete killing at 0.5-1 {micro}g/ml colistin concentrations. Thus, phages specific to clinical strains has a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.


title: Deceiving The Big Eaters: Salmonella Typhimurium SopB subverts host cell Xenophagy through the Akt-TFEB axis in macrophages

abstract: Salmonella, a stealthy facultative intracellular pathogen, harbors an array of host immune evasion strategies. This facilitates successful survival and replicative niches establishment in otherwise hostile host innate immune cells such as macrophages. Salmonella survives and utilizes macrophages for effective dissemination throughout the host causing systemic infection. One of the central host defense mechanisms in macrophages is bacterial xenophagy or macro-autophagy. Here we report for the first time that Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy through dual mechanisms. SopB is known to act as a phosphoinositide phosphatase and thereby can alter the phosphoinositide dynamics of the host cell. Here we demonstrate that this activity helps the bacterium escape autophagy by inhibiting terminal fusion of Salmonella containing vacuole (SCV) with both lysosomes and autophagosomes. We also report the second mechanism, wherein SopB downregulates overall lysosomal biogenesis through Akt- transcription factor EB (TFEB) axis. TFEB is a master regulator of lysosomal biogenesis and autophagy, and SopB restricts the nuclear localization of TFEB. This reduces the overall lysosome content inside host macrophages, further facilitating survival in macrophages and systemic dissemination of Salmonella in the host.


title: VE607 Stabilizes SARS-CoV-2 Spike In the "RBD-up" Conformation and Inhibits Viral Entry

abstract: SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its "up" conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.


title: Type B and Type A influenza polymerases have evolved distinct binding interfaces to recruit the RNA polymerase II CTD

abstract:
During annual influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs), can become predominant and cause severe morbidity and mortality. Phylogenetic analyses suggest that IAVs (primarily avian viruses) and IBVs (primarily human viruses) have diverged over long time scales. Identifying their common and distinctive features is an effective approach to increase knowledge about the molecular details of influenza infection. The virus-encoded RNA-dependent RNA polymerases (FluPolB and FluPolA) are PB1-PB2-PA heterotrimers that perform transcription and replication of the viral genome in the nucleus of infected cells. Initiation of viral mRNA synthesis requires a direct association of FluPol with the host RNA polymerase II (RNAP II), in particular the repetitive C-terminal domain (CTD) of the major RNAP II subunit, to enable "cap-snatching" whereby 5-capped oligomers derived from nascent RNAP II transcripts are pirated to prime viral transcription. Here, we present the first high-resolution co-crystal structure of FluPolB bound to a CTD mimicking peptide at a binding site crossing from PA to PB2. By performing structure-based mutagenesis of FluPolB and FluPolA followed by a systematic investigation of FluPol-CTD binding, FluPol activity and viral phenotype, we demonstrate that IBVs and IAVs have evolved distinct binding interfaces to recruit the RNAP II CTD, despite the CTD sequence being highly conserved across host species. We find that the PB2 627 subdomain, a major determinant of FluPol-host cell interactions and IAV host-range, is involved in CTD-binding for IBVs but not for IAVs, and we show that FluPolB and FluPolA bind to the host RNAP II independently of the CTD. Altogether, our results strongly suggest that the CTD-binding modes of IAV and IBV represent avian- and human-optimized binding modes, respectively, and that their divergent evolution was shaped by the broader interaction network between the FluPol and the host transcriptional machinery.

Authors summaryDuring seasonal influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs) and can cause severe outcomes. The influenza polymerase is a key drug target and it is therefore important to understand the common and distinctive molecular features of IBV and IAV polymerases. To achieve efficient transcription and replication in the nucleus of infected cells, influenza polymerases closely cooperate with the cellular RNA polymerase II (RNAP II) and interact with the repetitive C-terminal domain (CTD) of its major subunit. Here we gained new insights into the way IBV and IAV polymerases interact with the CTD of RNAP II. High-resolution structural data was used to perform structure-based mutagenesis of IBV and IAV polymerases followed by a systematic investigation of their interaction with RNAP II, transcription/replication activity and viral phenotype. Strikingly, we found that IBVs and IAVs have evolved distinct interfaces to interact with the host transcriptional machinery, in particular with the CTD of RNAP II. We provide evidence that these differences may have evolved as a consequence of the differences in IBV and IAV host range. Our findings are of significant importance with regard to the development of broad-spectrum antivirals that target the virus-host interface.


