title: A new framework for analysis of three-dimensional shape and architecture of human skeletal muscles

abstract: A new framework is presented for comprehensive analysis of the three-dimensional shape and architecture of human skeletal muscles from magnetic resonance and diffusion tensor imaging data. The framework comprises three key features: (1) identification of points on the surface of and inside a muscle that have a correspondence to points on and inside another muscle, (2) reconstruction of average muscle shape and average muscle fibre orientations, and (3) utilization of data on between-muscle variation to visualize and make statistical inferences about changes or differences in muscle shape and architecture. The general use of the framework is demonstrated by its application to three datasets. Analysis of data obtained before and after eight weeks of strength training revealed there was little regional variation in hypertrophy of the vastus medialis and vastus lateralis, and no systematic change in pennation angle. Analysis of passive muscle lengthening revealed heterogeneous changes in shape of the medial gastrocnemius, and confirmed the ability of the methods to detect subtle changes in muscle fibre orientation. Analysis of the medial gastrocnemius of children with unilateral cerebral palsy showed that muscles in the more-affected limb were shorter, thinner and less wide than muscles in the less-affected limb, and had slightly more pennate muscle fibres in the central and proximal part of the muscle. Amongst other applications, the framework can be used to explore the mechanics of muscle contraction, investigate adaptations of muscle architecture, build anatomically realistic computational models of skeletal muscles, and compare muscle shape and architecture between species.


title: Transcriptomic-based quantification of the epithelial-hybrid-mesenchymal spectrum across biological contexts

abstract: Epithelial-mesenchymal plasticity (EMP) underlies embryonic development, wound healing, and cancer metastasis and fibrosis. Cancer cells exhibiting EMP often have more aggressive behavior, characterized by drug resistance, and tumor-initiating and immuno-evasive traits. Thus, the EMP status of cancer cells can be a critical indicator of patient prognosis. Here, we compare three distinct transcriptomic-based metrics - each derived using a different gene list and algorithm - that quantify the EMP spectrum. Our results for 96 cancer-related RNA-seq datasets reveal a high degree of concordance among these metrics in quantifying the extent of EMP. Moreover, each metric, despite being trained on cancer expression profiles, recapitulates the expected changes in EMP scores for non-cancer contexts such as lung fibrosis and cellular reprogramming into induced pluripotent stem cells. Thus, we offer a scoring platform to quantify the extent of EMP in vitro and in vivo for diverse biological applications including cancer.

category: bioinformatics


title: Developing an enzyme selection tool supporting multiple hosts contexts

abstract: Engineering biological organisms that allow the integration of alternative metabolic pathways to natural ones is one of the goals of synthetic biology. Based on this, some of the most attractive applications in terms of synthetic organisms manufacture include the production of a wide range of pharmacologically useful metabolites produced in a sustainable and environmentally friendly way. Also, the biostable molecules green-production involves different types of therapeutic processes, e.g. prostheses and grafts stabilisation. Regarding the viability of genetically modified organisms, metabolic pathways must be first properly designed, taking into consideration the type of host organism that will be involved in metabolic production, as well as its biochemical and environmental conditions. To ensure the correct growth of these synthetic organisms, the enzyme selection must guarantee both the organism survival (and proliferation) and the optimal production of the desired metabolite. Developing enzyme selection tools is essential to enhance and make cost-effective the metabolic pathways design. This technical note presents the update of Selenzyme, the enzyme selection tool which is based on organisms taxonomic compatibility and allows appropriate enzyme selection considering its amino acid sequence. The purpose of the update is to allow multiple host input, in order to perform an affinity comparison between target organisms and each host. The affinity differences will depend on which host to be considered, allowing the user to select the optimal host for the enzyme concerned.


