title: Adding context to the pneumococcal core genesA bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae

abstract: Whole genome sequencing offers great opportunities for linking genotypes to phenotypes aiding in our understanding of human disease and bacterial pathogenicity. However, these analyses often overlook non-coding intergenic regions (IGRs). By disregarding the IGRs, crucial information is lost, as genes have little biological function without expression. In this study, we present the first complete pangenome of the important human pathogen Streptococcus pneumoniae (pneumococcus), spanning both the genes and IGRs. We show that the pneumococcus species retains a small core genome of IGRs that are present across all isolates. Gene expression is highly dependent on these core IGRs, and often several copies of these core IGRs are found across each genome. Core genes and core IGRs show a clear linkage as 81% of core genes are associated with core IGRs. Additionally, we identify a single IGR within the core genome that is always occupied by one of two highly distinct sequences, scattered across the phylogenetic tree. Their distribution indicates that this IGR is transferred between isolates through horizontal regulatory transfer independent of the flanking genes and that each type likely serves different regulatory roles depending on their genetic context.

category: bioinformatics


title: dynDeepDRIM: a dynamic deep learning model to infer direct regulatory interactions using single cell time-course gene expression data

abstract: Time-course single-cell RNA sequencing (scRNA-seq) data have been widely applied to reconstruct the cell-type-specific gene regulatory networks by exploring the dynamic changes of gene expression between transcription factors (TFs) and their target genes. The existing algorithms were commonly designed to analyze bulk gene expression data and could not deal with the dropouts and cell heterogeneity in scRNA-seq data. In this paper, we developed dynDeepDRIM that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. dynDeepDRIM integrated the primary image, neighbor images with time-course into a four-dimensional tensor and trained a convolutional neural network to predict the direct regulatory interactions between TFs and genes. We evaluated the performance of dynDeepDRIM on five time-course gene expression datasets. dynDeepDRIM outperformed the state-of-the-art methods for predicting TF-gene direct interactions and gene functions. We also observed gene functions could be better performed if more neighbor images were involved.


title: Identifying SNP associations and predicting disease risk from Genome-wide association studies using LassoNet

abstract: In this paper, we show that under certain conditions LassoNet [1] should outperform threshold of significance (P-value) methods for identifying multi-SNP disease associations and predicting disease risk using data from Genome Wide Association Studies. To demonstrate this, we built a genotype-phenotype simulation to comprehensively benchmark each methods performance in variant selection and in predicting disease risk. Our results suggest that LassoNet, with its ease of implementation, should be added to the biomedical informaticians toolkit. We release code to replicate our results.


title: Spatio-temporal feature based deep neural network for cell lineage analysis in microscopy images

abstract:
BackgroundTime-lapse microscopy has been widely used in biomedical experiments because it can visualize the molecular activities of living cells in real time. However, biomedical researchers are still conducting cell lineage analysis manually. Developing automatic lineage tracing algorithms is a challenging task. In the past two decades, deep neural networks (DNNs) became have shown outstanding performance on computer vision tasks. They can learn complex visual features, capture long-range temporal dependencies, and have the potential to be used for automatic cell lineage analysis.

MethodsIn this study, we propose a multi-task spatio-temporal feature based deep neural network for cell lineages analysis (Cell-STN). The Cell-STN extracts spatio-temporal features from microscopy image sequences by leveraging our convolutional long short-term memory based core block. And the proposed Cell-STN utilized a task specific network to predict the cell location, the mitosis event, and the apoptosis event in a multi-task manner.

ResultsWe evaluated the Cell-STN on three in-house datasets (MCF7, U2OS, and HCT116) and one public dataset (Fluo-N2DL-HeLa). For cell tracking, we used peak-wise precision, track-wise precision, end-peak precision, and spatial distance as metrics. The overall results showed the Cell-STN models outperform other state-of-the-art cell trackers. For mitosis and apoptosis tasks, we used accuracy, F1-score, temporal distance, and spatial distance as metrics. The Cell-STN models achieved the highest performance on all datasets.

ConclusionThis study presented a novel DNNs approach for cell lineage analysis in microscopy images. The Cell-STN showed outstanding performance on the four datasets. Additionally, the Cell-STN required minimal training data and can be adapted to new biological event detection tasks by appending task-specific layers. This algorithm has the potential to be used in real-world biomedical research.


title: Comparing the efficiency of monofilament and traditional nets for capturing bats

abstract: Traditional mist nets used for capturing bats have several drawbacks, particularly that they are inefficient at sampling many insectivorous species. One possible alternative is to use monofilament nets, whose netting is made of single strands of yarn instead of several as regular nets, making them less detectable. To date, no study has quantified the capture efficiency of monofilament nets compared to regular mist nets for the study of bats. Here we compare capture efficiency of monofilament and regular mist nets, focusing on bat abundance and species diversity at a lowland tropical forest in southwestern Costa Rica. During our sampling period, we captured 90 individuals and 14 species in regular nets and 125 individuals and 20 species in monofilament nets. The use of monofilament nets increased overall capture rates, but most notably for insectivorous species. Species accumulation curves indicate that samples based on regular nets are significantly underestimating species diversity, most notably as these nets fail at sampling rare species. We show that incorporating monofilament nets into bat studies offers an opportunity to expand records of different guilds and rare bat species and to improve our understanding of poorly-known bat assemblages while using a popular, relatively cheap and portable sampling method.

category: zoology


title: Large-scale differences in functional organization of left- and right-handed individuals using whole-brain, data-driven analysis of connectivity

abstract: Handedness influences differences in lateralization of language areas as well as dominance of motor and somatosensory cortices. However, differences in whole brain functional organization due to handedness have been relatively understudied beyond pre-specified networks of interest. Functional connectivity offers the ability to unravel differences in the functional organization of the whole brain. Here, we compared connectivity profiles of left- and right-handed individuals using data-driven parcellations of the whole brain. We explored differences in connectivity profiles of previously established regions of interest, and showed functional organization differences between primarily left- and primarily right-handed individuals in the motor, somatosensory, and language areas using functional connectivity. We then proceeded to investigate these differences in the whole brain and found that the functional organization of left- and right-handed individuals are not specific to regions of interest. In particular, we found that connections between and within-hemispheres and the cerebellum show distinct patterns of connectivity. Together these results shed light on regions of the brain beyond those traditionally explored that contribute to differences in the functional organization of left- and right-handed individuals.

category: neuroscience


title: Resting-state network topology characterizing callous-unemotional traits in adolescence

abstract:
BackgroundCallous-unemotional (CU) traits, a youth antisocial phenotype, are hypothesized to associate with aberrant connectivity (dis-integration) across the salience (SAL), default mode (DMN), and frontoparietal (FPN) networks. However, CU traits have a heterogeneous presentation and previous research has not modeled individual heterogeneity in resting-state connectivity amongst adolescents with CU traits. The present study models individual-specific network maps and examines topological features of individual and subgroup maps in relation to CU traits.

