title: High-performance Saccharomyces cerevisiae-based biosensor for heavy metal detection

abstract:
Heavy metals, i.e., Cu(II), are harmful to the environment. There is an increasing demand to develop inexpensive detection methods for heavy metals. Here, we developed a yeast biosensor with reduced-noise and improved signal output for potential on-site copper ion detection. The copper-sensing circuit was achieved by employing a secondary genetic layer to control the galactose-inducible (GAL) system in Saccharomyces cerevisiae. The reciprocal control of the Gal4 activator and Gal80 repressor under copper-responsive promoters resulted in a low-noise and ultrasensitive yeast biosensor for the copper ion detection. Furthermore, we developed a betaxanthin-based colorimetric assay, as well as 2-phenylethanol and styrene-based olfactory outputs for the copper ion detection. Notably, our engineered yeast sensor confers a narrow range switch-like behavior, which can give a "yes/no" response when coupled with betaxanthin-based visual phenotype. Taken together, we envision that the design principle established here might be applicable for developing other sensing systems for various chemical detections.

IMPORTANCEThe accumulation of heavy metals in the environment led to an imbalance with environmental and ecological impacts, and there is an increasing demand for inexpensive methods that permit on-site detection of heavy metals. In this study, a yeast biosensor with reduced-noise and improved signal output was developed. We used a betaxanthin-based colorimetric assay for monitoring the copper contamination in water samples, which confers a switch-like behavior with a "yes/no" response. In addition, we also explored the 2-phenylethanol and styrene-based outputs for copper detection, which might be useful for olfactory detection of heavy metals.


title: A novel framework for analysis of the shared genetic background of correlated traits

abstract: We propose a novel effective framework for analysis of the shared genetic background for a set of genetically correlated traits using SNP-level GWAS summary statistics. This framework called SHAHER is based on the construction of a linear combination of traits by maximizing the proportion of its genetic variance explained by the shared genetic factors. SHAHER requires only full GWAS summary statistics and matrices of genetic and phenotypic correlations between traits as inputs. Our framework allows both shared and unshared genetic factors to be to effectively analyzed. We tested our framework using simulation studies, compared it with previous developments, and assessed its performance using three real datasets: anthropometric traits, psychiatric conditions and lipid concentrations. SHAHER is versatile and applicable to summary statistics from GWASs with arbitrary sample sizes and sample overlaps, allows incorporation of different GWAS models (Cox, linear and logistic) and is computationally fast.

category: bioinformatics


title: SBP-SITA: A sequence-based prediction tools for S-itaconation

abstract: As a recently-reported post-translational modification, S-itaconation plays an important role in inflammation suppression. In order to understand its regulatory mechanism in many life activities, the essential step is the recognition of S-itaconation. However, it is difficult to identify S-itaconation in the proteome for the high cost, which limits further investigation. In this study, we constructed an ensemble algorithm based on Soft Voting Classifier. The area under the ROC curve (AUC) value 0.73 for ensemble model. Accordingly, we constructed the on-line prediction tool dubbed SBP-SITA for easily identifying Cystine sites. SBP-SITA is available at http://www.bioinfogo.org/sbp-sita.


title: PLaBAse: A comprehensive web resource for analyzing the plant growth-promoting potential of plant-associated bacteria

abstract: Plant-beneficial microorganisms are gaining importance for sustainable plant production and phytosanitary practices. Yet there is a lack of computational approaches targeting bacterial traits associated with plant growth-promotion (PGP), which hinders the in-silico identification, comparison, and selection of phytostimulatory bacterial strains. To address this problem, we have developed the new web resource PLaBAse (v1.01, http://plabase.informatik.uni-tuebingen.de/pb/plabase.php), which provides a number of services, including (i) a database for screening 5,565 plant-associated bacteria (PLaBA-db), (ii) a tool for predicting plant growth-promoting traits (PGPTs) of single bacterial genomes (PGPT-Pred), and (iii) a tool for the prediction of bacterial plant-association by marker gene identification (PIFAR-Pred). The latter was developed by Martinez-Garcia et al. and is now hosted at University of Tuebingen. The PGPT-Pred tool is based on our new PGPT ontology, a literature- and OMICs-curated, comprehensive, and hierarchical collection of [~]6,900 PGPTs that are associated with 6,965,955 protein sequences. To study the distribution of the PGPTs across different environments, we applied it to 70,540 bacterial strains associated with (i) seven different environments (including plants), (iii) five different plant spheres (organs), and (iii) two bacteria-induced plant phenotypes. This analysis revealed that plant-symbiotic bacteria generally have a larger genome size and a higher count of PGPT-annotated protein encoding genes. Obviously, not all reported PGPTs are restricted to -or only enriched in-plant-associated and plant symbiotic bacteria. Some also occur in human- and animal-associated bacteria, perhaps due to the transmission of PGP bacteria (PGPBs) between environments, or because some functions are involved in adaption processes to various environments. Here we provide an easy-to-use approach for screening of PGPTs in bacterial genomes across various phyla and isolation sites, using PLaBA-db, and for standardized annotation, using PGPT-Pred. We believe that this resource will improve our understanding about the entire PGP processes and facilitate the prediction of PGPB as bio-inoculants and for biosafety strategies, so as to help to establish sustainable and targeted bacteria-incorporated plant production systems in the future.


title: GREPore-seq: A Robust Workflow to Detect Changes after Gene Editing through Long-range PCR and Nanopore Sequencing

abstract: To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, pipelined, large-scale and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR-Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq).


title: Slow conformational changes of blue light sensor BLUF proteins in milliseconds

abstract:
BLUF (blue light sensor using flavin) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits red-shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red-shift is complete within nanoseconds. Contrary to this commonly accepted scheme, in this study, slow reaction kinetics were discovered in milliseconds ({tau}1- and {tau}2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics ({tau}2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the {tau}1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Graphical Abstract

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category: biophysics


title: Genuine Selective Caspase-2 Inhibition with new Irreversible Small Peptidomimetics

abstract: Caspase-2 (Casp2) is a promising therapeutic target in several human diseases including nonalcoholic steatohepatitis (NASH) and Alzheimers disease (AD). However, the design of active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics shows that these new compounds, such as LJ2 or its specific isomer LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. According to Casp2 role in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~ 5 500 000 M-1s-1) and the most selective inhibitor, LJ3a, has a near to 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that spatial configuration of C at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with {beta}-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

category: pharmacology and toxicology


title: Noninvasive Stimulation of Peripheral Nerves using Temporally-Interfering Electrical Fields

abstract: Electrical stimulation of peripheral nerves is a cornerstone of bioelectronic medicine. Effective ways to accomplish peripheral nerve stimulation noninvasively without surgically implanted devices is enabling for fundamental research and clinical translation. Here we demonstrate how relatively high frequency sine-wave carriers (3 kHz) emitted by two pairs of cutaneous electrodes can temporally interfere at deep peripheral nerve targets. The effective stimulation frequency is equal to the offset frequency (0.5 - 4 Hz) between the two carriers. We validate this principle of temporal interference nerve stimulation (TINS) in vivo using the murine sciatic nerve model. Effective actuation is delivered at significantly lower current amplitudes than standard transcutaneous electrical stimulation. Further, we demonstrate how flexible and conformable on-skin multielectrode arrays can facilitate precise alignment of TINS onto a nerve. Our method is simple, relying on repurposing of existing clinically-approved hardware. TINS opens the possibility of precise noninvasive stimulation with depth and efficiency previously impossible with transcutaneous techniques.