title: A General Framework for Representing and Annotating Multifaceted Cell Heterogeneity in Human Cell Atlas

abstract: The goal of big projects like Human Cell Atlas (HCA) and Human BioMedical Atlas Program (HuBMAP) is to build maps that comprehensively define and describe all cell types and their molecular features in a healthy human being. Just like geographical maps must have coordinates, a key task in building cell maps is to provide coordinate systems for cells. A well-designed coordinate system helps better understand the highly orchestrated function and organization of different cells. Cells could be depicted by external information like their spatial locations in the body and organ, the sex and race of the donor, and multiple endogenous attributes of cells such as their types, states, functions, developing trajectory, etc. These heterogeneities are encoded in or can be predicted with transcriptomics and other omics data. Cell heterogeneities are multifaceted, including three major types: continuous values or scores, categorical groups and structured annotations. Here we propose to a unified multidimensional coordinate system UniCoord to represent the multifaceted heterogeneities of cells. It is based on a general deep learning framework, with a supervised VAE structure to learn the mapping relationship between gene expressions and the generated coordinates in a low-dimensional space that encode multiple cell attributes of the three types. Experiment results on several datasets showed that UniCoord was able to represent a variety of cell heterogeneous properties that are discrete, continuous or of hierarchical structures. The trained UniCoord model can be used to automatically label attributes of cells and generate the corresponding expression data. Experiments showed that UniCoord is a feasible coordinates framework for representing multifaceted cell heterogeneity in comprehensive cell atlases.


title: Identifying potential natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach

abstract:
BackgroundBrucellosis is an infectious disease caused by bacteria of the genus Brucella. Although it is the most common zoonosis worldwide, there are increasing reports of drug resistance and cases of relapse after long term treatment with the existing drugs of choice. This study therefore aims at identifying possible natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach.

MethodsUsing PyRx 0.8 virtual screening software, the target was docked against a library of natural compounds obtained from edible African plants. The compound, 2-({3-[(3,5-dichlorobenzyl) amino] propyl} amino) quinolin-4(1H)-one (OOU) which is a co-crystallized ligand with the target was used as the reference compound. Screening of the molecular descriptors of the compounds for bioavailability, pharmacokinetic properties, and bioactivity was performed using the SWISSADME, pkCSM, and Molinspiration web servers respectively. The Fpocket and PLIP webservers were used to perform the analyses of the binding pockets and the protein ligand interactions. Analysis of the time-resolved trajectories of the Apo and Holo forms of the target was performed using the Galaxy and MDWeb servers. The lead compounds, Strophanthidin and Isopteropodin are present in Corchorus olitorius and Uncaria tomentosa (cat-claw) plants respectively.

ResultsIsopteropodin had a binding affinity score of -8.9 kcal / ml with the target and had 17 anti-correlating residues in pocket 1 after molecular dynamics simulation. The complex formed by Isopteropodin and the target had a total RMSD of 4.408 and a total RMSF of 9.8067. However, Strophanthidin formed 3 hydrogen bonds with the target at ILE21, GLY262 and LEU294, and induced a total RMSF of 5.4541 at Pocket 1.

ConclusionOverall, Isopteropodin and Strophanthidin were found to be better drug candidates than OOU and they showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1, hence abilities to treat brucellosis. In vivo and in vitro investigations are needed to further evaluate the efficacy and toxicity of the lead compounds.

Author SummaryO_LIStrophanthidin and Isopteropodin showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1
C_LIO_LIBoth compounds can be used to treat brucellosis.
C_LIO_LIBoth compounds showed potentials of being safe to use in humans.
C_LI


title: Nascent ribosomal RNA acts as surfactant that suppresses growth of fibrillar centers in nucleolus

abstract: Liquid-liquid phase separation (LLPS) has been thought to be the assembly mechanism of the multiphase structure of nucleolus, the site of ribosomal biogenesis. Condensates assembled by LLPS increase their size to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their size does not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the growth of FCs, we here construct a minimal theoretical model by taking into account the nascent pre-rRNA transcripts tethered to the surfaces of FCs by RNA polymerase I. Our theory predicts that nascent pre-rRNA transcripts generate the lateral osmotic pressure that counteracts the surface tension of the microphases and this suppresses the growth of the microphases over the optimal size. The optimal size of the microphases decreases with increasing the transcription rate and decreasing the rate of RNA processing. This prediction is supported by our experiments showing that the size of FCs increased with increasing the dose of transcription inhibitor. This theory may provide insight in the general mechanism of the size control of nuclear bodies.