MethodsParticipants aged 13-17 completed resting-state functional connectivity and the inventory of callous-unemotional traits as part of the Nathan Klein Rockland study. A sparse network approach (GIMME) was used to derive individual-level and subgroup maps of all participants. We then examined heterogeneous network features associated with CU traits.

ResultsHigher rates of CU traits increased probability of inclusion in one subgroup, which had the highest mean level of CU traits. Analysis of network features reveals less density within the FPN and greater density between DMN-FPN associated with CU traits.

DiscussionFindings indicate heterogeneous person-specific connections and some subgroup connections amongst adolescents associate with CU traits. Higher CU traits associate with lower density in the FPN, which has been associated with attention and inhibition, and higher density between the DMN-FPN, which have been linked with cognitive control, social working memory, and empathy. Our findings suggest less efficiency in FPN function which, when considered mechanistically, could result in difficulty suppressing DMN when task positive networks are engaged. This is an area for further exploration but could explain cognitive and socio-affective impairments in CU traits.


title: Mussel memory: bivalves learn to fear parasites

abstract: Fear plays a crucial role in predator-prey interactions and can have cascading impacts on the structure of whole ecosystems. Comparable fear effects have recently been described for hosts and their parasites but our understanding of the underlying mechanisms remains limited by the lack of empirical examples. Here, we experimentally tested if bivalves Mytilus edulis can  learn to fear the infective transmission stages (cercariae) of the trematode Himasthla elongata and if experienced mussels change their parasite-avoidance behaviour accordingly. Our results show that previous experience with parasites, but not infection per se, lead to a reduced filtration activity in mussels in the presence of cercariae compared to parasite-naive conspecifics. This reduction in filtration activity resulted in lower infection rates in mussels. Since parasite avoidance comes at the cost of lower feeding rates, mussels likely benefit from the ability to adjust their defence behaviour when infection risks are high. Overall, these dynamic learning processes of avoidance behaviour can be expected to play a significant role in regulating the bivalves ecosystem engineering function in coastal habitats.

category: ecology


title: Policy-relevant indicators for invasive alien species assessment and reporting

abstract: Invasive alien species are repeatedly shown to be amongst the top threats to biodiversity globally. Robust indicators for measuring the status and trends of biological invasions are lacking, but essential for monitoring biological invasions and the effectiveness of interventions. Here, we formulate and demonstrate three such indicators that capture the key dimensions of species invasions, each a significant and necessary advance to inform invasive alien species policy targets: 1) Rate of Invasive Alien Species Spread, which provides modelled rates of ongoing introductions of species based on invasion discovery and reporting. 2) Impact Risk, that estimates invasive alien species impacts on the environment in space and time and provides a basis for nationally targeted prioritization of where best to invest in management efforts. 3) Status Information on invasive alien species, that tracks improvement in the essential dimensions of information needed to guide relevant policy and data collection and in support of assessing invasive alien species spread and impact. We show how proximal, model-informed status and trend indicators on invasive alien species can provide more effective global (and national) reporting on biological invasions, and how countries can contribute to supporting these indicators.


title: Leveraging epigenomes and three-dimensional genome organization for interpreting regulatory variation

abstract: Understanding the impact of regulatory variants on complex phenotypes is a significant challenge because the genes and pathways that are targeted by such variants are typically unknown. Furthermore, a regulatory variant might influence a particular genes expression in a cell type or tissue-specific manner. Cell-type specific long-range regulatory interactions that occur between a distal regulatory sequence and a gene offers a powerful framework for understanding the impact of regulatory variants on complex phenotypes. However, high-resolution maps of such long-range interactions are available only for a handful of model cell lines. To address this challenge, we have developed L-HiC-Reg, a Random Forests based regression method to predict high- resolution contact counts in new cell lines, and a network-based framework to identify candidate cell line-specific gene networks targeted by a set of variants from a Genome-wide association study (GWAS). We applied our approach to predict interactions in 55 Roadmap Epigenome Consortium cell lines, which we used to interpret regulatory SNPs in the NHGRI GWAS catalogue. Using our approach, we performed an in-depth characterization of fifteen different phenotypes including Schizophrenia, Coronary Artery Disease (CAD) and Crohns disease. In CAD, we found differentially wired subnetworks consisting of known as well as novel gene targets of regulatory SNPs. Taken together, our compendium of interactions and associated network-based analysis pipeline offers a powerful resource to leverage long-range regulatory interactions to examine the context-specific impact of regulatory variation in complex phenotypes.


title: The OPAQUE1/DISCORIDA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize

abstract: Asymmetric divisions produce cells with different fates and are critical for development. Here we show that a maize myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells prior to and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 occurs during late cytokinesis, when the plant-specific phragmoplast -- made of microtubules, actin and other proteins - forms the nascent cell plate. The phragmoplast becomes misguided and does not meet the previously established division site. Initial phragmoplast guidance is correct in o1. However, as phragmoplast expansion continues, phragmoplasts in o1 stomatal precursor cells become misguided and do not meet the cortex at the established division site. To understand how this myosin protein contributes to phragmoplast guidance, we identified O1-interacting proteins. Other myosins, specific actin-binding proteins, and maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs) interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

category: plant biology


title: The OPAQUE1/DISCORDIA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize


title: Peer teaching as bioinformatics training strategy: incentives, challenges, and benefits.

abstract: As biomedical research becomes more data-intensive, bioinformatics is becoming essential to understanding biological processes, systems, and diseases. In this paper we describe the use of a series of peer teaching workshops as a strategy to respond to the bioinformatics training needs at a research-intensive institution. In addition to the data collected from the workshops, we also used personal experiences of researchers who participated as peer teachers to understand the incentives, challenges, and benefits of peer teaching. Developing communication skills such as confidence in teaching, explaining complex concepts, and better understanding of the topic emerged as primary benefits that the teachers obtained from this experience. Lack of time for teaching and the struggles of classroom management were identified as two major challenges. We suggest that peer teaching can be beneficial not only to train researchers in bioinformatics, but also as a professional development opportunity for graduate students and postdoctoral trainees.

category: scientific communication and education


title: Simple Model for Encoding Natural Images by Retinal Ganglion Cells with Nonlinear Spatial Integration

abstract:
A central goal in sensory neuroscience is to understand the neuronal signal processing involved in the encoding of natural stimuli. A critical step towards this goal is the development of successful computational encoding models. For ganglion cells in the vertebrate retina, the development of satisfactory models for responses to natural visual scenes is an ongoing challenge. Standard models typically apply linear integration of visual stimuli over space, yet many ganglion cells are known to show nonlinear spatial integration, in particular when stimulated with contrast-reversing gratings. We here study the influence of spatial nonlinearities in the encoding of natural images by ganglion cells, using multielectrode-array recordings from isolated salamander and mouse retinas. We assess how responses to natural images depend on first- and second-order statistics of spatial patterns inside the receptive field. This leads us to a simple extension of current standard ganglion cell models. We show that taking not only the weighted average of light intensity inside the receptive field into account but also its variance over space can partly account for nonlinear integration and substantially improve response predictions of responses to novel images. We find that response predictions for salamander ganglion cell classes with large receptive fields profit most from including spatial contrast information. Finally, we demonstrate how this model framework can be used to assess the spatial scale of nonlinear integration. Our results underscore that nonlinear spatial stimulus integration in the salamander retina translates to stimulation with natural images. Furthermore, the introduced model framework provides a simple, yet powerful extension of standard models and may serve as a benchmark for the development of more detailed models of the nonlinear structure of receptive fields.