title: Candidate undecaprenyl phosphate translocases enable conditional microbial fitness and pathogenesis

abstract:
The mechanisms that enable adaptation of peptidoglycan, the structural unit of the bacterial cell wall, to shifting extracellular conditions such as pH remain largely unknown. Here, we identify a DUF368-containing membrane protein in the cholera pathogen Vibrio cholerae that is critical for pathogenesis and alkaline fitness. V. cholerae and Staphylococcus aureus lacking their cognate DUF368-containing protein have pH-dependent cell wall defects consistent with surface accumulation of undecaprenyl phosphate (C55-P), an essential lipid carrier for the biogenesis of peptidoglycan and other key bacterial cell surface polymers. In both species, DUF368-containing proteins exhibit synthetic genetic interactions with putative transporters from the DedA family, suggesting these proteins represent complementary long-sought C55-P translocases that enable envelope maintenance functions critical for microbial fitness within and outside the host.

One-Sentence SummaryDUF368-containing and DedA-family proteins are undecaprenyl phosphate transporter candidates and are required for bacterial alkaline fitness and pathogenesis.


title: Viviparity is sufficient for the evolution of genomic imprinting

abstract: Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms.

category: molecular biology


title: Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia


title: During replicative aging, changes in DNA double-strand break repair correlate with an increased rate of mutation.

abstract:
A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). During replicative aging in the unicellular eukaryotic organism, S. cerevisiae, the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we show that DNA resection decreased early in replicative aging as did the recovery of long-range resection factors, Dna2-Sgs1 and Exo1. As aging progressed, the retention of Ku70 then decreased and irreparable DSBs accumulated with the pore complex at the nuclear periphery. End-bridging remained intact as HR and NHEJ declined, but eventually it became disrupted in cells of older replicative age. In all, our work provides insight about age-related molecular changes in DSB repair, where both canonical pathways become disrupted. HR declines first, leading to a transient reliance on NHEJ, which then also declines as cells repair through a mechanism yielding products with larger deletions and 1-2bp of microhomologies at break junctions.

HighlightsO_LIDecreased DNA resection at DSBs is an early event of replicative aging
C_LIO_LIEnd-joining repair increases as resection decreases at DSBs in older cells
C_LIO_LIIn older cells the products of DSB repair contain deletions and microhomologies
C_LIO_LIDSBs associate with the NPC at the nuclear periphery more in older cells
C_LIO_LIOld Cell Enrichment method suitable for molecular biology approaches in budding yeast
C_LI


title: ZNF598 responds to mitochondrial stress to abort stalled translation on mitochondrial outer membrane and maintain tissue homeostasis

abstract: Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination and its level is upregulated upon mitochondrial stress in mammalian cells and Drosophila. Overexpression of ZNF598 protects against mitochondrial stress. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborted stalled translation of mitochondrial outer membrane-associated mRNAs and removed faulty translation products causal of disease. These results shed lights on the regulation of ZNF598 and its important role in mitochondrial homeostasis.

category: cell biology


title: An Inducible Cre Mouse with Preferential Activity in Vascular Smooth Muscle Evades a Previously Lethal Intestinal Phenotype

abstract: All smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a new tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed limited activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice displayed high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains, including Myh11-CreERT2, where SMC gene loss results in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.

category: genetics


title: ACME: an Affinity-based Cas9 Mediated Enrichment method for targeted nanopore sequencing

abstract:
Targeted sequencing significantly improves accuracy and coverage and aids in providing the depth necessary to detect rare alleles in a heterogenous population of cells. Until the introduction of nanopore Cas9 Targeted-Sequencing (nCATS), a lack of efficient long-read compatible targeting techniques made it difficult to study specific regions of interest on long-read platforms. Existing nCATS-based strategies are currently limited by the per molecule target lengths capturable (<30kb), requiring several Cas9 guides to tile across larger regions of interest, ultimately reducing the number of targets that can be surveyed per reaction. Also, longer read lengths help reduce mapping errors, making it more likely that complex structural rearrangements can be resolved. Absence of a background reduction step in nCATS also increases the competition between non-target and target fragments in the sequencing pool for pore occupancy, decreasing the overall percentage of on-target reads. To address this, we introduce ACME - an Affinity-based Cas9-Mediated Enrichment method - that helps reduce background reads, increasing on-target coverage and size of target regions that can be spanned with single reads to 100kb.