category: neuroscience


title: The contrasting shape representations that support object recognition in humans and CNNs

abstract:
1The success of Convolutional Neural Networks (CNNs) in classifying objects has led to a surge of interest in using these systems to understand human vision. Recent studies have argued that when CNNs are trained in the correct learning environment, they can emulate a key property of human vision - learning to classify objects based on their shape. While showing a shape-bias is indeed a desirable property for any model of human object recognition, it is unclear whether the resulting shape representations learned by these networks are human-like. We explored this question in the context of a well-known observation from psychology showing that humans encode the shape of objects in terms of relations between object features. To check whether this is also true for the representations of CNNs, we ran a series of simulations where we trained CNNs on datasets of novel shapes and tested them on a set of controlled deformations of these shapes. We found that CNNs do not show any enhanced sensitivity to deformations which alter relations between features, even when explicitly trained on such deformations. This behaviour contrasted with human participants in previous studies as well as in a new experiment. We argue that these results are a consequence of a fundamental difference between how humans and CNNs learn to recognise objects: while CNNs select features that allow them to optimally classify the proximal stimulus, humans select features that they infer to be properties of the distal stimulus. This makes human representations more generalisable to novel contexts and tasks.

Author summaryThe human visual system is highly adept at recognising and reasoning about objects under a wide variety of viewing conditions. Research in vision sciences has shown that humans largely rely on shape to recognise objects, but extracting object shape from a retinal image is a challenge (indeed, it is an ill-posed problem), and until recently, models of vision have been poor at classifying naturalistic images of objects. This has changed with the development of Convolutional Neural Networks (CNNs) that perform as well as humans on certain recognition tasks. Thus it makes sense to ask if we can gain insight into how humans extract shape by studying CNNs. Here, we show that there is a fundamental difference between the two systems. While humans pay particular attention to relations between an objects parts and features, we show that the shape extracted by CNNs is akin to a template, where all diagnostic features are equally important. Thus, despite their success, these results show that CNNs are currently not good models of human object recognition and machine learning models must bridge this gap in order to capture the robustness and generalisability of human vision.


title: Innate heuristics and fast learning support escape route selection in mice

abstract: When faced with imminent danger, animals must rapidly take defensive actions to reach safety. Mice can react to innately threatening stimuli in less than 250 milliseconds [1] and, in simple environments, use spatial memory to quickly escape to shelter [2,3]. Natural habitats, however, often offer multiple routes to safety which animals must rapidly identify and choose from to maximize the chances of survival [4]. This is challenging because while rodents can learn to navigate complex mazes to obtain rewards [5,6], learning the value of different routes through trial-and-error during escape from threat would likely be deadly. Here we have investigated how mice learn to choose between different escape routes to shelter. By using environments with paths to shelter of varying length and geometry we find that mice prefer options that minimize both path distance and path angle relative to the shelter. This choice strategy is already present during the first threat encounter and after only ~10 minutes of exploration in a novel environment, indicating that route selection does not require experience of escaping. Instead, an innate heuristic is used to assign threat survival value to alternative paths after rapidly learning the spatial environment. This route selection process is flexible and allows quick adaptation to arenas with dynamic geometries. Computational modelling of different classes of reinforcement learning agents shows that the observed behavior can be replicated by model-based agents acting in an environment where the shelter location is rewarding during exploration. These results show that mice combine fast spatial learning with innate heuristics to choose escape routes with the highest survival value. They further suggest that integrating priors acquired through evolution with knowledge learned from experience supports adaptation to changing environments while minimizing the need for trial-and-error when the errors are very costly.


title: Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice

abstract: The suprachiasmatic nuclei (SCN) of the anterior hypothalamus host the circadian pacemaker that synchronizes mammalian rhythms with the day-night cycle. SCN neurons are intrinsically rhythmic, thanks to a conserved cell-autonomous clock mechanism. In addition, circuit-level emergent properties confer a unique degree of precision and robustness to SCN neuronal rhythmicity. However, the multicellular functional organization of the SCN is not yet fully understood. Although SCN neurons are well coordinated, experimental evidences indicate that some neurons oscillate out of phase in SCN explants, and possibly to a larger extent in vivo. Here, we used microendoscopic Ca2+i imaging to investigate SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca2+i spikes superimposed upon slow changes in baseline Ca2+i levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca2+i were well coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent interactions at the multicellular level. Hence, fast Ca2+i spikes and slow changes in baseline Ca2+i levels highlighted how diverse activity patterns could articulate within the temporal network unity of the SCN in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.


title: Sex-specific topology of the nociceptive circuit shapes dimorphic behavior in C. elegans

abstract: How sexually dimorphic behavior is encoded in the nervous system is poorly understood. Here, we characterize the dimorphic nociceptive behavior in C. elegans and study the underlying circuits, which are composed of the same neurons but are wired differently. We show that while sensory transduction is similar in the two sexes, the downstream network topology markedly shapes behavior. We fit a network model that replicates the observed dimorphic behavior in response to external stimuli, and use it to predict simple network rewirings that would switch the behavior between the sexes. We then show experimentally that these subtle synaptic rewirings indeed flip behavior. Strikingly, when presented with aversive cues, rewired males were compromised in finding mating partners, suggesting that network topologies that enable efficient avoidance of noxious cues have a reproductive "cost". Our results present a deconstruction of the design of a neural circuit that controls sexual behavior, and how to reprogram it.


title: Milking it for all it's worth: The effects of environmental enrichment on maternal nurturance, lactation quality, and offspring social behavior

abstract:
Breastfeeding confers robust benefits to offspring development in terms of growth, immunity, and neurophysiology. Similarly, improving environmental complexity (i.e., environmental enrichment; EE) contributes developmental advantages to both humans and laboratory animal models. However, the impact of environmental context on maternal care and milk quality has not been thoroughly evaluated, nor are the biological underpinnings of EE on offspring development understood. Here, Sprague-Dawley rats were housed and bred in either EE or standard-housed (SD) conditions. EE dams gave birth to a larger number of pups, and litters were standardized and cross-fostered across groups on postnatal day (P)1. Maternal milk samples were then collected on P1 (transitional milk phase) and P10 (mature milk phase) for analysis. While EE dams spent less time nursing, postnatal enrichment exposure was associated with heavier offspring bodyweights. Milk from EE mothers had increased triglyceride levels, a greater microbiome diversity, and a significantly higher abundance of bacterial families related to bodyweight and energy metabolism. These differences reflected comparable transcriptomic changes at the genome-wide level. In addition to changes in lactational quality, we observed elevated levels of cannabinoid receptor 1 in the hypothalamus of EE dams, and sex- and time-dependent effects of EE on offspring social behavior. Together, these results underscore the multidimensional impact of the combined neonatal and maternal environments on offspring development and maternal health. Moreover, they highlight potential deficiencies in the use of "gold standard" laboratory housing in the attempt to design translationally relevant animal models in biomedical research.