Author SummaryFor understanding how sensory systems operate in the natural environment, an important goal is to develop models that capture neuronal responses to natural stimuli. For retinal ganglion cells, which connect the eye to the brain, current standard models often fail to capture responses to natural visual scenes. This shortcoming is at least partly rooted in the fact that ganglion cells may combine visual signals over space in a nonlinear fashion. We here show that a simple model, which not only considers the average light intensity inside a cells receptive field but also the variance of light intensity over space, can partly account for these nonlinearities and thereby improve current standard models. This provides an easy-to-obtain benchmark for modeling ganglion cell responses to natural images.


title: Degradation intermediates as an indicator of mRNA and cell metabolism

abstract: Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints1,2. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent3,4, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

category: molecular biology


title: Optimisation of unsteady flight in learned avian perching manoeuvres

abstract: Perching at speed is amongst the most challenging flight behaviours that birds perform1,2, and beyond the capability of current autonomous vehicles. Smaller birds may touchdown by hovering3-8, but larger birds typically swoop upward to perch1,2 - presumably because the adverse scaling of their power margin prohibits slow flapping flight9, and because swooping transfers excess kinetic to potential energy1,2,10. Perching is risky in larger birds6,11, demanding precise control of velocity and pose12-15, but it is unknown how they optimize this challenging manoeuvre. More generally, whereas cruising flight behaviours such as migration and commuting are adapted to minimize cost-of-transport or time-of-flight16, the optimization of unsteady flight manoeuvres remains largely unexplored7,17. Here we show that swooping minimizes neither the time nor energy required to perch safely in Harris hawks Parabuteo unicinctus, but instead minimizes the distance flown under hazardous post-stall conditions. By combining motion capture data from 1,563 flights with flight dynamics modelling, we found that the birds choice of where to transition from powered dive to unpowered climb minimizes the distance from the landing perch over which very high lift coefficients are required. Time and energy are therefore invested to maintain the control authority needed to execute a safe landing, rather than being minimized continuously as in technical applications of autonomous perching under nonlinear feedback control13 and deep reinforcement learning18,19. Naive birds learn this behaviour on-the-fly, so our findings suggest an alternative reward function for reinforcement learning of autonomous perching in air vehicles.

category: animal behavior and cognition


title: Optimization of avian perching manoeuvres


title: Optimized extraction method enables quantitative analysis of surface metabolite recovery for exposomics and behavioral studies

abstract: Workplace chemical exposures are a major source of occupational injury. Although over half of these are skin exposures, exposomics research often focuses on chemical levels in the air or in worker biofluids such as blood and urine. Until now, one limitation has been the lack of methods to quantitatively measure surface chemical transfer. Outside the realm of harmful chemicals, the small molecules we leave behind on surfaces can also reveal important aspects of human behavior. In this study, we developed a swab-based quantitative approach to determine small molecule concentrations across common surfaces. We demonstrate its utility using one drug, cyclobenzaprine, and two human-derived metabolites, carnitine and phenylacetylglutamine, on four common surfaces: linoleum flooring, plastified laboratory workbench, metal and Plexiglass. This approach enabled linear small molecule recovery and quantification of molecule abundance on workplace built environment surfaces. Overall, this method paves the way for future quantitative exposomics studies.

category: biochemistry


title: A novel post-developmental role of the Hox genes underlies normal adult behaviour

abstract: The molecular mechanisms underlying the stability of mature neurons and neural circuits are poorly understood. Here we explore this problem and discover that the Hox genes are a component of the genetic programme that maintains normal neural function in adult Drosophila. We show that post-developmental downregulation of the Hox gene Ultrabithorax (Ubx) in adult neurons leads to substantial anomalies in flight. Mapping the cellular basis of these effects reveals that Ubx is required within a subset of dopaminergic neurons, and cell circuitry analyses and optogenetics allow us to link these dopaminergic neurons to flight control. Functional imaging experiments show that Ubx is necessary for normal dopaminergic activity, and neuron-specific RNA-sequencing defines two previously uncharacterised ion channel-encoding genes as potential mediators of Ubx behavioural roles. Our study thus reveals a novel role of the Hox system in controlling adult behaviour and neural function.


title: Exo1-protected DNA nicks direct crossover formation in meiosis

abstract: In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is critical for the viability of haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday Junction (dHJ) intermediates. This dHJ resolution step involves the actions Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. At present little is known about how these factors act in meiosis at the molecular level. Here we show that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 required for interactions with DNA, such as bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase specifically reduced the crossover levels of exo1 DNA binding mutants to levels approaching the exo1 null. In addition, our work identified a role for Exo1 in crossover interference that appears independent of its resection activity. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.


title: Structure modeling hints at a granular organization of the Vertebrate Golgi ribbon

abstract: Vertebrate cells display a specific Golgi apparatus architecture, known as the "ribbon", where the functional subunits, the mini-stacks, are linked into a tridimensional network. The importance of the ribbon architecture is underscored by evidence of its disruption in a host of diseases, but just how it relates to the biological Golgi functions remains unclear. Are all the connections between mini-stacks functionally equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, due to the lack of a secretory cargo providing a quantifiable readout of the functional output of ribbon-embedded mini-stacks. Endothelial cells produce rod-shaped secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. In these cells, the Golgi apparatus exerts a dual control on WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF  boluses" while the ribbon architecture allows their linear co-packaging at the trans-Golgi network generating WPBs of different lengths. This Golgi/WPB size relationship lends itself to mathematical analysis. Here, different ribbon structures were modeled and their predicted effects on WPB size distribution compared to the ground truth of experimental data. Strikingly, the best-fitting model describes a Golgi ribbon made by linked subunits corresponding to differentially functioning monomer and dimer ministacks. These results raise the intriguing possibility that the fine-grained structure of the Golgi ribbon is more complex than previously thought.

category: cell biology


title: ZetaSuite, A Computational Method for Analyzing Multi-dimensional High-throughput Data, Reveals Genes with Opposite Roles in Cancer Dependency

abstract: The rapid advance of high-throughput technologies has enabled the generation of two-dimensional or even multi-dimensional high-throughput data, e.g., genome-wide siRNA screen (1st dimension) for multiple changes in gene expression (2nd dimension) in many different cell types or tissues or under different experimental conditions (3rd dimension). We show that the simple Z-based statistic and derivatives are no longer suitable for analyzing such data because of the accumulation of experimental noise and/or off-target effects. Here, we introduce ZetaSuite, a statistical package designed to score and rank hits from two-dimensional screens, construct regulatory networks based on response similarities, and eliminate off-targets. Applying this method to two large cancer dependency screen datasets, we identify not only genes critical for cell fitness, but also those required for constraining cell proliferation. Strikingly, most of those cancer constraining genes function in DNA replication/repair checkpoint, suggesting that cancer cells also need to protect their genomes for long-term survival.