category: biophysics


title: Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

abstract: The proton electrochemical gradient drives substrate transport across the cell membrane via a diverse set of secondary active transporters. Proton coupled peptide transporters (POTs) are important for peptide transport in prokaryotes and eukaryotic cells, where they mediate the uptake of di- and tri-peptides in addition to drug and pro-drug molecules. Previously, we captured a POT transporter from Staphylococcus hominis, PepTSh, in a cytoplasm-facing, inward open state (Minhas et al., 2018). Biochemical experiments have further revealed several critical residues for proton coupled transport; however, the precise role played by these residues in coupling proton binding to conformational changes as well as the timescales for proton transfers have remained obscure. Here, we employed multiscale modeling, including classical molecular dynamics, reactive molecular dynamics, and enhanced free energy sampling to characterize proton coupling within this transporter. We show directly that proton binding to a glutamate on TM7 opens the extracellular gate. The inward proton flow is found to induce movement of the peptide towards the cytosol by varying the protonation state of a second conserved glutamate on TM10. We also show that proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible, revealing a mechanism for proton movement inside the transporter.


title: Prevention of Drug-Induced Lung Fibrosis via Inhibition of the MRTF/SRF Transcription Pathway

abstract: Drug-induced lung fibrosis is a debilitating disease, linked to high morbidity and mortality. A number of drugs can cause fibrosis, many of which are used to treat cancer, including chemotherapy agents and immune checkpoint inhibitors. The MRTF/SRF transcription pathway has been proposed as a potential therapeutic target, as it is critical for myofibroblast differentiation, a hallmark of fibrosis. In human lung fibroblasts, the MRTF/SRF pathway inhibitor, CCG-257081, effectively decreased mRNA levels of downstream genes: smooth muscle actin and connective tissue growth factor, with IC50s of 4 and 15 M, respectively. The ability of CCG-257081 to prevent inflammation and fibrosis, measured via pulmonary collagen content and histopathology, was tested in a murine model of chemotherapy-induced lung fibrosis. Animals were given intraperitoneal bleomycin for four weeks, and concurrently dosed with CCG-257081 (0, 10, 30, and 100 mg/kg PO), a clinical anti-fibrotic (nintedanib), or clinical standard of care (prednisolone). Mice treated with 100 mg/kg CCG-257081 gained weight vs. vehicle-treated control mice, while those receiving nintedanib and prednisolone lost significant weight. Hydroxyproline content and histological findings in tissue of animals on 100 mg/kg CCG-257081 were not significantly different from naive tissue, indicating successful prevention. Measures of tissue fibrosis were comparable between CCG-257081 and nintedanib, but only the MRTF/SRF inhibitor decreased plasminogen activator inhibitor-1 (PAI-1), a marker linked to fibrosis, in bronchoalveolar lavage fluid. Prednisolone led to marked increases in lung fibrosis. This study demonstrates the potential use of MRTF/SRF inhibitors to prevent drug-induced lung fibrosis in a clinically relevant model of drug-induced disease.

category: pharmacology and toxicology


title: Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

abstract: Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

category: plant biology


title: Children with amblyaudia show less flexibility in auditory cortical entrainment to periodic non-speech sounds

abstract:
ObjectiveWe investigated auditory temporal processing in children with amblyaudia (AMB), a subtype of auditory processing disorder, via cortical neural entrainment.

Design and study samplesEvoked responses were recorded to click-trains at slow vs. fast (8.5 vs. 14.9/sec) rates in n=14 children with AMB and n=11 age-matched controls. Source and time-frequency analyses decomposed EEGs into oscillations (reflecting neural entrainment) stemming from the bilateral auditory cortex.

ResultsPhase-locking strength in AMB depended critically on the speed of auditory stimuli. In contrast to age-matched peers, AMB responses were largely insensitive to rate manipulations. This rate resistance was seen regardless of the ear of presentation and in both cortical hemispheres.