Significance StatementMaternal care quality is different between environmentally enriched (EE) and standard laboratory housed (SD) dams. SD rat dams spend more time nursing their young. This may result in overfeeding which can program offspring neural stress responses. Alternatively, metabolic differences in milk may affect neurodevelopmental outcomes, which are different between EE and SD animals. To test this, we evaluated milk and offspring behavior. Milk from EE dams had elevated triglyceride levels and microbiome diversity. EE offspring had heavier body weights and increased social behavior which was lost with crossfostering into SD housing. These data identify potential deficiencies in the quality of "gold standard" laboratory housing and its impact on the welfare and design of translationally relevant animal models in biomedical research.


title: GZMKhigh CD8+ T effector memory cells are associated with CD15high neutrophil abundance in early-stage colorectal tumors and predict poor clinical outcome.

abstract:
Tumor contexture has emerged as a major prognostic determinant and tumor infiltrating CD8+ T cells have been associated with a better prognosis in several solid tumors, including early-stage colorectal cancer (CRC). However, the tumor immune infiltrate is highly heterogeneous and understanding how the interplay between different immune cell compartments impacts on the clinical outcome is still in its infancy.

Here, we describe in a prospective cohort a novel CD8+ T effector memory population, which is characterized by high levels of Granzyme K (GZMKhigh CD8+ TEM) and is correlated with CD15high tumor infiltrating neutrophils. We provide both in vitro and in vivo evidence of the role of stromal cell-derived factor 1 (CXCL12/SDF-1) in driving functional changes on neutrophils at the tumor site, promoting their retention and increasing the crosstalk with CD8+ T cells. Mechanistically, as a consequence of the interaction with neutrophils, CD8+ T cells are skewed towards a CD8+ TEM phenotype and produce high levels of GZMK, which in turn decreases E-cadherin pathway. The correlations of GZMKhigh CD8+ TEM and neutrophils with both tumor progression in mice and early relapse in CRC patients demonstrate the role of GZMKhigh CD8+ TEM in promoting malignancy. Indeed, a gene signature defining GZMKhigh CD8+ TEM was associated with worse prognosis on a larger independent cohort of CRC patients and a similar analysis was extended to lung cancer (TCGA).

Overall, our results highlight the emergence of GZMKhigh CD8+ TEM in early-stage CRC tumors as a hallmark driven by the interaction with neutrophils, which could implement current patient stratification and be targeted by novel therapeutics.

category: immunology


title: Genetic, parental and lifestyle factors influence telomere length

abstract: The average length of telomere repeats (TL) declines with age and is considered to be a marker of biological ageing. Here, we measured TL in six blood cell types from 1,046 individuals using the clinically validated Flow-FISH method. We identified remarkable cell-type-specific variations in TL. Host genetics, environmental, parental and intrinsic factors such as sex, parental age, and smoking are associated to variations in TL. By analysing the genome-wide methylation patterns, we identified that the association of maternal, but not paternal, age to TL is mediated by epigenetics. Coupling these measurements to single-cell RNA-sequencing data for 62 participants revealed differential gene expression in T-cells. Genes negatively associated with TL were enriched for pathways related to translation and nonsense-mediated decay. Altogether, this study addresses cell-type-specific differences in telomere biology and its relation to cell-type-specific gene expression and highlights how perinatal factors play a role in determining TL, on top of genetics and lifestyle.

category: cell biology


title: Fast, live-cell imaging of 15 intracellular compartments by deep learning segmentation of super-resolution data

abstract: The number of colors that can be used in fluorescence microscopy to image the live-cell anatomy and organelles interactions is far less than the number of intracellular organelles and compartments. Here, we report that deep convolutional neuronal networks can predict 15 subcellular structures from super-resolution spinning-disk microscopy images using only one dye, one laser excitation, and two detection channels. Comparing to the colocalization images, this method achieves pixel accuracies of over 91.7%, which not only bypasses the fundamental limitation of multi-color imaging but also accelerates the imaging speed by more than one order of magnitude.


title: Selection-driven adaptation to the extreme Antarctic environment in the Emperor penguin

abstract: The eco-evolutionary history of penguins is profoundly influenced by their shift from temperate to cold environments. Breeding only in Antarctica during the winter, the Emperor penguin appears as an extreme outcome of this process, with unique features related to insulation, heat production and energy management. However, whether this species actually diverged from a less cold-adapted ancestor, thus more similar in ecology to its sister species, the King penguin, is still an open question. As the Antarctic niche shift likely resulted in vast changes in selective pressure experienced by the Emperor penguin, the identification and relative quantification of the genomic signatures of selection, unique to each of these sister species, could answer this question. Applying a suite of phylogeny-based methods on 7,651 orthologous gene alignments of seven penguins and 13 other birds, we identified a set of candidate genes showing significantly different selection regimes either in the Emperor or in the King penguin lineage. Our comparative approach unveils a more pervasive selection shift in the Emperor penguin, supporting the hypothesis that its extreme cold adaptation is a derived state from a more King penguin-like ecology. Among the candidate genes under selection in the Emperor penguin, four genes (TRPM8, LEPR, CRB1, and SFI1) were identified before in other cold adapted vertebrates, while, on the other hand, 161 genes can be assigned to functional pathways relevant to cold adaptation (e.g., cardiovascular system, lipid, fatty acid and glucose metabolism, insulation, etc.). Our results show that extreme cold adaptation in the Emperor penguin largely involved unique genetic options which, however, affect metabolic and physiological traits common to other cold-adapted homeotherms.

category: evolutionary biology


title: Reliable hypotheses testing in animal social network analyses: global index, index of interactions and residual regression

abstract: Animal social network analyses (ASNA) have led to a foundational shift in our understanding of animal sociality that transcends the disciplinary boundaries of genetics, spatial movements, epidemiology, information transmission, evolution, species assemblages and conservation. However, some analytical protocols (i.e., permutation tests) used in ASNA have recently been called into question due to the unacceptable rates of false negatives (type I error) and false positives (type II error) they generate in statistical hypothesis testing. Here, we show that these rates are related to the way in which observation heterogeneity is accounted for in association indices. To solve this issue, we propose a method termed the "global index" (GI) that consists of computing the average of individual associations indices per unit of time. In addition, we developed an "index of interactions" (II) that allows the use of the GI approach for directed behaviours. Our simulations show that GI: 1) returns more reasonable rates of false negatives and positives, with or without observational biases in the collected data, 2) can be applied to both directed and undirected behaviours, 3) can be applied to focal sampling, scan sampling or "gambit of the group" data collection protocols, and 4) can be applied to first- and second-order social network measures. Finally, we provide a method to control for non-social biological confounding factors using linear regression residuals. By providing a reliable approach for a wide range of scenarios, we propose a novel methodology in ASNA with the aim of better understanding social interactions from a mechanistic, ecological and evolutionary perspective.