title: Local biodiversity change reflects interactions among changing abundance, evenness and richness

abstract: Biodiversity metrics often integrate data on the presence and abundance of multiple species. Yet understanding covariation of changes to the numbers of individuals, the evenness of species relative abundances, and the total number of species remains limited. Using individual-based rarefaction curves, we introduce a conceptual framework to understand how expected positive relationships among changes in abundance, evenness and richness arise, and how they can break down. We then examined interdependencies between changes in abundance, evenness and richness in more than 1100 assemblages sampled either through time or across space. As predicted, richness changes were greatest when abundance and evenness changed in the same direction, and countervailing changes in abundance and evenness acted to constrain the magnitude of changes in species richness. Site-to-site changes in abundance, evenness, and richness were often decoupled, and pairwise relationships between changes in these components across assemblages were weak. In contrast, changes in species richness and relative abundance were strongly correlated for assemblages varying through time. Temporal changes in local biodiversity showed greater inertia and stronger relationships between the component changes when compared to site-to-site variation. Local variation in assemblage diversity was rarely due to a passive sample from a more or less static species abundance distribution. Instead, changing species relative abundances often dominated local variation in diversity. Moreover, how changing relative abundances combined with changes to total abundance frequently determined the magnitude of richness changes. Embracing the interdependencies between changing abundance, evenness and richness can provide new information for better understanding biodiversity change in the Anthropocene.


title: Biosynthetic gene cluster profiling predicts the positive association between antagonism and phylogeny in Bacillus

abstract: Understanding the driving forces and intrinsic mechanisms of microbial competition is a fundamental question in microbial ecology. Despite the well-established negative correlation between exploitation competition and phylogenetic distance, the process of interference competition that is exemplified by antagonism remains controversial. Here, we studied the genus Bacillus, a commonly recognized producer of multifarious antibiotics, to explore the role of phylogenetic patterns of biosynthetic gene clusters (BGCs) in mediating the relationship between antagonism and phylogeny. Comparative genomic analysis revealed a positive association between BGC distance and phylogenetic distance. Antagonistic tests demonstrated that the inhibition phenotype positively correlated with both phylogenetic and predicted BGC distance, especially for antagonistic strains possessing abundant BGCs. Mutant-based verification showed that the antagonism was dependent on the BGCs that specifically harbored by the antagonistic strain. These findings highlight that BGC-phylogeny coherence regulates the positive correlation between congeneric antagonism and phylogenetic distance, which deepens our understanding of the driving force and intrinsic mechanism of microbial interactions.


title: Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2

abstract: Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinsons disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.


title: Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase

abstract:
Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of -synuclein, huntingtin or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Recruitment and clustering of Hsc70 on the fibrils appear to be necessary for disassembly.

Here we use atomic force microscopy (AFM) to show that segments of in vitro assembled -synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography reveals fibrils with regions of densely bound chaperones extending from the fibre surface, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of [~]4 subunits per second.


title: EVALUATION OF STED SUPER-RESOLUTION IMAGE QUALITY BY IMAGE CORRELATION SPECTROSCOPY (QuICS)

abstract: Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.

category: biophysics


title: Hydrozoan sperm-specific H2B histone variants stabilize chromatin and block transcription without enhancing chromatin condensation

abstract: Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.

category: developmental biology


title: The control of transcriptional memory by stable mitotic bookmarking

abstract: To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is thought to be mediated, in part, by mitotic bookmarking by transcription factors (TF). However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that the pioneer-like transcription factor GAF acts as stable mitotic bookmarker during zygotic genome activation. We report that GAF remains associated to a large fraction of its interphase targets including at cis-regulatory sequences of key developmental genes, with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we provide evidence that GAF binding establishes competence for rapid activation upon mitotic exit.


title: The pigtail macaque (Macaca nemestrina) model of COVID-19 reproduces diverse clinical outcomes and reveals new and complex signatures of disease

abstract: The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over four million people worldwide as of July 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.

category: immunology


title: Regulation of eIF2α Phosphorylation by MAPKs Influences Polysome Stability and Protein Translation

abstract: Regulation of protein translation occurs primarily at the level of initiation and is mediated by multiple signaling pathways, majorly mechanistic target of rapamycin complex 1 (mTORC1), mitogen-activated protein kinases (MAPKs), and the eukaryotic translation initiation factor eIF2. While mTORC1 and eIF2 influence the polysome stability, MAPKs influence the phosphorylation of the cap-binding protein eIF4E and are known to influence translation of only a small set of mRNAs. Here, we demonstrate that p38 MAPK and ERK1/2 regulate translation through integrated stress response (ISR) pathways. Dual inhibition (dual-Mi) of p38 MAPK and ERK1/2 caused substantial phosphorylation of eIF2 in a synergistic manner, resulting in near-absolute collapse of polysomes. This regulation was independent of Mnk1/2, a well-studied mediator of translation regulation by the MAPKs. Dual-Mi-induced polysome dissociation was far more striking than that caused by sodium arsenite, a strong inducer of ISR. Interestingly, induction of ISR caused increased p38 phosphorylation, and its inhibition resulted in stronger polysome dissociation, indicating the importance of p38 in the translation activities. Thus, our studies demonstrate a major, unidentified role for ERK1/2 and more particularly p38 MAPK in the maintenance of homeostasis of polysome association and translation activities.


title: Allosteric regulation of 3CL protease of SARS-CoV-2 and SARS-CoV observed in the crystal structure ensemble

abstract: The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)-disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.


title: Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

abstract:
SARS-CoV-2 main protease (Mpro) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based Flip-GFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/458041v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b0c310org.highwire.dtl.DTLVardef@d652deorg.highwire.dtl.DTLVardef@da8d0corg.highwire.dtl.DTLVardef@62449b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Flip-GFP and Protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate the reported SARS-CoV-2 Mpro inhibitors.


title: Immunostimulatory activity of Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia, Saccharomyces boulardii and their derivatives on RAW 264.7 macrophages

abstract: Yeasts are a group of microorganisms with structural and metabolic characteristics that influence their recognition by immune cells resulting in a species-specific response. Although Saccharomyces boulardii is a widely studied probiotic yeast, immunostimulation by non-Saccharomyces yeasts still underexplored. Therefore, the aim of this study was to characterize the response induced in macrophages stimulated by yeasts Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia and their derivatives: heat-killed cells, supernatant and DNA. RAW 264.7 murine macrophages were stimulated in vitro for 24 h and the response generated was evaluated by analyzing mRNA transcription of cytokines (IL2, IL4, IL10, IL13, IL23, TNF-), transcription factors (Bcl6, NF{kappa}{beta}, STAT3), Toll-like receptor 2 (TLR2) and YM1 protein. Viable and heat-killed cells of P. kluyveri and H. uvarum were responsible for high levels of relative mRNA transcription of transcription factors and TLR2 (between 2 - 8-fold increase), however were able to induce only low transcription levels for analyzed cytokines ([≤] 2-fold increase). Viable cells of C. intermedia were able to stimulate a significant transcription of IL4 (7.6-fold increase) and Bcl6 (4-fold increase), while heat-killed cells stimulated the highest level of TNF- (2.4-fold increase) among yeasts and their derivatives. Furthermore, supernatant from C. intermedia culture induced significant (p < 0.05) levels of TLR2 (4.4-fold increase), being the only one among supernatants to present high levels of relative mRNA transcription of TLR2. Data found in this work arouse interest in further studies on interaction between non-Saccharomyces yeasts and immune system cells, mainly referring to immunomodulatory capacity.