ACME uses a HisTag-based isolation and pulldown of Cas-9 bound non-target reads, reducing the background noise in sequencing. We designed a panel of guide RNAs targeting 10 genes to enrich for specific regions of the cancer genome and tested them in two breast cell lines - MCF 10A and SK-BR-3. These gene targets spanned different size ranges (10kb to 150kb) allowing us to identify the largest target sizes that could be optimally captured by single molecules spanning the entire region. When compared with using just nCATS, the ACME method for background reduction increased the overall coverage across the entire length of all targets by 2-fold to 25-fold. By using ACME to eliminate smaller competing non-targets from the sequencing library, we saw a 3- to 7-fold increase in the number of reads spanning 100% of the gene targets when compared to nCATS. For one of our larger targets, BRCA2, we observed >60-fold target enrichment, close to 70x coverage, and 3-20 reads spanning the entire 95kb target. We observed an increase in enrichment, depth, and number of whole gene spanning reads for other genes on the panel as well across both cell lines, with enrichment as high as 4000-fold for some genes. Furthermore, ACME identified all SVs previously called within our targets by ONT and PacBio whole genome sequencing and performed on par with these platforms for SNP detection when compared with Illumina short-read whole genome sequencing.

category: genomics


title: Chimeric a-subunit isoforms generate functional yeast V-ATPases with altered regulatory properties in vitro and in vivo

abstract: V-ATPases are highly regulated, multi-subunit proton pumps that acidify organelles. The V-ATPase a-subunit is a two-domain protein containing a C-terminal transmembrane domain that participates in proton transport and a N-terminal cytosolic domain (aNT) that acts as a regulatory hub, integrating environmental inputs to regulate assembly, localization, and V-ATPase activity. Tissue- and organelle-specific a-subunit isoforms exist in most organisms, but how regulatory inputs are decoded by aNT isoforms is unknown. The yeast S. cerevisiae encodes only two organelle-specific a-isoforms, Stv1 in the Golgi and Vph1 in the vacuole. Based on recent structures, we designed chimeric yeast aNTs in which the globular proximal and distal ends are exchanged. The Vph1 proximal-Stv1 distal (VPSD) aNT chimera binds to the glucose-responsive RAVE assembly factor in vitro but exhibits little binding to phosphoinositide lipids that activate V-ATPases. The Stv1 proximal-Vph1 distal (SPVD) aNT lacks RAVE binding but binds more tightly to phosphoinositides than Vph1 or Stv1. When attached to the Vph1 C-terminal domain in vivo, both chimeras complement growth defects of a vph1{Delta} mutant, but only the SPVD chimera exhibits wild-type V-ATPase activity. Cells containing the SPVD chimera adapt more slowly to a poor carbon source than wild-type cells but grow more rapidly than wild-type after a shift to alkaline pH. This is the first example of a "redesigned" V-ATPase with altered regulatory properties and adaptation to specific stresses.

category: biochemistry


title: Analysis of altered pre-mRNA splicing patterns caused by a mutation in the RNA binding protein hnRNPA1 linked to amyotrophic lateral sclerosis

abstract: Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in a number of ALS patients. Here, we generate two cell models to investigate how a mutation in the C-terminal low complexity domain of hnRNPA1 affects global pre-mRNA splicing patterns and RNA binding. We show that a single amino acid change in the C-terminal low complexity domain (D262V) leads to changes in splicing of thousands of transcripts whose genes are linked to the DNA damage response, cilia organization and translation. We also show that there are changes in RNA binding of the mutant hnRNPA1 protein to transcripts whose splicing patterns change. Finally, we show that cells expressing the hnRNPA1 D262V mutation exhibit an aggregation phenotype, markedly reduced growth rates and changes in stress granules. This study shows that global changes in pre-mRNA splicing patterns caused by a single mutation in the hnRNPA1 protein lead to phenotypes related to ALS and that specific cellular pathways are affected.


title: Antibody affinity and concentration of convalescent sera provide context for reduced SARS-CoV-2 Omicron spike affinity of therapeutic antibodies

abstract: We assessed the affinities of the therapeutic monoclonal antibodies (mAbs) cilgavimab, tixagevimab, sotrovimab, casirivimab, and imdevimab to the receptor binding domain (RBD) of wild type, Delta, and Omicron spike. The Omicron RBD affinities of cilgavimab, tixagevimab, casirivimab, and imdevimab decreased by at least two orders of magnitude relative to their wild type equivalents, whereas sotrovimab binding was less severely impacted. These affinity reductions correlate with reduced antiviral activities of these antibodies, suggesting that simple affinity measurements can serve as an indicator for activity before challenging and time-consuming virus neutralization assays are performed. We also compared the properties of these antibodies to serological fingerprints (affinities and concentrations) of wild type RBD specific antibodies in 74 convalescent sera. The affinities of the therapeutic mAbs to wild type and Delta RBD were in the same range as the polyclonal response in the convalescent sera indicative of their high antiviral activities against these variants. However, for Omicron RBD, only sotrovimab retained affinities that were within the range of the polyclonal response, in agreement with its high activity against Omicron. Serological fingerprints thus provide important context to affinities and antiviral activity of mAb drugs and could guide the development of new therapeutics.