ConclusionChildren with AMB show a stark inflexibility in auditory cortical entrainment to rapid sounds. In addition to reduced capacity to integrate information between the ears, we identify more rigid tagging of external auditory stimuli. Our neurophysiological findings may account for certain temporal processing deficits commonly observed in AMB and related auditory processing disorders (APDs) behaviorally. More broadly, our findings may inform communication strategies and future rehabilitation programs; increasing the rate of stimuli above a normal (slow) speech rate is likely to make stimulus processing more challenging for individuals with AMB/APD.

category: neuroscience


title: Humans reconfigure target and distractor processing to address distinct task demands

abstract: When faced with distraction, we can focus more on goal-relevant information (targets) or focus less goal-conflicting information (distractors). How people decide to distribute cognitive control across targets and distractors remains unclear. To help address this question, we developed a parametric attentional control task that can index both target discriminability and distractor interference. We find that participants exert independent control over target and distractor processing. We measured control adjustments through the influence of incentives and previous conflict on target and distractor sensitivity, finding that these have dissociable influences on control. Whereas incentives preferentially led to target enhancement, conflict on the previous trial preferentially led to distractor suppression. These distinct drivers of control altered sensitivity to targets and distractors early in the trial, promptly followed by reactive reconfiguration towards task-appropriate feature sensitivity. Finally, we provide a process-level account of these findings by show that these control adjustments are well-captured by an evidence accumulation model with attractor dynamics over feature weights. These results help establish a process-level account of control configuration that provides new insights into how multivariate attentional signals are optimized to achieve task goals.


title: Spontaneous alpha-band oscillations bias subjective contrast perception

abstract:
Perceptual decisions depend both on the features of the incoming stimulus and on the ongoing brain activity at the moment the stimulus is received. Specifically, trial-to-trial fluctuations in cortical excitability have been linked to fluctuations in the amplitude of pre-stimulus alpha oscillations ({approx}8-13 Hz), which are in turn are associated with fluctuations in subjects tendency to report the detection of a stimulus. It is currently unknown whether alpha oscillations bias post-perceptual decision making, or even bias subjective perception itself. To answer this question, we used a contrast discrimination task in which subjects reported which of two gratings - one in each hemifield - was perceived as having a stronger contrast. Our EEG analysis showed that subjective contrast was reduced for the stimulus in the hemifield represented in the hemisphere with relatively stronger pre-stimulus alpha amplitude, reflecting reduced cortical excitability. Furthermore, the strength of this spontaneous hemispheric lateralization was strongly correlated with the magnitude of individual subjects biases, suggesting that the spontaneous patterns of alpha lateralization play a role in explaining the intersubject variability in contrast perception. These results indicate that spontaneous fluctuations in cortical excitability, indicted by patterns of pre-stimulus alpha amplitude, affect perceptual decisions by altering the phenomenological perception of the visual world.

Significance StatementOur moment to moment perception of the world is shaped by the features of the environment surrounding us, as much as by the constantly evolving states that characterize our brain activity. Previous research showed how the ongoing electrical activity of the brain can influence whether a stimulus has accessed conscious perception. However, evidence is currently missing on whether these electrical brain states can be associated to the subjective experience of a sensory input. Here we show that local changes in patterns of electrical brain activity preceding visual stimulation can bias our phenomenological perception. Importantly, we show that the strength of these variations can help explaining the great inter-individual variability in how we perceive the visual environment surrounding us.


title: Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

abstract: Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

category: immunology


title: Comparative analysis of L-fucose utilization and its impact on growth and survival of Campylobacter isolates

abstract:
Campylobacter jejuni and Campylobacter coli were previously considered asaccharolytic, but are now known to possess specific saccharides metabolization pathways, including L-fucose. To investigate the influence of the L-fucose utilization cluster on Campylobacter growth, survival and metabolism, we performed comparative genotyping and phenotyping of the C. jejuni reference isolate NCTC11168 (human isolate), C. jejuni Ca1352 (chicken meat isolate), C. jejuni Ca2426 (sheep isolate), and C. coli Ca0121 (pig manure isolate), that all possess the L-fucose utilization cluster.