category: microbiology


title: Global distribution of anaerobic dichloromethane degradation potential

abstract:
Anthropogenic activities and natural processes release dichloromethane (DCM), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising eight to ten genes with predicted 79.6 - 99.7% amino acid identity. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2 = 0.71 - 0.85) indicating their value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting DCM turnover in diverse habitats. The broad distribution of anaerobic DCM catabolic potential suggests a relevant control function for emissions to the atmosphere, and a role for DCM as a microbial energy source in critical zone environments. The findings imply that the global DCM flux might be far greater than emission measurements suggest.

ImportanceDichloromethane (DCM) is an increasing threat to stratospheric ozone with both anthropogenic and natural emission sources. Anaerobic bacterial metabolism of DCM has not yet been taken into consideration as a factor in the global DCM cycle. The discovery of the mec gene cassette associated with anaerobic bacterial DCM metabolism and its widespread distribution in environmental systems highlight a strong attenuation potential for DCM. Knowledge of the mec cassette offers new opportunities to delineate DCM sources, enables more robust estimates of DCM fluxes, supports refined DCM emission modeling and simulation of the stratospheric ozone layer, reveals a novel, ubiquitous C1 carbon metabolic system, and provides prognostic and diagnostic tools supporting bioremediation of groundwater aquifers impacted by DCM.


title: Microbiomes in drinking water treatment and distribution: a critical review and meta-analysis from source to tap

abstract: A meta-analysis of existing and available Illumina 16S rRNA datasets from drinking water source, treatment and drinking water distribution systems (DWDS) were collated to compare changes in abundance and diversity throughout. Samples from bulk water and biofilm were used to assess principles governing microbial community assembly and the value of amplicon sequencing to water utilities. Individual phyla relationships were explored to identify competitive or synergistic factors governing DWDS microbiomes. The relative importance of stochasticity in the assembly of the DWDS microbiome was considered to identify the significance of source and treatment in determining communities in DWDS. Treatment of water significantly reduces overall species abundance and richness, with chlorination of water providing the most impact to individual taxa relationships. The assembly of microbial communities in the bulk water of the source, primary treatment process and DWDS is governed by more stochastic processes, as is the DWDS biofilm. DWDS biofilm is significantly different from bulk water in terms of local contribution to beta diversity, type and abundance of taxa present. Water immediately post chlorination has a more deterministic microbial assembly, highlighting the significance of this process in changing the microbiome, although elevated levels of stochasticity in DWDS samples suggest that this may not be the case at customer taps. 16S rRNA sequencing is becoming more routine, and may have several uses for water utilities, including: detection and risk assessment of potential pathogens such as those within the genera of Legionella and Mycobacterium; assessing the risk of nitrification in DWDS; providing improved indicators of process performance and monitoring for significant changes in the microbial community to detect contamination. Combining this with quantitative methods like flow cytometry will allow a greater depth of understanding of the DWDS microbiome.


title: CryoEM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition

abstract:
Viruses use a plethora of mechanisms to evade immune responses. A new example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000 [A]2 binding surface comprised of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. An atomic understanding of this novel pathogen-host interaction may contribute to the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B.

One-Sentence SummaryThese studies show how a conserved viral nucleotide metabolism protein is repurposed to inhibit a potent antiviral factor.


title: Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape

abstract:
At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. Although a small, systematic trend towards NAb destabilization not observed for Delta or Gamma was detected for Omicron, and despite bearing significantly more RBD mutations, the epistatic landscape of the Omicron variant closely resembles that of Gamma. These results suggest that, although Omicron poses new risks not observed with Delta, structural constraints on the RBD hamper continued evolution towards more complete vaccine escape. The modest ensemble of mutations relative to the WT that are currently known to reduce vaccine efficacy is likely to comprise the majority of all possible escape mutations for future variants, predicting continued efficacy of the existing vaccines.

SignificanceEmergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. At the time of writing, December, 2021, Omicron is poised to replace Delta as the dominant variant worldwide. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the WT and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for the Delta variant, structural constraints on the RBD make continued evolution towards more complete vaccine escape unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.


title: Intrinsic variation in the vertically transmitted insect-specific core virome of Aedes aegypti

abstract: Since 2009, local outbreaks of dengue (serotypes 1-3) mediated by Aedes aegypti mosquitoes have occurred in the United States, particularly in Florida (FL). In 2016 and 2017, dengue virus serotype 4 was found alongside several insect-specific viruses (ISVs) in pools of A. aegypti from sites in Manatee County, FL, in the absence of an index case. Although ISVs have been characterized in A. aegypti globally, the constitution of a core virome in natural populations remains unclear. Using mosquitoes sampled from the same area in 2018, we compared baseline ovary viromes of field (G0) and lab (Orlando) A. aegypti via metagenomic RNA sequencing. Across all samples, virome composition varied by sample type (field- or colony-derived). Four ISVs comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a previously described toti-like virus (Totiviridae), unclassified Riboviria, and four previously described orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the toti-like virus, partiti-like virus, and one orthomyxo-like virus were assembled and analyzed phylogenetically. Multigenerational maintenance of these ISVs was confirmed orthogonally by RT-PCR in G0 and G7 mosquitoes, indicating vertical transmission as the mechanism for ISV sustentation. This study provides fundamental information regarding ISV ecology, persistence, and variation in A. aegypti in nature.


title: May a Strain Chlamydia Isolated From SARS Patient's Autopsy Issues Inhibit the Proliferation of SARS-CoV? An Early Observation in Vitro.

abstract: We found that a strain chlamydia isolated from SARS patients autopsy issues could decrease the proliferation of SARS-CoV in vitro; The inhibitory factors distribute both in the extracellular and intracellular cultures.


title: Relationship between COVID-19 infection and neurodegeneration: Computational insight into interactions between the SARS-CoV-2 spike protein and the monoamine oxidase enzymes

abstract:
Although COVID-19 has been primarily associated with pneumonia, recent data show that its causative agent, the SARS-CoV-2 coronavirus, can infect many vital organs beyond the lungs, including the heart, kidneys and the brain. The literature agrees that COVID-19 is likely to have long-term mental health effects on infected individuals, which signifies a need to understand the role of the virus in the pathophysiology of brain disorders that is currently unknown and widely debated. Our docking and molecular dynamic simulations show that the affinity of the spike protein from the wild type (WT) and the South African B.1.351 (SA) variant towards the MAO enzymes is comparable to that for its ACE2 receptor. This allows for the WT/SA...MAO complex formation, which changes MAO affinities for their neurotransmitter substrates, thus consequently impacting the rates of their metabolic conversion and misbalancing their levels. Knowing that this fine regulation is strongly linked with the etiology of various brain pathologies, these results are the first to highlight the possibility that the interference with the brain MAO catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection, thus placing a neurobiological link between these two conditions in the spotlight. Since the obtained insight suggests that a more contagious SA variant causes even larger disturbances, and with new and more problematic strains likely emerging in the near future, we firmly advise that the presented prospect of the SARS-CoV-2 induced neurological complications should not be ignored, but rather requires further clinical investigations to achieve an early diagnosis and timely therapeutic interventions.