title: Serological fingerprints link antiviral activity of therapeutic antibodies to affinity and concentration


title: AAA+ protease-adaptor structures reveal altered conformations and ring specialization

abstract: ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here, we present high-resolution cryo-EM structures of ClpAPS complexes showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two structural classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated and tucked away from the channel, and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies demonstrate new structures and functions for key AAA+ elements. In addition to ClpAPS delivery, pore-loop tucking may be used broadly by AAA+ unfoldases, for example during enzyme pausing/unloading.

category: biophysics


title: KMT2C deficiency promotes APOBEC mutagenesis and genomic instability in multiple cancers

abstract: Histone methyltransferase KMT2C is among the frequently mutated epigenetic modifier genes in cancer. It has additional roles in DNA replication, but the effects of KMT2C deficiency on genomic instability during tumorigenesis are unclear. Analyzing 9,663 tumors from 30 cohorts, we report that KMT2C mutant tumors have a significant excess of APOBEC mutational signatures in several cancer types. We show that KMT2C deficiency promotes APOBEC expression and deaminase activity, and compromises DNA replication speed and delays fork restart, facilitating APOBEC mutagenesis targeting ssDNA near stalled forks. APOBEC-mediated mutations primarily accumulate during early replication, and tend to cluster along the genome and also in 3D nuclear contexts. Excessive APOBEC mutational signatures in KMT2C mutant tumors correlate with elevated genomic instability and signatures of homologous recombination deficiency. We propose that in multiple cancer types KMT2C deficiency is a likely driver of APOBEC mutagenesis, which promotes further genomic instability during cancer progression.

category: cancer biology


title: Minimal RNA replicons for targeted gene silencing based on an asymptomatic viroid

abstract: Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and disrupt plant homeostasis by interfering with endogenous cell mechanisms. To circumvent this issue, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids. In particular, Eggplant latent viroid (ELVd), an asymptomatic viroid, was manipulated with insertions between 21 to 42 nucleotides and our results show that larger insertions are tolerated but secondary structure is critical for their stability. Additionally, these ELVd constructs are able of local and systemic spread and can silence a target gene in eggplant. Inspired by the design of artificial microRNAs, we have developed a standardized procedure to generate stable insertions into the ELVd genome capable of silencing the desired target gene. Analogously to VIGS, we have termed our approach Viroid Induced Gene Silencing (VdIGS) and demonstrate that is a promising tool for dissecting gene functions in eggplant. Overall, this represents the use of minimal circular replicating RNAs able to spread systemically combined with the production of a tailored sRNA for targeted silencing.

category: plant biology


title: Genome-wide identification, characterization, and comparative analysis of NLR resistance genes in Coffea spp.

abstract: The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.


title: Science in motion: A qualitative analysis of journalists' use and perception of preprints

abstract: This qualitative study explores how and why journalists use preprints -- unreviewed research papers -- in their reporting. Through thematic analysis of interviews conducted with 19 health and science journalists in the second year of the COVID-19 pandemic, it applies a theoretical framework that conceptualizes COVID-19 preprint research as a form of post-normal science, characterized by high scientific uncertainty and societal relevance, urgent need for political decision-making, and value-related policy considerations. Findings suggest that journalists approach the decision to cover preprints as a careful calculation, in which the potential public benefits and the ease of access preprints provided were weighed against risks of spreading misinformation. Journalists described viewing unreviewed studies with extra skepticism and relied on diverse strategies to find, vet, and report on them. Some of these strategies represent standard science journalism, while others, such as labeling unreviewed studies as preprints, mark a departure from the norm. However, journalists also reported barriers to covering preprints, as many felt they lacked the expertise or the time required to fully understand or vet the research. The findings suggest that coverage of preprints is likely to continue post-pandemic, with important implications for scientists, journalists, and the publics who read their work.