category: animal behavior and cognition


title: Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

abstract:
The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

category: microbiology


title: Genomic determinants of Furin cleavage in diverse European SARS-related bat coronaviruses

abstract: The furin cleavage site in SARS-CoV-2 is unique within the Severe acute respiratory syndrome-related coronavirus (SrC) species. We re-assessed diverse SrC from European horseshoe bats and reveal molecular determinants such as purine richness, RNA secondary structures and viral quasispecies potentially enabling furin cleavage. Furin cleavage thus likely emerged from the SrC bat reservoir via molecular mechanisms conserved across reservoir-bound RNA viruses, supporting a natural origin of SARS-CoV-2.


title: Evidence for a mouse origin of the SARS-CoV-2 Omicron variant

abstract: The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting the possibility of host-jumping. The molecular spectrum (i.e., the relative frequency of the twelve types of base substitutions) of mutations acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients, but was highly consistent with spectra associated with evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.


title: A comprehensive examination of ACE-2 receptor and prediction of spike glycoprotein and ACE-2 interaction based on in silico analysis of ACE-2 receptor

abstract: ACE-2 receptor plays a vital role not only in the SARS-CoV-induced epidemic but also in some diseases. Studies have been carried out on the interactions of ACE-2-SARS-CoV proteins. However, comprehensive research has not been conducted on ACE2 protein by using bioinformatic tools. The present study especially two places, G104 and L108 points, which are effective in protecting the structure of the ACE-2 protein, play a critical role in the biological functioning of this protein, and play an essential role in determining the chemicalphysical properties of this protein, and play a crucial role for ACE-2 protein-SARS CoV surface glycoprotein, were determined. It was also found that the G104 and L108 regions were more prone to possible mutations or deletions than the other ACE-2 protein regions. Moreover, it was determined that all possible mutations or deletions in these regions affect the chemical-physical properties, biological functions, and structure of the ACE-2 protein. Having a negative GRAVY value, one transmembrane helix, a significant molecular weight, a long-estimated half-life as well as most having unstable are results of G104 and L108 points mutations or deletions. Finally, it was determined that LQQNGSSVLS, which belong to the ACE-2 protein, may play an active role in binding the spike protein of SARS-CoV. All possible docking score results were estimated. It is thought that this study will bring a different perspective to ACE-2 _SARS-CoV interaction and other diseases in which ACE-2 plays an important role and will also be an essential resource for studies on ACE-2 protein.


title: Learning Single-Cell Perturbation Responses using Neural Optimal Transport

abstract: The ability to understand and predict molecular responses towards external perturbations is a core question in molecular biology. Technological advancements in the recent past have enabled the generation of high-resolution single-cell data, making it possible to profile individual cells under different experimentally controlled perturbations. However, cells are typically destroyed during measurement, resulting in unpaired distributions over either perturbed or non-perturbed cells. Leveraging the theory of optimal transport and the recent advents of convex neural architectures, we learn a coupling describing the response of cell populations upon perturbation, enabling us to predict state trajectories on a single-cell level. We apply our approach, CO_SCPLOWELLC_SCPLOWOT, to predict treatment responses of 21,650 cells subject to four different drug perturbations. CO_SCPLOWELLC_SCPLOWOT outperforms current state-of-the-art methods both qualitatively and quantitatively, accurately capturing cellular behavior shifts across all different drugs.


title: Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2

abstract:
The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

SIGNIFICANCEThe ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. In this present study, we screened two compound libraries of 2,864 molecules and identified a potent inhibitor (TS-984) for blocking the coronavirus S-protein and the human cell ACE2 receptor. TS-984 might have the potential to be developed into an effective anti-coronavirus drug for treating COVID-19.


title: Connectome-wide association analysis identified significant brain functional alteration in adults with attention-deficit/hyperactivity disorder

abstract: Adults with attention-deficit/hyperactivity disorder (ADHD), as an extreme-phenotype of ADHD, is still facing problems of inconsistency and undeciphered mechanisms for its neuropathology. To address this matter, our present study performed connecotome-wide voxel-based analyses with the resting-state fMRI data of 84 adults with ADHD and 89 healthy controls. We found that functional connectivity patterns of the left precuneus and the left middle temporal significantly altered in ADHD populations serving as potential neural biomarkers to distinguish ADHD with healthy controls, with subsequent seed-based analysis revealing the dysfunction of functional connections both intra- and inter-default mode and attention networks, among which middle temporal gyrus plays the key role of  bridge linking the default mode and attention networks. After cognitive behavioral therapy, two of these ADHD-altered functional connections ameliorated accompanied with improvement of ADHD core symptoms. Additionally, imaging genetic analyses also revealed close relationships between the observed brain functional alterations and ADHD-risk genes. Taken together, our findings suggested the interference of default mode on attention networks in adults with ADHD, which would be severing as a potential biomarker for both ADHD pathogenesis and treatment effects.


title: Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

abstract: Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.


title: Viral infection engenders bona fide and bystander lung memory B cell subsets through permissive selection

abstract: Lung-resident memory B cells (MBCs) provide localized protection against reinfection in the respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.


title: Striking Antibody Evasion Manifested by the Omicron Variant of SARS-CoV-2

abstract: The Omicron (B.1.1.529) variant of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 (coronavirus disease 2019) vaccines and antibody therapies4. This concern is amplified by the findings from our study. We found B.1.1.529 to be markedly resistant to neutralization by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 17 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.


title: From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer

abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection rates approaching 40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2, WTD fawns shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD. Infectious virus was recovered from respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

Author summaryThe high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 over time and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that - if confirmed as a new reservoir of infection - may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans.


title: Rehydration of freeze substituted brain tissue for preembedding immuno-electron microscopy

abstract: Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.


title: Effects of habitat destruction on coevolving metacommunities

abstract: Habitat destruction is a growing threat to biodiversity and ecosystem services. The ecological consequences of habitat loss and fragmentation involve reductions in species abundance and even the extinction of species and interactions. However, we do not yet understand how habitat loss can alter the coevolutionary trajectories of the remaining species or how coevolution, in turn, affects their response to habitat loss. To investigate this, we develop a spatially explicit model which couples metacommunity and coevolutionary dynamics. We show that, by changing the size, composition and structure of local networks, habitat destruction increases the diversity of coevolutionary outcomes across the landscape. Furthermore, we show that while coevolution dampens the negative effects of habitat destruction in mutualistic networks, its effects on the persistence of antagonistic communities are less predictable.