TABLE OF CONTENTS ENTRY

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/458208v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1976c81org.highwire.dtl.DTLVardef@54bb35org.highwire.dtl.DTLVardef@cea31aorg.highwire.dtl.DTLVardef@364100_HPS_FORMAT_FIGEXP  M_FIG C_FIG Docking and molecular dynamic simulations highlight the possibility that the interference with the brain monoamine oxidase (MAO) catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection.


title: Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model

abstract:
RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 23cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5 and 3 sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity.

Complementarily, by screening of a tRF&tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.


title: Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates

abstract:
Ice-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival of sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more ice-binding proteins than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an ice-binding protein was significantly greater in intertidal species as compared to those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk, that there is an array of uncharacterized ice binding proteins and highlight the need for broader laboratory-based surveys of the diversity of ice binding activity across diverse taxonomic and ecological groups.

Summary statementIntertidal invertebrates have a disproportionate number of putative ice-binding proteins relative to other habitats. These putative proteins are highly similar to antifreeze glycoproteins and type II antifreeze proteins from fish.


title: Exploring the conformational landscape and stability of Aurora A using ion-mobility mass spectrometry and molecular modelling

abstract: Protein kinase inhibitors are proving highly effective in helping treat a number of non-communicable diseases driven by aberrant kinase signaling. They are also extremely valuable as chemical tools to help delineate cellular roles of kinase signaling complexes. The binding of small molecule inhibitors induces conformational effects on kinase dynamics; evaluating the effect of such interactions can assist in developing specific inhibitors and is deemed imperative to understand both inhibition and resistance mechanisms. Using gas-phase ion mobility-mass spectrometry (IM-MS) we characterized changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations: one highly-populated compact conformer similar to that observed in most crystal structures, a second highly-populated conformer possessing a more open structure that is infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Comparison of active (phosphorylated) and inactive (non-phosphorylated) forms of Aur A revealed that the active enzyme has different conformer weightings and is less stable than the inactive enzyme. Notably, inhibitor binding shifts conformer balance towards the more compact configurations adopted by the unbound enzyme, with both IM-MS and modelling revealing inhibitor-mediated stabilisation of active Aur A. These data highlight the power of IM-MS in combination with molecular dynamics simulations to probe and compare protein kinase structural dynamics that arise due to differences in activity and as a result of compound binding.


title: (Machine) Learning the mutation signatures of SARS-CoV-2: a primer for predictive prognosis

abstract:
MotivationContinuous emergence of new variants through appearance, accumulation and disappearance of mutations in viruses is a hallmark of many viral diseases. SARS-CoV-2 and its variants have particularly exerted tremendous pressure on global healthcare system owing to their life threatening and debilitating implications. The sheer plurality of the variants and huge scale of genome sequence data available for Covid19 have added to the challenges of traceability of mutations of concern. The latter however provides an opportunity to utilize SARS-CoV-2 genomes and the mutations therein as  big data records to comprehensively classify the variants through the (machine) learning of mutation patterns. The unprecedented sequencing effort and tracing of disease outcomes provide an excellent ground for identifying important mutations by developing machine learnt models or severity classifiers using mutation profile of SARS-CoV-2. This is expected to provide a significant impetus to the efforts towards not only identifying the mutations of concern but also exploring the potential of mutation driven predictive prognosis of SARS-CoV-2.

ResultsWe describe how a graduated approach of building various severity specific machine learning classifiers, using only the mutation corpus of SARS-CoV-2 genomes, can potentially lead to the identification of important mutations and guide potential prognosis of infection. We demonstrate the applicability of model derived important mutations and use of Shapley values in order to identify the significant mutations of concern as well as for developing sparse models of outcome classification. A total of 77,284 outcome traced SARS-CoV-2 genomes were employed in this study which represented a total corpus of 30346 unique nucleotide mutations and 18647 amino acid mutations. Machine learning models pertaining to graduated classifiers of target outcomes namely  Asymptomatic, Mild, Symptomatic/Moderate, Severe and Fatal were built considering the TRIPOD guidelines for predictive prognosis. Shapley values for model linked important mutations were employed to select significant mutations leading to identification of less than 20 outcome driving mutations from each classifier. We additionally describe the significance of adopting a  temporal modeling approach to benchmark the predictive prognosis linked with continuously evolving pathogens. A chronologically distinct sampling is important in evaluating the performance of models trained on  past data in accurately classifying prognosis linked with genomes of future (observed with new mutations). We conclude that while machine learning approach can play a vital role in identifying relevant mutations, caution should be exercised in using the mutation signatures for predictive prognosis in cases where new mutations have accumulated along with the previously observed mutations of concern.

Contactsharmila.mande@tcs.com

Supplementary informationSupplementary data are enclosed.


title: Plus and minus ends of microtubule respond asymmetrically to kinesin binding by a long range directionally driven allosteric mechanism

abstract: Many members in the kinesin superfamily walk predominantly towards the plus end of the micro-tubule (MT) in a hand-over-hand manner. Despite great progress in elucidating the mechanism of stepping kinetics, the origin of stepping directionality is not fully understood. To provide quantitative insights into this important issue, we represent the structures of conventional kinesin (Kin1), MT, and the Kin1-MT complex using the elastic network model, and calculate the residue-dependent responses to a local perturbation in these constructs. Fluctuations in the residues in the {beta} domain of the /{beta}-tubulin are distinct from the  domain. Surprisingly, the Kin1-induced asymmetry, which is more pronounced in /{beta}-tubulin in the plus end of MT than in the minus end, propagates spatially across multiple /{beta}-tubulin dimers. Kin1 binding expands the MT lattice by mechanical stresses, resulting in a transition in the cleft of /{beta} tubulin dimer between a closed (CC for closed cleft) state (not poised for Kin1 to bind) to an open (OC for open cleft) binding competent state. The long-range asymmetric responses in the MT, leading to the creation of OC states with high probability in several /{beta} dimers on the plus end of the bound Kin1, is needed for the motor to take multiple steps towards the plus end of the MT. Reciprocally, kinesin binding to the MT stiffens the residues in the MT binding region, induces correlations between switches I and II in the motor, and enhances fluctuations in ADP and the residues in the binding pocket. Our findings explain both the directionality of stepping and MT effects on a key step in the catalytic cycle of Kin1.