title: Beta-cell Cre expression and reduced Ins1 gene dosage protect mice from type 1 diabetes

abstract: Cre-mediated recombineering is the main tool of choice for cell type-specific analysis of gene function in pre-clinical models. In the type 1 diabetes research field, multiple lines of NOD mice have been generated that express Cre in pancreatic {beta}-cells using insulin promoter fragments, but tissue promiscuity remains a concern. The constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mouse on a C57/bl6-J background has been shown to have high {beta}-cell specificity and with no off-target effects reported. Therefore, we explored if Ins1Cre mice on an NOD background could be used as a novel tool to investigate {beta}-cell specific gene deletion in a type 1 diabetes setting. Here, we examine a new NOD mouse model in which Cre is inserted into the endogenous Ins1 locus for ideal {beta}-cell specificity. Fully wildtype (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null mice (Ins1Cre/Neo) littermate mice had either none, one or two Ins1 alleles replaced with Cre or a neomycin cassette. Female Ins1 Neo/WT mice exhibited significant protection from type 1 diabetes. Ins1Cre/WT knock-in mice were further protected. The effects of combined neo and Cre knock-in in Ins1Neo/Cre mice were additive and resulted in near-complete type 1 diabetes prevention up to one year of age. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced in insulitis at 12 weeks of age. We were unable to find significant differences in insulin auto-antibodies or in immune populations between genotypes in the pancreatic lymph node or spleen in 50 weeks old mice, suggesting a {beta}-cell specific mechanism. Collectively, these data confirm previous reports that the loss of Ins1 alleles protects NOD mice from diabetes and demonstrates, for the first time, that Cre itself may have additional protective effects. This has significant implication for the experimental design and interpretation of pre-clinical type 1 diabetes studies using {beta}-cell-specific Cre in NOD mice.


title: Inactivating Mast Cell Function Promotes Steady-State and Regenerative Hematopoiesis

abstract: Deeper understanding of the cellular and molecular pathways regulating hematopoiesis is critical to maximize the therapeutic potential of hematopoietic stem cells (HSCs) in curative procedures including hematopoietic stem cell transplantation (HST). We have recently identified mast cells (MCs) as therapeutically-targetable components of the HSC niche. Here, we demonstrate that mice lacking MCs display peripheral neutrophilia, expansion of bone marrow (BM) HSC populations, resistance to repeated 5-fluorouracil (5-FU) administration, and a BM genetic signature primed for hematopoietic proliferation. MC deficiency functionally altered both the hematopoietic and the stromal compartment of the BM as hematopoietic reconstitution was accelerated in wildtype mice that received MC deficient BM and in MC deficient recipients that received wildtype BM. Finally, we demonstrate that mice treated at steady state with the MC stabilizing agent ketotifen exhibit increased BM cellularity as well as expansion of phenotypic HSC populations. This work provides novel mechanistic rationale to explore mast cells as a target to enhance human BM transplants. Additionally, the potential of repurposing FDA approved mast cell targeting therapies to promote hematopoietic regeneration may provide well-tolerated treatment strategies at a fraction of the cost and development time.


title: Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

abstract:
Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA in viral replication organelles in infected cells and directly interacted with high affinity with DENV RNA in the 3 untranslated region in vitro (KD = 100-200 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.

AUTHOR SUMMARYDengue virus is a mosquito-transmitted virus endemic to the tropics and subtropics, affecting an estimated 390 million people yearly. While the mechanisms of infection, pathogenesis and immune evasion have been extensively studied in humans, replication in Aedes mosquitoes has received much less attention, despite being a critical step in the arbovirus transmission cycle. Here, we used a proteomic approach to identify Aedes mosquito proteins recruited by dengue virus non- structural proteins NS1 and NS5. In addition to previously established host proteins that interact with DENV in mammals, we identified Loquacious, a double-stranded RNA binding protein involved in the antiviral RNAi immune response of mosquitoes. Unexpectedly, our data showed Loquacious functions as a proviral factor that is recruited to replication organelles to facilitate viral RNA replication. We propose that DENV exploits host immune components, such as Loquacious, for its own benefit.


title: Testing the Efficacy of Transungual Drug Delivery System (Nail Lacquer) Containing Terbinafine Against Aspergillus brasiliensis for the Topical Treatment of Onychomycosis.

abstract: According to a survey of 42 epidemiological studies, the cases of Non-dermatophyte mould onychomycosis (NDMO) caused by Aspergillus species have been found to increase. The number ranges from <1 to 35% of all cases of onychomycosis in the general population and the number is higher in the diabetic population. Around7.7-100% of cases of NDMO are caused by Aspergillus species. Hence the attempt of investigation to test the efficacy of the drug by transungual drug permeation has been made. The antifungal agent  Terbinafine was incorporated in a topical dosage form (Nail lacquer). The formulation is optimized by a drug design matrix of 23 type using Design-Expert(R)software, version 11 (DX11). The antifungal assay was performed using the Kirby Bauer disk diffusion method and other evaluation parameters like Drying time, Viscosity are evaluated. Optimized formulation with the highest desirability was selected out of all the possible formulations which showed the best results with good physicochemical properties and antifungal activity. Thus, it can be concluded that medicated nail lacquers containing Terbinafine can be used as a successful method to treat non-dermatophyte mould onychomycosis (NDMO) caused by Aspergillus species.