category: ecology


title: Simulated visual hallucinations in virtual reality enhance cognitive flexibility

abstract: Historically, psychedelic drugs are known to modulate cognitive flexibility, a central aspect of cognition permitting adaptation to changing environmental demands. Despite proof suggesting phenomenological similarities between artificially-induced and actual psychedelic altered perception, experimental evidence is still lacking about whether the former is also able to modulate cognitive flexibility. To address this, we measure participants cognitive flexibility through behavioral tasks after the exposure to virtual reality panoramic videos and their hallucinatory-like counterparts generated by the DeepDream algorithm. Results show that the estimated semantic network has a flexible structure when preceded by altered videos. Crucially, following the simulated psychedelic exposure, individuals also show an attenuated contribution of the automatic process and chaotic dynamics underlying the decision process. This suggests that simulated altered perceptual phenomenology enhances cognitive flexibility, presumably due to a reorganization in the cognitive dynamics that facilitates the exploration of uncommon decision strategies and inhibits automated choices.


title: Combined SHP2 and ERK inhibition for the treatment of KRAS-driven Pancreatic Ductal Adenocarcinoma

abstract: Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

category: cancer biology


title: Individualized isometric neuromuscular electrical stimulation training promotes myonuclear accretion in mouse skeletal muscle

abstract: Skeletal muscle is a plastic tissue that adapts to exercise through fusion of muscle stem cells (MuSCs) with myofibers, a physiological process referred to as myonuclear accretion. However, it is still unclear whether myonuclear accretion is driven by increased mechanical loading per se, or occurs, at least in part, in response to exercise-induced muscle injury. Here, we developed a carefully monitored and individualized neuromuscular electrical stimulation (NMES) training protocol of the mouse plantar flexor muscles. Each NMES training session consisted of 80 isometric contractions at a submaximal mechanical intensity corresponding to ~ 15% of maximal tetanic force to avoid muscle damage. NMES trained mice were stimulated for 2 x 3 consecutive days separated by one day of rest, for a total of 6 sessions. Experiments were conducted on C57BL/6J and BALB/c males at 10-12 weeks of age. NMES led to a robust myonuclear accretion and higher MuSC content in gastrocnemius muscle of both mouse lines, without overt signs of muscle damage/regeneration or muscle hypertrophy or force improvement. This new mouse model of myonuclear accretion relying on the main function of skeletal muscles, i.e., force production in response to electrical stimuli, will be of utmost interest to further understand the role of MuSCs in skeletal muscle adaptations.

category: physiology


title: High content screening and computational prediction reveal viral genes that suppress innate immune response

abstract:
Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-{kappa}B and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

IMPORTANCEInfectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

category: systems biology


title: Prefrontal top-down projections control context-dependent strategy selection

abstract: The rules governing behavior often vary with behavioral contexts. As a consequence, an action rewarded in one context may be discouraged in another. Animals and humans are capable of switching between behavioral strategies under different contexts and acting adaptively according to the variable rules, a flexibility that is thought to be mediated by the prefrontal cortex (PFC)1-4. However, how the PFC orchestrates context-dependent switch of strategies remains unclear. Here we show that pathway-specific projection neurons in the medial PFC (mPFC) differentially contribute to context-instructed strategy selection. In a decision-making task in which mice have been trained to flexibly switch between a previously established rule and a newly learned rule in a context-dependent manner, the activity of mPFC neurons projecting to the dorsomedial striatum encodes the contexts, and further represents decision strategies conforming to the old and new rules. Moreover, the activity of these neuron is required for context-instructed strategy selection. In contrast, the activity of mPFC neurons projecting to the ventral midline thalamus does not discriminate between the contexts, and represents the old rule even if mice have adopted the new one; furthermore, these neurons act to prevent the strategy switch under the new rule. Our results suggest that the mPFC[->]striatum pathway promotes flexible strategy selection guided by contexts, whereas the mPFC[->]thalamus pathway favors fixed strategy selection by preserving old rules. Balanced activity between the two pathways may be critical for adaptive behaviors.


title: Considerable escape of SARS-CoV-2 variant Omicron to antibody neutralization

abstract: The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1,2. It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development3, and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies.


title: Link between aging and atheroprotection in Mif-deficient atherosclerotic mice

abstract: AbstractAtherosclerosis is a lipid-triggered chronic inflammatory condition of our arteries and the main underlying pathology of myocardial infarction and stroke. Pathogenesis is age-dependent, but the mechanistic links between disease progression, age, and atherogenic cytokines and chemokines are incompletely understood. Here, we studied the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) in atherogenic Apoe-/- mice across different stages of aging and cholesterol-rich high-fat diet (HFD). MIF promotes atherosclerosis by mediating atherogenic monocyte and T-cell recruitment, amplifying lesional inflammation, and suppressing atheroprotective B-cell responses. However, age-related links between atherogenesis and MIF and its role in advanced atherosclerosis in aged mice have not been systematically explored. We compared effects of global Mif-gene deficiency in 30-, 42-, and 48-week-old Apoe-/- mice on HFD for 24, 36, or 42 weeks, respectively, and in 52-week-old mice on a 6-week HFD. While a regio-specific atheroprotective phenotype of Mif-deficiency was observed in the 30/24-week-old group, atheroprotection was not detected in the 48/42- and 52/6-week-old groups, suggesting that atheroprotection afforded by global Mif-gene deletion differs across aging stages and atherogenic diet duration. We identify a combination of mechanisms that could explain this phenotype: i) Mif-deficiency promotes lesional Trem2+ macrophage numbers in younger but not aged mice; ii) Mif-deficiency favors formation of lymphocyte-rich stage-I/II ATLOs in younger mice but ATLO numbers equalize with those in Apoe-/- controls in the older mice; and iii) plasma anti-oxLDL-IgM antibody levels are decreased in aged Mif-deficient mice. Of note, these three markers (Trem2+ macrophages, ATLOs, anti-oxLDL-IgM antibodies) have been previously linked to atheroprotection. Together, our study thus suggests that regio-specific atheroprotection due to global Mif-gene deficiency in atherogenic Apoe-/- mice is lost upon advanced aging and identifies mechanisms that could explain this phenotype shift. These observations may have implications for translational MIF- directed strategies.


title: Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility.

abstract: Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y-27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model and we demonstrate that microtubules act to balance the actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.


title: Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation

abstract: Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much effort has been made to better understand the molecular mechanisms responsible for apical constriction, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging and tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance of the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

category: developmental biology


title: Using deep convolutional neural networks to forecast spatial patterns of Amazonian deforestation