title: The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

abstract: Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying MIDAS-UBL switching between strongly- and weakly-bound states is unknown. Here, we use single-molecule optical tweezers to investigate the force-dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to tenfold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1s MIDAS domain forms catch bonds with more than one UBL-substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.


title: Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering

abstract:
Coronavirus disease-2019 (COVID-19), a lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of the SARS-CoV-2 Spike protein, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the protein to the lipid leaflet in contact with the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

SIGNIFICANCEWe have recreated here important elements of the critical membrane fusion mechanism of the SARS-CoV-2 coronavirus by simplifying the system down to its core elements, amenable to experimental analysis by neutron scattering. Neutrons are well suited for the study of protein - membrane interactions under physiological conditions, since they allow structural and dynamics characterization at room temperature. Our results revealed strikingly different functions encoded in the viral Spike fusion domain and thereby provide a potential calcium-dependent cell entry mechanism for SARS-CoV-2. In particular, calcium drives the proteins N-terminal to harpoon through the host membrane, while removing calcium re-orients the protein so that it is able to bridge and dehydrate lipid membranes, facilitating their fusion.


title: A gap-free tomato genome built from complementary PacBio and Nanopore long DNA sequences reveals extensive linkage drag during breeding

abstract: The assembly and scaffolding of plant crop genomes facilitates the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of Tobacco Mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all twelve chromosomes were represented as twelve contiguous sequences (N50=68.5 Mbp). The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato assemblies that made use of genetic maps and HiC for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1 Mbp region of the S. peruvianum genome during tomato breeding. We show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near complete genome sequences.


title: Revealing the genetic components responsible for the unique photosynthetic stem capability of the wild almond Prunus arabica (Olivier) Meikle

abstract: Almond (Prunus dulcis (Mill.) D. A. Webb) is a major deciduous fruit tree crop worldwide. During dormancy, under warmer temperatures and inadequate chilling hours, the plant metabolic activity increases and may lead to carbohydrate deficiency. Prunus arabica (Olivier) Meikle is a bushy wild almond species known for its green, un-barked stem, which stays green even during the dormancy period. Our study revealed that P. arabica green stems assimilate significantly high rates of CO2 during the winter as compared to P. dulcis cv. Um el Fahem (U.E.F), and may improve carbohydrate status throughout dormancy. To uncover the genetic inheritance and mechanism behind the P. arabica Stem Photosynthetic Capability (SPC), a segregated F1 population was generated by crossing P. arabica to U.E.F. Both parents whole genome was sequenced, and a single nucleotide polymorphism (SNP) calling identified 4,887 informative SNPs for genotyping. A robust genetic map for U.E.F and P. arabica was constructed (971 and 571 markers, respectively). QTL mapping and association study for the SPC phenotype revealed major QTL (log of odd (LOD)=20.8) on chromosome 7, and another minor but significant QTL on chromosome 1 (LOD=3.9). Finally, a list of 73 candidate genes was generated. This work sets the stage for future research to investigate the mechanism regulating the SPC trait, how it affects the trees physiology, and its importance for breeding new cultivars better adapted to high winter temperatures.


title: Effectiveness of surface-based North Atlantic right whale detection methods for vessel strike mitigation

abstract: Increasing commercial and recreational use of the worlds ocean leads to growing concerns on vessel and marine mammal encounters. For endangered species, like the North Atlantic right whale (NARW), vessel strikes can be responsible for the majority of the recorded deaths. Reducing the number of vessel strikes is key to improve North Atlantic right whale protection and a number of mitigation methods have been proposed and implemented. In this manuscript, we developed an agent-based model to assess the effectiveness of surface-based whale detection methods for vessel strike mitigation. We find that the effectiveness of such systems varies highly depending on the vessels speed and maneuverability. We also find that if vessel-based whale detection systems are used in conjunction with other mitigation measures such as general speed restrictions, they can be very effective and could lead to a significant decrease in vessel strikes when deployed at a large-scale.


title: Engineering yeast for de-novo synthesis of the insect repellent - nepetalactone

abstract: While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe a platform for de novo nepetalactone production in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.

category: synthetic biology


title: A novel effective live-attenuated human metapneumovirus vaccine candidate produced in the serum-free suspension DuckCelt-T17 cell platform

abstract: Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen for which there is currently no specific treatment or licensed vaccine. Different strategies have been evaluated to prevent this infection, including the use of live-attenuated vaccines (LAVs). However, further development of LAV approaches is often hampered by the lack of highly efficient and scalable cell-based production systems that support worldwide vaccine production. In this context, avian cell lines cultivated in suspension are currently competing with traditional cell platforms used for viral vaccine manufacturing. We investigated whether the DuckCelt(R)-T17 avian cell line (Vaxxel) we previously described as an efficient production system for several influenza strains could also be used to produce a new HMPV LAV candidate (Metavac(R)), an engineered SH gene-deleted mutant of the A1/C-85473 strain of HMPV. To that end, we characterized the operational parameters of multiplicity of infection (MOI), cell density, and trypsin addition to achieve optimal production of the LAV Metavac(R) in the DuckCelt(R)-T17 cell line platform. We demonstrated that the DuckCelt(R)-T17 cell line is permissive and is well adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac(R) vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt(R)-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, as well as its capacity to induce efficient neutralizing antibodies in a mouse model. Our results suggest that the DuckCelt(R)-T17 avian cell line is a very promising platform for scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac(R).


title: Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge

abstract: Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guerin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.


title: A Plasmid Network from the Gut Microbiome of Semi-isolated Human Groups Reveals Unique and Shared Metabolic and Virulence Traits

abstract: The plasmidome, the set of plasmids in gut microbiomes, has the potential to contribute to the microbiome assembly, as well as human health and physiology. Nevertheless, this niche remains poorly explored, likely due to the difficulties attributed to mining mobile elements from complex environments such as microbiomes. In general, most microbiome studies focus on urban-industrialized groups, but here, we studied semi-isolated groups, which represent a link between the ancestral and modern human groups. Based on metagenomic data, we characterized their plasmidome, including the set of accessory genes and functions from the gut microbiome of the Hadza, Matses, Tunapuco, and Yanomami. In silico analyzes revealed unique plasmid clusters and gene functions for each human group related to their diet and lifestyle. In addition, network analysis revealed a dozen plasmid clusters shared by these distinct groups but that are also circulating in other niches worldwide. Moreover, in these microbiomes, there are novel and unique plasmids associated with their resistome and virulome. A resistome encompassing six antibiotic classes and multiple metals, and a virulome with type VI secretion systems were identified. Functional analysis revealed pathways associated with urban-industrialized groups, such as lipopolysaccharide biosynthesis that was characterized in the Hadza plasmidome. These results demonstrate the richness of features in the semi-isolated human groups plasmidome and provide a snapshot of the forces that are acting in their gut microbiome. Their plasmidomes also represent an important source of information with biotechnological and/or pharmaceutical potential.