title: Model-free prediction of microbiome compositions

abstract: The recent recognition of the importance of the microbiome to the hosts health and well-being, has yielded efforts to develop therapies that aim to shift the microbiome from a disease-associated steady-state to a healthier one. Direct manipulation techniques of the species assemblage are currently available, e.g., using probiotics or narrow-spectrum antibiotics to introduce or eliminate specific taxa. However, predicting the species abundances at the new steady-state remains a challenge, mainly due to the difficulties of deciphering the delicate underlying network of ecological interactions or constructing a predictive model for such complex ecosystem. Here, we propose a model-free method to predict the species abundances at the new steady state based on their presence/absence configuration by utilizing a multi-dimensional k-nearest-neighbors (kNN) regression algorithm. By analyzing data from numeric simulations of ecological dynamics, we show that our predictions, which consider the presence/absence of all species holistically, outperform both the null model that uses the statistics of each species independently and a predictive neural network model. We analyze real metagenomic data of human-associated microbial communities and found that by relying on a small number of  neighboring samples, i.e., samples with similar species assemblage, the kNN predicts the species abundance better than the whole-cohort average. By studying both real metagenomic and simulated data, we show that the predictability of our method is tightly related to the dissimilarity-overlap relationship of the training data. Our results demonstrate how model-free methods can prove useful in predicting microbial communities and may facilitate the development of microbial-based therapies.


title: Air pollution induces Staphylococcus aureus USA300 respiratory tract colonisation mediated by specific bacterial genetic responses dependent on the global virulence gene regulators Agr and Sae

abstract:
Exposure to particulate matter (PM), a major component of air pollution, is associated with exacerbation of chronic respiratory disease, and infectious diseases such as community acquired pneumonia. Although PM can cause adverse health effects through direct damage to host cells, our previous study showed that PM can also impact bacterial behaviour by promoting in vivo colonisation. In this study we describe the genetic mechanisms involved in the bacterial response to exposure to black carbon (BC), a constituent of PM found in most sources of air pollution. We show that Staphylococcus aureus strain USA300 LAC grown in BC prior to inoculation showed increased murine respiratory tract colonisation and pulmonary invasion in vivo, as well as adhesion and invasion of human epithelial cells in vitro. Global transcriptional analysis showed that BC has a widespread effect on S. aureus transcriptional responses, altering the regulation of the major virulence gene regulators Sae and Agr and causing increased expression of genes encoding toxins, proteases, and immune evasion factors. Together these data describe a previously unrecognised causative mechanism of air pollution-associated infection, in that exposure to BC can increase bacterial colonisation and virulence factor expression by acting directly on the bacterium rather than via the host.

Originality-Significance StatementThis study shows that exposure to air pollution results in a global change in gene expression in bacteria. Specifically, our data show that in the important human pathogen Staphylococcus aureus, exposure to a major constituent of air pollution, black carbon (BC) results in widespread changes in global gene expression, altering the expression of key virulence determinants. Furthermore, S. aureus that are exposed to BC prior to inoculation show increased colonisation of the murine nasopharynx and lungs in vivo, and increased adhesion and invasion in lung epithelial cells in vitro. These findings indicate that air pollution has a significant and direct impact on bacteria, altering their behaviour and their potential to colonise and invade during infection. While many studies have taken a host-focussed approach to studying the impact of air pollution on human health, this study takes a pathogen-focussed approach to further the understanding of these fundamental interactions to identify new causative mechanisms of the detrimental effects of air pollution. This is critical for understanding the adverse health effects caused by exposure to air pollution, the single largest environmental risk to human health in the world.


title: Mechanical control of the mammalian circadian clock via YAP/TAZ and TEAD

abstract: Circadian rhythms are a key survival mechanism that dictates biological activity according to the day-night cycle1. In animals, cell-autonomous circadian clocks can be found in nearly every cell type and are subjected to multi-layered regulation2-4. Although these peripheral clocks are remotely controlled by the master clock in the brain, they are also sensitive to their immediate physical microenvironment through mechanisms that are still unknown5,6. Here we show that the circadian clock in fibroblasts is regulated mechanically through YAP/TAZ and TEAD. We use high-throughput analysis of single-cell circadian rhythms and apply controlled mechanical, biochemical, and genetic perturbations to study the expression of the core clock gene Rev-erb7. We observe that Rev-erb circadian oscillations are disrupted concomitantly with the translocation of YAP/TAZ to the nucleus. By targeted mutations and tuning expression levels of YAP we identify TEAD as the transcriptional effector of this mechanosensitive regulatory pathway. Our findings establish a mechanism that links cell mechanobiology and the circadian clock, which could contribute to explain the circadian impairment observed in cancer and ageing, where the regulation of the mechanical environment and YAP/TAZ is lost8-11.