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_LITropical forests are subject to diverse deforestation pressures but their conservation is essential to achieve global climate goals. Predicting the location of deforestation is challenging due to the complexity of the natural and human systems involved but accurate and timely forecasts could enable effective planning and on-the-ground enforcement practices to curb deforestation rates. New computer vision technologies based on deep learning can be applied to the increasing volume of Earth observation data to generate novel insights and make predictions with unprecedented accuracy.
C_LIO_LIHere, we demonstrate the ability of deep convolutional neural networks to learn spatiotemporal patterns of deforestation from a limited set of freely available global data layers, including multispectral satellite imagery, the Hansen maps of historic deforestation (2001-2020) and the ALOS JAXA digital surface model, to forecast future deforestation (2021). We designed four original deep learning model architectures, based on 2D Convolutional Neural Networks (2DCNN), 3D Convolutional Neural Networks (3DCNN), and Long Short-Term Memory (LSTM) Recurrent Neural Networks (RNN) to produce spatial maps that indicate the risk to each forested pixel (~30 m) in the landscape of becoming deforested within the next year. They were trained and tested on data from two ~80,000 km2 tropical forest regions in the Southern Peruvian Amazon.
C_LIO_LIWe found that the networks could predict the likely location of future deforestation to a high degree of accuracy. Our best performing model - a 3DCNN - had the highest pixel-wise accuracy (80-90%) when validated on 2020 deforestation based 2014-2019 training. Visual examination of the forecasts indicated that the 3DCNN network could automatically discern the drivers of forest loss from the input data. For example, pixels around new access routes (e.g. roads) were assigned high risk whereas this was not the case for recent, concentrated natural loss events (e.g. remote landslides).
C_LIO_LICNNs can harness limited time-series data to predict near-future deforestation patterns, an important step in using the growing volume of satellite remote sensing data to curb global deforestation. The modelling framework can be readily applied to any tropical forest location and used by governments and conservation organisations to prevent deforestation and plan protected areas.
C_LI


title: The blackgrass genome reveals patterns of divergent evolution of non-target site resistance to herbicides.

abstract: Globally, weedy plants result in more crop yield loss than plant pathogens and insect pests combined. Much of the success of weeds rests with their ability to rapidly adapt in the face of human-mediated environmental management and change. The evolution of resistance to herbicides is an emblematic example of this rapid adaptation. Here, we focus on Alopecurus myosuroides (blackgrass), the most impactful agricultural weed in Europe. To gain insights into the evolutionary history and genomic mechanisms underlying adaptation in blackgrass, we assembled and annotated its large, complex genome. We show that non-target site herbicide resistance is oligogenic and likely evolves from standing genetic variation. We present evidence for divergent selection of resistance at the level of the genome in wild, evolved populations, though at the transcriptional level, resistance mechanisms are underpinned by similar patterns of up-regulation of stress- and defence-responsive gene families. These gene families are expanded in the blackgrass genome, suggesting that the large, duplicated, and dynamic genome plays a role in enabling rapid adaptation in blackgrass. These observations have wide significance for understanding rapid plant adaptation in novel stressful environments.

category: genomics


title: Computational analysis of the effect of SARS-CoV-2 variant Omicron Spike protein mutations on dynamics, ACE2 binding and propensity for immune escape

abstract: The recently reported Omicron (B.1.1.529) SARS-CoV-2 variant has a large number of mutations in the Spike (S) protein compared to previous variants. Here we evaluate the potential effect of Omicron S mutations on S protein dynamics and ACE2 binding as contributing factors to infectivity as well as propensity for immune escape. We define a consensus set of mutations from 77 sequences assigned as Omicron in GISAID as of November 25. We create structural models of the Omicron S protein in the open and closed states, as part of a complex with ACE2 and for each of 77 complexes of S bound to different antibodies with known structures. We have previously utilized Dynamical Signatures (DS) and the Vibrational Entropy Score (VDS) to evaluate the propensity of S variants to favour the open state. Here, we introduce the Binding Influence Score (BIS) to evaluate the influence of mutations on binding affinity based on the net gain or loss of interactions within the protein-protein interface. BIS shows excellent correlation with experimental data (Pearson correlation coefficient of 0.87) on individual mutations in the ACE2 interface for the Alpha, Beta, Gamma, Delta and Omicron variants combined. On the one hand, the DS of Omicron highly favours a more rigid open state and a more flexible closed state with the largest VDS of all variants to date, suggesting a large increase in the chances to interact with ACE2. On the other hand, the BIS shows that apart from N501Y, all other mutations in the interface reduce ACE2 binding affinity. VDS and BIS show opposing effects on the overall effectiveness of Omicron mutations to promote binding to ACE2 and therefore initiate infection. To evaluate the propensity for immune escape we calculated the net change of favourable and unfavourable interactions within each S-antibody interface. The net change of interactions shows a positive score (a net increase of favourable interactions and decrease of unfavourable ones) for 41 out of 77 antibodies, a nil score for 15 and a negative score for 21 antibodies. Therefore, in only 28% of S-antibody complexes (21/77) we predict some level of immune escape due to a weakening of the interactions with Omicron S. Considering that most antibody epitopes and the mutations are within the S-ACE2 interface our results suggest that mutations within the RBD of Omicron may give rise to only partial immune escape, which comes at the expense of reduced ACE2 binding affinity. However, this reduced ACE2 affinity appears to have been offset by increasing the occupancy of the open state of the Spike protein.


title: The structural repertoire of Fusarium oxysporum f. sp. lycopersici effectors revealed by experimental and computational studies

abstract: Plant pathogens secrete proteins, known as effectors, that function in the apoplast and inside plant cells to promote virulence. Effectors can also be detected by cell-surface and cytosolic receptors, resulting in the activation of defence pathways and plant immunity. Our understanding of fungal effector function and detection by immunity receptors is limited largely due to high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. Recent studies have demonstrated that fungal effectors can be grouped into structural classes despite significant sequence variation. Using protein x-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class. Using AlphaFold ab initio protein structure prediction, benchmarked against the experimentally determined structures, we demonstrate SIX6 and SIX13 are FOLD effectors. We show that the conserved N-domain of Avr1 and Avr3 is sufficient for recognition by their corresponding, but structurally-distinct, immunity receptors. Additional structural prediction and comparison indicate that 11 of the 14 SIX effectors group into four structural families. This revealed that genetically linked effectors are related structurally, and we provide direct evidence for a physical association between one divergently-transcribed effector pair. Collectively, these data indicate that Fol secretes groups of structurally-related molecules during plant infection, an observation that has broad implications for our understanding of pathogen virulence and the engineering of plant immunity receptors.

category: plant biology


title: Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5

abstract: The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC is formed when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. Both the increase in C5 conversion and sMAC generation were linked to the expression of lipopolysaccharide (LPS) O-Antigen in the bacterial outer membrane. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Release of MAC precursors from bacteria induced lysis of bystander human erythrocytes in the absence of other serum components. However, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this bystander lysis. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.


title: Pharmacokinetics and Pharmacodynamics of Vancomycin derivative LYSC98 in a Murine Thigh Infection Model against Staphylococcus aureus

abstract:
LYSC98 is a vancomycin derivative used for gram-positive bacterial infections therapy. We reported the pharmacokinetic/pharmacodynamic (PK/PD) targets of LYSC98 against Staphylococcus aureus using a murine thigh infection model. Three Staphylococcus aureus strains were utilized. Single-dose plasma pharmacokinetics of LYSC98 were determined in infected mice after the tail vein injection of 2, 4, and 8mg/kg. The results showed maximum plasma concentration (Cmax) 11466.67 - 48866.67 ng/mL, area under the concentration-time curve from 0 to 24 h(AUC0-24) 14788.42 -91885.93 ng/mL{middle dot}h, and elimination half-life(T1/2) 1.70-2.64 h, respectively. The Cmax (R2 0.9994) and AUC0-24 (R2 0.981) were positively correlated with the dose of LYSC98 in the range of 2-8 mg/kg.