All isolates showed enhanced survival and prolonged spiral cell morphology in aging cultures up to day seven in L-fucose-enriched MEM medium (MEMF) compared to MEM. HPLC analysis indicated L-fucose utilization linked to acetate, lactate, pyruvate and succinate production, confirming the activation of the L-fucose pathway in these isolates. Highest consumption of L-fucose by C. coli Ca0121, is conceivably linked to its enhanced growth performance up to day 7, reaching 9.3 log CFU/ml compared to approximately 8.3 log CFU/ml for the C. jejuni isolates. Genetic analysis of their respective L-fucose clusters revealed several differences, including a 1 bp deletion in the Cj0489 gene of C. jejuni NCTC11168, causing a frameshift in this isolate resulting in two separate genes, Cj0489 and Cj0490, while no apparent phenotype could be linked to the presumed frameshift in the NCTC11168 isolate. Additionally, we found that the L-fucose cluster of C. coli Ca0121 was most distant from C. jejuni NCTC11168, but confirmation of links to L-fucose metabolism associated phenotypic traits in C. coli versus C. jejuni isolates requires further studies.

ImportanceCampylobacter is the leading cause of gastroenteritis in humans worldwide, with increasing incidence and prevalence in recent years. The most prevalent species are Campylobacter jejuni and C. coli with 83% and 10% of all Campylobacter cases, respectively. Previously it was found that the majority of Campylobacter isolates are able to metabolize L-fucose (fuc+ isolates), a sugar that is widely present in the human gut. Putative roles for L-fucose in fuc+ C. jejuni isolates were found in growth, biofilm formation and virulence. Despite this, relatively little is known about L-fucose metabolism and the impact on growth and survival in fuc+ Campylobacter isolates. The results from our comparative genotyping and phenotyping study demonstrate that L-fucose, in both C. jejuni and C. coli fuc+ isolates, is involved in enhanced survival, prolonged spiral cell morphology and changes in the general metabolism. Possible links between phenotypes and differences in respective L-fucose gene clusters are discussed.

category: microbiology


title: The ribbon architecture of the Golgi apparatus is not restricted to vertebrates

abstract:
The Golgi apparatus plays a central role as a processing and sorting station along the secretory pathway. In multicellular organisms, this organelle displays two structural organizations, whereby its functional subunits, the mini-stacks, are either dispersed throughout the cell or linked into a centralized structure, called Golgi "ribbon". The Golgi ribbon is considered to be a feature typical of vertebrate cells. Here we report that this is not the case. We show that sea urchin embryonic cells assemble Golgi ribbons during early development. Sea urchins are deuterostomes, the bilaterian animal clade to which chordates, and thus vertebrates, also belong.

Far from being a structural innovation of vertebrates, the Golgi ribbon therefore appears to be an ancient cellular feature evolved before the split between echinoderms and chordates. Evolutionary conservation of the ribbon architecture surmises that it must play fundamental roles in the biology of deuterostomes.

category: cell biology


title: Genetic variation in ALDH4A1 predicts muscle health over the lifespan and across species

abstract: Environmental stress can negatively impact organismal aging, however, the long-term impact of endogenously derived reactive oxygen species from normal cellular metabolism remains less clear. Here we define the evolutionarily conserved mitochondrial enzyme ALH-6/ALDH4A1 as a biomarker for age-related changes in muscle health by combining C. elegans genetics and a gene-wide association study (GeneWAS) from aged human participants of the US Health and Retirement Study (HRS)1-4. In a screen for mutations that activate SKN-1-dependent oxidative stress responses in the muscle of C. elegans5-7, we identified 96 independent genetic mutants harboring loss-of-function alleles of alh-6, exclusively. These genetic mutations map across the ALH-6 polypeptide, which lead to age-dependent loss of muscle health. Intriguingly, genetic variants in ALDH4A1 differentially impact age-related muscle function in humans. Taken together, our work uncovers mitochondrial alh-6/ALDH4A1 as a critical component of normal muscle aging across species and a predictive biomarker for muscle health over the lifespan.