title: Apropos of Universal Epitope Discovery for COVID-19 Vaccines: A Framework for Targeted Phage Display-Based Delivery and Integration of New Evaluation Tools

abstract: Targeted bacteriophage (phage) particles are potentially attractive yet inexpensive platforms for immunization. Herein, we describe targeted phage capsid display of an immunogenically relevant epitope of the SARS-CoV-2 Spike protein that is empirically conserved, likely due to the high mutational cost among all variants identified to date. This observation may herald an approach to developing vaccine candidates for broad-spectrum, towards universal, protection against multiple emergent variants of coronavirus that cause COVID-19.


title: Template strand deoxyuridine promoter recognition by a viral RNA polymerase

abstract: Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control and host antiviral defense evasion - it encodes two unique DNA-dependent RNA polymerases (RNAPs) that function at different stages of virus morphogenesis in the cell, and its double stranded (ds) DNA genome contains uracils instead of thymines throughout1,2. Unlike any known RNAP, the AR9 non-virion RNAP (nvRNAP), which transcribes late phage genes, recognizes promoters in the template strand of dsDNA and efficiently differentiates obligatory uracils from thymines in its promoters3. Here, using structural data obtained by cryo-electron microscopy and X-ray crystallography on the AR9 nvRNAP core, holoenzyme, and a promoter complex, and a variety of in vitro transcription assays, we elucidate a unique mode of uracil-specific, template strand-dependent promoter recognition. It is achieved by a tripartite interaction between the promoter specificity subunit, the core enzyme, and DNA adopting a unique conformation. We also show that interaction with the non-template strand plays a critical role in the process of AR9 nvRNAP promoter recognition in dsDNA, and that the AR9 nvRNAP core and a part of its promoter specificity subunit that interacts with the core are structurally similar to their bacterial RNAP counterparts. Our work demonstrates the extent to which viruses can evolve new functional mechanisms to control acquired multisubunit cellular enzymes and make these enzymes serve their needs.


title: The exosomes derived from urine of premature infants target MAPK8 via miR-30a-5p to protect cisplatin-induced acute kidney injury in mice

abstract:
AimAcute kidney injury (AKI), a global public health issue, not only causes millions of deaths every year, but is also a susceptible factor for chronic kidney disease (CKD). Nephrotoxic drugs are an important cause of AKI. There is still a lack of effective and satisfactory prevention method in clinical practice. This study investigated the protective effect of the exosomes derived from urine of premature infants on cisplatin-induced acute kidney injury.

MethodsIsolation of exosomes from fresh urine of premature infants: The characteristics of exosomes were determined by flow cytometry, transmission electron microscopy and Western blotting. A C57BL/6 mice model of cisplatin-induced acute kidney injury was established. The mice in the experimental group were given 100ug exosomes dissolved in 200ul solution. The mice in the control group were given normal saline (200ul). These treatments were performed 24 hours after AKI was induced by intraperitoneal injection of cisplatin. To evaluate renal function, blood was drawn 24 hours after AKI model was established and serum creatinine (sCr) was measured. The mice were euthanized 72 hours after exosome treatment. The kidneys were collected for pathological examination, RNA and protein extraction, and the evaluation of renal tubular damage and apoptosis. In the in-vitro experiment, human renal cortex/proximal tubular cells (HK2) was induced by cisplatin to assess the protective ability of the exosomes derived from urine of premature infants. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blotting were used to evaluate the effect of exosomes treatment on the apoptosis of HK2 cells induced by cisplatin. Exosome microRNA sequencing technology and bioinformatics analysis method were applied to investigate the miRNAs enriched in exosomes and their target genes. The dual luciferase gene reporter system was used to detect the interaction of target genes.

ResultsTreatment of exosomes derived from urine of premature infants could decrease the level of serum creatinine and the apoptosis of renal tubular cell, inhibit the infiltration of inflammatory cell, protect mice from acute kidney injury induced by cisplatin and reduce mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes derived from urine of premature infants. It protected HK2 cells from cisplatin-induced apoptosis by targeting and down-regulating the 3UTR of mitogen-activated protein kinases (MAPK8) mRNA.

ConclusionsAccording to our results, the exosomes derived from urine of premature infants alleviated cisplatin-induced acute kidney injury in mice and inhibited the apoptosis of human proximal tubular cells (HK2) induced by cisplatin in vitro. MiR-30a-5p in exosomes inhibited cisplatin-induced MAPK activation, ameliorated apoptosis, and protected renal function. The exosomes derived from urine of premature infants provided a promising acellular therapy for AKI.


title: HIF-1α stabilization modified by glutaredoxin-1 is critical for intestinal angiogenesis in NEC pathogenesis

abstract:
BackgroundHypoxia inducible factor (HIF-1) are essential in the pathogenesis of necrotizing enterocolitis (NEC), which is stabilized by Grx1 deletion. Until now, the mechanism of HIF-1 in the intestinal microcirculation in NEC is not well defined. We intend to investigate the role of HIF-1 in the development of NEC in regulating the microcirculation and the following vasodilatory signal, VEGF.

Materials and methodsExperimental NEC was induced in full-term C57BL/6 mouse and Grx1-/- pups through the formula gavage and hypoxia technique. The HIF-1 signal was blocked utilizing the HIF-1 inhibitor, YC-1. Intestinal tissues were collected at predetermined time points for the assessment of intestinal microcirculation and the HIF-1 activity involved signal.

ResultsWe found that NEC inducement impaired the intestinal microcirculation, but intestinal blood flow and capillary density were ameliorated in Grx1-/- mice, which was associated with the GSH-protein adducts of HIF-1 in the intestinal tissue. Grx1 ablation could also promote vascular endothelial growth factor (VEGFA) production in the intestinal tissue. This intestinal microvascular improvement was not found in the HIF-1 inhibited mice, suggesting the HIF-1 dependent manner for intestinal microcirculatory perfusion.

ConclusionThe current data demonstrated that HIF-1 signaling is involved in the intestinal microvascular modification during the pathogenesis of NEC, suggesting that targeting with HIF-1 might be a promising strategy for NEC treatment.


title: Translation of MT-ATP6 pathogenic variants reveals distinct regulatory consequencesfrom the co-translational quality control of mitochondrial protein synthesis.

abstract: Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogenous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.


title: Flanking sequences regulate the toxicity, non-ATG translation, and aggregation of RNA with tandem CAG Repeats

abstract: Nucleotide repeat-expansions cause several neurodegenerative disorders, including Huntingtons disease and spinocerebellar ataxia. The expanded repeat-containing RNA transcribed from the affected loci agglomerate in the nucleus as pathogenic foci. Here we demonstrate that depending on their surrounding sequence context, RNAs with expanded CAG repeats can also undergo nuclear export and aggregate in the cytoplasm. Cytoplasmic aggregation of repeat-containing RNA coincides with several disease hallmarks, including repeat-associated non-AUG (RAN) translation, mislocalization of RNA binding proteins, and cell toxicity. Interestingly, the repeat-containing RNA co-aggregate with RAN translation products. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and also alleviates cell toxicity. Our findings provide a cogent explanation for aberrant cytoplasmic localization of RNA binding proteins and implicate cis-acting flanking sequences in mediating RAN translation and disease.