Dose fractionation studies using total doses of 2 to 8 mg/kg administered with q6h, q8h, q12h, and q24h were performed to evaluate the correlation of different PK/PD indices with efficacy. Sigmoid model analysis showed Cmax/MIC (R2 0.8941) was the best PK/PD index to predict the efficacy of LYSC98. In the dose ranging studies, two Methicillin-resistant Staphylococcus aureus (MRSA) clinical strains were used to infect the mice and 2-fold-increasing doses (1 to 16 mg/kg) of LYSC98 were administered. The magnitude of LYSC98 Cmax/MIC associated with net stasis, 1, 2, 3 and 4 - log10 kill were 5.78, 8.17, 11.14, 15.85 and 30.58, respectively. The results of this study showed LYSC98 a promising antibiotic with in vivo potency against MRSA, and will help in the dose design of phase one study for LYSC98.


title: Differential impact of rhamnolipids and polysorbate80 on composition and functionality of the gut microbiota in the SHIME system.

abstract:
Dietary emulsifiers have been shown to affect the composition and function of the gut microbial community, both in vivo and in vitro. Yet, several knowledge gaps remain to be addressed: the impact from a longer timeframe exposure on the gut microbiota, interindividual variability in microbiome response and the putative impact from novel clean label alternatives for current food emulsifiers.

In the present study, the impact of one conventional dietary emulsifier, TWEEN80, and one potential novel alternative, rhamnolipids, on the human gut microbiota was investigated using the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). The faecal microbiota from two human donors, with high and low responsiveness to the emulsifiers, were exposed to 0,05 m% and 0,5 m% of the emulsifiers for 7 days.

The results confirmed previous observations that the effects on the composition and functionality are both emulsifier- and donor dependent. The effects reached an equilibrium after about 3 days of exposure. Overall, TWEEN80 and rhamnolipids displayed opposite effects: TWEEN80 increased cell counts, reduced propionate concentration, increased butyrate levels, increased a.o. Faecalibacterium, Blautia and Hungatella abundance, while rhamnolipids did the opposite. Rhamnolipids also sharply increased the abundance of unclassified Lachnospiraceae. On the other hand, both emulsifiers increased the relative abundance of unclassified Enterobacteriaceae. Both emulsifiers also altered the microbial metabolome in different ways and a pathway enrichment analysis tool revealed that the metabolome alterations could be reminiscent of gut issues and obesity.

Overall, the impact from the rhamnolipids was larger than that of TWEEN80 at similar concentrations, indicating that the former may not necessarily be a safer alternative for the latter. The microbiotas response also depended on its original composition and the sensitivity status for which the faecal donors were selected, was preserved. Whether the same donor-diversity and longitudinal impact can be expected in the human colon as well and what impact this has on the host will have to be further investigated.


title: Linear plasmids in Klebsiella and other Enterobacteriaceae

abstract:
Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function, however each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Significance as a BioResource to the communityThis study provides the first report of linear plasmids identified within the Klebsiella pneumoniae species complex and the first report in Enterobacteriaceae besides Salmonella.We present the first comparative analysis of linear plasmid sequences in Enterobacteriaceae, however whilst this family is highly clinically significant, the functional and/or evolutionary importance of these plasmids is not yet clear. To facilitate future studies to address these questions, we have publicly deposited (i) the collection of linear plasmid sequence data; (ii) isolates representative of each of the distinct linear plasmid phylogroups.

Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary information

O_LIWhole genome sequence reads from Klebsiella pneumoniae isolates sequenced in this study have been deposited in NCBI SRA under the accession numbers listed in Table S1.
C_LIO_LIRepresentative annotated sequences of one linear plasmid per phylogroup have been deposited in FigShare, doi 10.26180/16729126.
C_LIO_LIA copy of all linear plasmid sequences that we assembled from publicly available genome sequence reads are available in FigShare, doi 10.26180/16531365. Read accessions for these are given in Table S1.
C_LIO_LIEleven representative K. pneumoniae isolates harbouring linear plasmids described in this study have been deposited with the National Collection of Type Cultures (NCTC) and are available for purchase under the NCTC accession numbers listed in Table S1.K. pneumoniae 1194/11 (representative of phylogroup B) has been deposited in the Microorganisms Collection Center, Adolfo Lutz Institute, Sao Paulo, Brazil. To request strain 1194/11 (IAL 3063, SISGEN ABBF09B), contact: Microorganisms Collection Center
Culture Collection Laboratory
Instituto Adolfo Lutz, Sao Paulo State Department of Health
Address: Av Dr Arnaldo, 351, 10 floor, room 1020
Phone number: +55 11 3068-2884
Zip code 01246-000, Sao Paulo, Brazil
E-mail: colecaoial@ial.sp.gov.br

C_LIO_LIAlignments of terminal inverted repeat sequences for each phylogroup can be found in Data S1, available on FigShare, doi 10.26180/16531371.
C_LI


title: Generating Electricity from Water Evaporation Through Microbial Biofilms

abstract: Sustainable strategies for energy production are required to reduce reliance on fossil fuels and to power electronics without generating toxic waste.1-7 Generating electricity from water evaporation through engineered materials is a promising approach,8,9 but power outputs have been low and the materials employed were not sustainably produced. Microorganisms can be mass produced with renewable feedstocks. Here, we demonstrate that it is possible to engineer microbial biofilms as a cohesive, flexible material for long-term continuous electricity production from evaporating water. The biofilm sheets were the functional component in devices that continuously produced power densities ([~]1 W/cm2) higher than that achieved with non-biological materials. Current production scaled directly with biofilm-sheet size and skin-patch devices harvested sufficient electricity from the moisture on skin to continuously power wearable devices. The results demonstrate that appropriately engineered biofilms can perform as robust functional materials without the need for further processing or maintaining cell viability. Biofilm-based hydroelectric current production was comparable to that achieved with similar sized biofilms catalyzing current production in microbial fuel cells,10,11 without the need for an organic feedstock or maintaining cell viability. The ubiquity of biofilms in nature suggests the possibility of additional sources of biomaterial for evaporation-based electricity generation and the possibility of harvesting electricity from diverse aqueous environments.