category: genetics


title: Joint Registration of Multiple Point Clouds for Fast Particle Fusion in Localization Microscopy

abstract:
We present a fast particle fusion method for particles imaged with single-molecule localization microscopy. The state-of-the-art approach based on all-to-all registration has proven to work well but its computational cost scales unfavourably with the number of particles N, namely as N2. Our method overcomes this problem and achieves a linear scaling of computational cost with N by making use of the Joint Registration of Multiple Point Clouds (JRMPC) method. Straightforward application of JRMPC fails as mostly locally optimal solutions are found. These usually contain several overlapping clusters, that each consist of well-aligned particles, but that have different poses. We solve this issue by repeated runs of JRMPC for different initial conditions, followed by a classification step to identify the clusters, and a connection step to link the different clusters obtained for different initializations. In this way a single well-aligned structure is obtained containing the majority of the particles.

We achieve reconstructions of experimental DNA-origami datasets consisting of close to 400 particles within only 10 min on a CPU, with an image resolution of 3.2 nm. In addition, we show artifact-free reconstructions of symmetric structures without making any use of the symmetry. We also demonstrate that the method works well for poor data with a low density of labelling and for 3D data.


title: scBasset: Sequence-based modeling of single cell ATAC-seq using convolutional neural networks

abstract: 1Single cell ATAC-seq (scATAC) shows great promise for studying cellular heterogeneity in epigenetic landscapes, but there remain significant challenges in the analysis of scATAC data due to the inherent high dimensionality and sparsity. Here we introduce scBasset, a sequence-based convolutional neural network method to model scATAC data. We show that by leveraging the DNA sequence information underlying accessibility peaks and the expressiveness of a neural network model, scBasset achieves state-of-the-art performance across a variety of tasks on scATAC and single cell multiome datasets, including cell type identification, scATAC profile denoising, data integration across assays, and transcription factor activity inference.


title: Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers

abstract: Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is determined by "sticky" interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean field "stickers-and-spacers" model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and non-specific polymer-solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multi-associative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid-liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps, or a flip in the slope of the tie-lines belonging to a single coexistence region.


title: Data cleaning for image-based profiling enhancement

abstract: With the advent of high-throughput assays, a large number of biological experiments can be carried out. Image-based assays are among the most accessible and inexpensive technologies for this purpose. Indeed, these assays have proved to be effective in characterizing unknown functions of genes and small molecules. Image analysis pipelines have a pivotal role in translating raw images that are captured in such assays into useful and compact representation, also known as measurements. CellProfiler is a popular and commonly used tool for this purpose through providing readily available modules for the cell/nuclei segmentation, and making various measurements, or features, for each cell/nuclei. Single cell features are then aggregated for each treatment replica to form treatment "profiles." However, there may be several sources of error in the CellProfiler quantification pipeline that affects the downstream analysis that is performed on the profiles. In this work, we examined various preprocessing approaches to improve the profiles. We consider identification of drug mechanisms of action as the downstream task to evaluate such preprocessing approaches. Our enhancement steps mainly consist of data cleaning, cell level outlier detection, toxic drug detection, and regressing out the cell area from all other features, as many of them are widely affected by the cell area. We also examined unsupervised and weakly-supervised deep learning based methods to reduce the feature dimensionality, and finally suggest possible avenues for future research.


title: pH-dependent polymorphism of the structure of SARS-CoV-2 nsp7

abstract: The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical bundle folding at neutral pH. Remarkably, the 4 helix shows gradual dislocation from the core 2-3 structure as pH increases from 6.5 to 7.5. The protonation state of residue H36 contributes to the change of nsp7s intramolecular interactions, and thus, to the structural variation near-neutral pH. Spin-relaxation results revealed that all three loop regions in nsp7 possess dynamic properties associated with this structural variation.