title: Single cell transcriptomic and spatial landscapes of the developing human pancreas

abstract: The progress made in directed differentiation of stem cells has shown that understanding human pancreas development can provide cues for generating unlimited amounts of insulin-producing beta cells for transplantation therapy in diabetes. However, current differentiation protocols have not been successful in reproducibly generating functional human beta cells in vitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single cell RNA sequencing with spatial transcriptomics at multiple developmental timepoints and revealed distinct temporal-spatial gene cascades in the developing human pancreas. Cell trajectory inference identified endocrine progenitor populations and novel branch-specific genes as the progenitors differentiate towards alpha or beta cells, indicating that transcriptional maturation occurred over this developmental timeframe. Spatial differentiation trajectories indicated that immature Schwann cells are spatially co-located with endocrine progenitors and contribute to beta cell maturation via the L1CAM-EPHB2 pathway. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for interrogating human pancreas development for the research community.

category: developmental biology


title: Interactions between evolved pesticide resistance and pesticide exposure on immunity against pathogens

abstract: Chemical and microbial pesticides are both important tools in combating agricultural pests and disease vectors. However, the interactive effects of chemical and microbial agents on insect physiology could result in either antagonism or facilitation among control strategies, as both can activate stress, detoxification, and immune responses. In natural populations, moreover, the evolution of resistance to chemical pesticides is common and often involves differential regulation of mechanisms involved in the primary responses to both pathogens and pesticides, adding an additional degree of complexity for predicting net control outcomes. To investigate the interactive effects of chemical pesticide resistance, exposure, and bacterial infection on host outcomes, we experimentally evolved resistance to two different classes of pesticides (organophosphates and pyrethroids) in the red flour beetle, Tribolium castaneum. We exposed pesticide susceptible and resistant lines to pesticides, the entomopathogen and biocontrol agent Bacillus thuringiensis (Bt), or both. Pesticide resistance and Bt exposure were individually associated with slower development, indicating sub-lethal fitness costs to resistance and infection, respectively. After organophosphate exposure, however, beetles developed more quickly and were more likely to survive if also exposed to Bt. We used RNAseq to examine the interactive effects of pesticide resistance, pesticide exposure, and Bt exposure on gene expression. Pyrethroid-resistant insects exhibited dampened immune responses to Bt infection relative to susceptible ones. In a similar vein, simultaneous exposure to organophosphates and Bt resulted in muted stress-associated transcriptional responses compared to exposure with only one factor. Our data suggest that evolved resistance, pesticide exposure, and biopesticide infection all exert contrasting effects on life history parameters and host physiology that could ultimately influence the demography and control of pest populations in complex ways.

category: evolutionary biology


title: Cryomilling Tethered Chromatin Conformation Capture reveal new insights into inter-chromosomal interactions

abstract: Traditional methods used to map the three-dimensional organization of chromatin in-situ generally involve chromatin conformation capture by formaldehyde crosslinking, followed by detergent solubilization and enzymatic digestion of DNA. Ligation of proximal DNA fragments followed by next generation sequencing (NGS) generates contact information that enables a global view of the chromatin conformation. Here, we explore the use of cryomilling to physically fragmentize the cells under cryogenic conditions to probe chromatin interactions in the cryomilled cell fragments by the tethered chromatin conformation capture (TCC). Our results show that cryomilling TCC (CTCC) can generate a global contact map similar to that obtained with in-situ Hi-C. This result suggests that summation of chromatin interactions mapped in individual subcellular fragments can reconstitute the global contact map of intact cells in an ensemble manner, paving the way for chromatin conformation analyses of solid tissue by CTCC. Compared with the conventional in-situ methods such as Hi-C, CTCC shows more uniform access to different subcompartments of the folded genome. On the other hand, most inter-chromosomal (trans) contacts are diminished or lost in CTCC except for a group of unique trans contacts that remain intact throughout the cryomilling and in- vitro crosslinking steps. These apparently ultra-stable trans interactions have much enhanced signal in CTCC due to the elimination of signals of most, presumably weak and transient trans interactions. Systematic and comparative analyses between CTCC and in-situ Hi-C provide further insights into the chromatin structure organization and reveal a generally unentangled chromosome interface and the existence of stable inter-chromosomal contacts that may represent intermingled inter-chromosomal interfaces.