title: A world of viruses nested within parasites: Unraveling viral diversity within parasitic flatworms (Platyhelminthes)

abstract: Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host-shifts. Yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein we uncover viruses of flatworms (Phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of Neodermatans to a parasitic lifestyle. Most Neodermatan viruses seem to co-diversify with their host, with the exception of rhabdoviruses which may switch host more often, based on phylogenetic relationships. Neodermatan rhabodviruses also have an ancestral position to vertebrate-associated viruses, including Lyssaviruses, suggesting that vertebrate rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases.


title: Bacterial origin of thymidylate and folate metabolism in Asgard Archaea

abstract: Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group. We also experimentally validated the functionality of the lateral gene transfer of Psyn thymidylate synthase ThyX. This enzyme uses bacterial-like folates efficiently and is inhibited by mycobacterial ThyX inhibitors. Our data raise the possibility that the thymidylate metabolism, required for de novo DNA synthesis, originated in bacteria and has been independently transferred to archaea and eukaryotes. In conclusion, our study has revealed that recent prevalent lateral gene transfer has markedly shaped the evolution of Asgard archaea by allowing them to adapt to specific ecological niches.


title: The African Swine Fever Isolate ASFV-Kenya-1033-IX is highly virulent and stable after growth in the wild boar cell line WSL

abstract: In this study, we describe an African swine fever genotype IX virus (ASFV-Kenya-1033-IX), which was isolated from a domestic pig in Western Kenya during a reported outbreak, including efficiency of virus replication, in vivo virulence, and genome stability in pulmonary alveolar macrophages (PAM) and in a wild boar cell line (WSL). The ASFV-Kenya-1033-IX stock, which underwent multiple passages in WSL (more than 20), retained its ability to replicate in primary macrophages and it also retained the virulence in vivo. At the genomic level, only a few single nucleotide differences were observed between the macrophage and WSL-grown virus. Thus, we propose that the WSL cell line is suitable to produce live attenuated ASFV vaccine candidates based on this isolate and probably of similar viruses. The genome sequences for ASFV-Kenya-1033-IX grown in macrophages and in WSL cells was submitted to GenBank and a challenge model based on this isolate was set up, which will aid the development of vaccines against genotype IX ASFV circulating in Eastern and Central Africa.


title: Development and Characterization of Recombinant Vesicular Stomatitis Virus (rVSV)-based Bivalent Vaccine Against COVID-19 Delta Variant and Influenza Virus

abstract: COVID-19 and influenza are both highly contagious respiratory diseases with a wide range of severe symptoms and cause great disease burdens globally. It has become very urgent and important to develop a bivalent vaccine that is able to target these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant VSV (rVSV) vaccine candidates. These rVSV-based vaccines co-express SARS-CoV-2 Delta variant spike protein (SP) or the receptor binding domain (RBD) and four copies of the highly conserved M2 ectodomain (M2e) of influenza A fused with the Ebola glycoprotein DC-targeting/activation domain. Animal studies have shown that immunization with these bivalent rVSV vaccines induced efficient but variable levels of humoral and cell-mediated immune responses against both SARS-CoV-2 and influenza M2e protein. Significantly, our vaccine candidates induced production of high levels of neutralizing antibodies that protected cells against SARS-CoV-2 Delta and other SP-pseudovirus infections in culture. Furthermore, vaccination with the bivalent VSV vaccine via either intramuscular or intranasal route efficiently protected mice from the lethal challenge of H1N1 and H3N2 influenza viruses and significantly reduced viral load in the lungs. These studies provide convincing evidence for the high efficacy of this bivalent vaccine to prevent influenza replication and initiate robust immune responses against SARS-CoV-2 Delta variants. Further investigation of its efficacy to protect against SARS-CoV-2 Delta variants will provide substantial evidence for new avenues to control two contagious respiratory infections, COVID-19 and influenza.


title: METTL3 facilitates hepatic fibrosis progression via m6A-YTHDF2 dependent silencing of GPR161

abstract:
Hepatic fibrosis (HF) is a very common condition seen in millions of patients with various liver diseases. N6-methyladenosine (m6A) plays critical roles in various biological and pathological processes. However, the role of m6A and its main methyltransferase METTL3 in HF remains obscure. Here, we reported that METTL3 expression was elevated in HSCs from CCl4 induced fibrotic liver. METTL3 knockdown in HSCs mediated by recombinant adeno-associated-virus serotype 9 packed short hairpin RNA against METTL3 alleviated liver injury and fibrosis compared to empty carrier group. Mechanistically, the decreased liver fibrosis in CCl4-treated HSC-specific METTL3 knockdown mice was due to the increased GPR161 that is a suppressor of Hedgehog pathway, a well-known pathway to activate in liver injury and regeneration. As expect, GPR161 transferred into HSCs alleviated liver fibrosis and HSC activation. Forced GPR161 expression inhibited Gli3 activated form nuclear accumulation and subsequently suppressed fibrosis-associate gene expression. Conclusion, HSC-specific deletion of METTL3 inhibits liver fibrosis via elevated GPR161 expression, which subsequently suppressed Hedgehog pathway activation and fibrosis-associated genes expression, providing novel therapeutic targets for HF therapy.

Synopsis

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/472749v1_ufig1.gif" ALT="Figure 1">
View larger version (78K):
org.highwire.dtl.DTLVardef@1245f3aorg.highwire.dtl.DTLVardef@e7effdorg.highwire.dtl.DTLVardef@13ce319org.highwire.dtl.DTLVardef@61be8b_HPS_FORMAT_FIGEXP  M_FIG C_FIG The role of RNA m6A methyltransferase METTL3 in the progression of hepatic fibrosis is unclear. Here, we reveal that suppresses m6A modification by depletion of METTL3 in Hepatic Stellate Cells (HSCs), stabilizes the GPR161 transcripts, that involved in suppressing Hedgehog signaling pathway, through YTHDF2, thereby alleviating the fibrosis feature of CCl4-induced liver injury.

O_LIDisruption of m6A methyltransferases METTL3 in HSCs alleviates liver fibrosis feature.
C_LIO_LIMETTL3 depletion in liver HSCs suppresses its activation by suppressing Hedgehog signaling pathway through stabilizing GPR161 transcripts in an m6A-YTHDF2 dependent manner.
C_LIO_LIGPR161 activates PKA that mediates phosphorylation and degradation of full-length Gli3 (Gli3-FL) protein, then the short Gli3 (Gli3-R) translocated into the nucleus and suppresses the fibrosis associated gene transcription.
C_LIO_LIThe elevated METTL3 expression is attribute to its promoter H3K27ac modification.
C_LI

category: molecular biology


title: NAD+ capping of RNA in Archaea and Mycobacteria

abstract: Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD+ capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD+ capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD+-capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD+-capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD+ into RNA, and that the genes of NAD+-capped transcripts are preceded by promoter elements compatible with {sigma}A/{sigma}F dependent expression. Taken together, our findings demonstrate that NAD+ capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD+-capped RNA landscape in mycobacteria, providing a basis for its future exploration.