title: Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

abstract: Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA secondary structure and nucleotide modifications can both interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We find that OTTR robustly captures full-length tRNAs in budding yeast and in mouse testis, with relatively low levels of premature termination at known barriers - 1-methylguanine, N2,N2-dimethylguanine, and 1-methyladenine - to typical reverse transcriptases. Moreover, these and several other nucleotide modifications leave misincorporation signatures in OTTR datasets which enables their detection without any additional protocol steps. Turning to analysis of small RNAs such as tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRFs - including both 5 and 3 tRNA halves derived from the majority of tRNAs - than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.


title: Effect of compound Tinglizi Decoction on non-classical pathway of pyroptosis in rats with chronic obstructive pulmonary disease related pulmonary hypertension

abstract:
ObjectiveTo observe the effect of compound Tinglizi Decoction on non-classical pathway of cell death in rats with pulmonary hypertension, and to explore its mechanism of action in the treatment of COPD-related pulmonary hypertension.

Methods60 male SD rats were randomly divided into Western group, normal group, model group, high-dose group, medium dose group and low-dose group, with 10 rats per group. We detected the protein expression levels of caspase-11, GSDMD and IL-6 by Western blot, detected serum TNF- by ELISA, and observed the morphology of lung tissue by hematoxylin and eosin (HE) staining.

ResultsIn the expression of caspase11, GSDMD, IL-6 mRNA and protein in lung tissue, the model group was significantly higher than the normal group (P < 0.01), and the high-dose group, middledose group, low-dose group and western medicine group were significantly lower than the model group (P < 0.01). The levels of serum TNF- and serum creatinine in the model group were significantly higher than those in the normal group (P < 0.01); and those in the high-dose group, middle-dose group, low-dose group and western medicine group were significantly lower than those in the model group (P < 0.05).

ConclusionThe mechanism of compound Tinglizi Decoction in the intervention of COPD-related pulmonary hypertension rats is related to the inhibition of non-classical pathway of cell death.


title: Membrane-catalyzed IAPP aggregation is dominated by secondary nucleation

abstract: Type II diabetes is characterized by the loss of pancreatic {beta}-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, i.e. the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent {beta}-cell death in type II diabetes.


title: Membrane-catalyzed aggregation of islet amyloid polypeptide is dominated by secondary nucleation


title: Evaluation of Nutritional Potential and Effect of Processing on Improving Nutrient Content of Cassava (Mannihot esculenta crantz) Root and Leaves

abstract: Cassava (Manihot esculenta crantz) is a major food crop in Sub-Saharan Africa, Asia, the Pacific, and South America, where it feeds around 800 million people. Its roots are an excellent source of energy, and its leaves are rich in minerals, vitamins, and protein, which could substantially supplement the current starchy diets if properly detoxified since they contain some toxic anti-nutritional factors. The objective of this study was to provide information on the nutritional profile of cassava root and leaves and role of processing in enhancing and stabilizing their valuable nutrients. Two cassava varieties, kello and qulle, obtained from Areca Agricultural Research Center were used in this study. Roots and leaves were processed (fermented, boiled, and fluid bed dried), and nutritional, anti-nutritional, and functional properties were assessed using AOAC (Association of Official Analytical Chemists) standard procedures. As compared to leaves, roots had higher carbohydrate contents, ranging from 81.0 to 87.1 g/100g, whereas leaves had higher protein (21.2 to 28.4g/100g), total carotenoid (234.1 to 987.9 g/g), fiber (16.1 to 22.9 g/100g), Ca (499.8 to 545.4 mg/100g), K (1193.4 to 1211.0 mg/100g), Mg (274.5 to 340.5 mg/100g) and Fe (129.1 to 146.1 mg/kg) contents. Anti-nutritional factors were slightly higher in the leaves than in the roots, with toxic cyanides ranging from 10.1 to 61.0 mg/kg in the leaves and from 1.8 to 47.5 mg/kg in the roots. However, the processing of leaves decreased cyanide content from 61.0 mg/kg to 10.1 mg/kg. Similarly, other anti-nutritional factors, such as condensed tannins, oxalates, and phytate were reduced from 52.0 to 21.0 mg/100g, 156.5 to 25.9 mg/100g, and 35.9 to 4.0 mg/100g), respectively. Hence, the fermentation of leaves and roots followed by boiling and drying is an interesting approach to reduce anti-nutritional factors significantly and ensure their nutritional quality. This study revealed that variety kello had a relatively better nutritional profile than variety qulle, for both root and leaves, except for total carotenoid content, which was higher in the latter. Genetic background and processing methods can greatly affect the nutritional profile of cassava varieties, so it is critical to analyze diverse cassava germplasm and refine the processing method to identify highly nutritious and healthy varieties.

category: scientific communication and education