category: biochemistry


title: Phomoarcherin B as a novel HIV-1 reverse transcriptase RNase H activity inhibitor: conclusions from comprehensive computational analysis

abstract:
The HIV epidemic has claimed more than 32.7 million live since its emergence in 1981, while many ART and HAART therapies are available and provide relief and control for patients, most of these therapeutics come with long-term side effects, resistance, socio-economical barriers, and other obstacles. In this study, genomic analysis was performed on 98 HIV-1 genomes to determine the most coherent target that could be utilized to restrict and cease the viral replication, the reverse transcriptase enzyme. Following the identification of the target protein, the RNase H activity of the reverse transcriptase was nominated as the potent target given the limited research associated with it. A library of 94 thousand small molecule inhibitors was generated and virtual screening was performed to identify hits, based on the reproducibility of the screening results, 4 compounds with the best scores were considered and their interaction within the active site was analyzed. Subsequently, all-atom molecular dynamics simulations and MM-PBSA was performed to validate the stability and binding free energy of the hits within the RNase H active. In silico ADMET assays were performed on the hit compounds to analyze their drug-likeness, physicochemical and pharmacological properties. Phomoarcherin B, a pentacyclic aromatic sesquiterpene naturally found in the endophytic fungus Phomopsis archeri, known for its anticancer properties scored the best in all the experiments and was nominated as a potential inhibitor of the HIV-1 reverse transcriptase RNase H activity.

Author SummaryThe Human immunodeficiency virus 1 (HIV-1) has remained a global public health issue for the past 4 decades with millions of patients around the globe affected. Long years of research in the field of anti-viral therapies had introduced several drugs to combat the viral infection, however, most of these drugs come with their shortcomings. In this study, computational drug discovery approaches were utilized to research novel drugs that could be used as anti-viral agents against the former virus, the results had revealed Phomoarcherin B, a natural compound from an endophytic fungus to hold promising therapeutic potentials.


title: Active growth signalling promotes cancer cell sensitivity to the CDK7 inhibitor ICEC0942

abstract:
CDK7 has a central role in promoting cellular proliferation, through the activation of the mitotic CDKs, and by driving global gene expression, through targeting RNA polymerase II. Several recently developed CDK7 inhibitors (CDK7i) have been shown to be non-toxic and to limit tumour growth for a number of cancer cell types and are now in Phase I/II clinical trials. However, the mechanisms underlying the sensitivity of particular cancer cells to CDK7 inhibition remain largely unknown.

To improve the outcome of individual patients and increase the chances of successful CDK7i approval, we assessed which fundamental cellular processes determine sensitivity to CDK7 inhibition, using the highly specific CDK7 inhibitor ICEC0942. Our data shows that selective CDK7 inhibition acutely arrests cells in the G1 phase of the cell cycle, which over time leads to senescence. Through a genome-wide CRISPR knock-out chemogenetic screen we identified active mTOR (mammalian target of rapamycin) signalling, as an important determinant of ICEC0942-induced senescence and show that a cancer-associated mutation that promotes cell growth can increase sensitivity to ICEC0942. Our work indicates that cellular growth is an important predictive marker for sensitivity to CDK7i.

category: cancer biology


title: Targeting the Cx26/NANOG/Focal Adhesion Kinase Complex via Cell Penetrating Peptides in Triple Negative Breast Cancers

abstract:
PurposeTriple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype and has limited treatment options. We previously identified a protein complex unique to TNBC cancer stem cells composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of Cx26 designed to target the complex could attenuate tumor growth in pre-clinical models.

Experimental DesignHistological assessment was employed to verify expression of complex members. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Peptides with high affinity were engineered with a cell-penetrating sequence and assessed in functional assays including cell proliferation, self-renewal, and in vivo tumor growth, and downstream signaling changes were measured.

ResultsBinding studies revealed that the Cx26 cytoplasmic C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar to micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. An antennapedia cell penetrating peptide sequence was engineered to the Cx26 C-terminal tail and confirmed intracellular localization. The cell-penetrating Cx26 C-terminal tail peptide (aCx26-pep) disrupted self-renewal as assessed by the tumorsphere formation assay and inhibited NANOG target gene expression in TNBC cells but not in luminal mammary epithelial cells. In a pre-clinical setting, aCx26-pep reduced tumor growth and proliferation and induced cell death.

ConclusionsWe provide proof-of-concept that a Cx26 peptide-based strategy can inhibit TNBC growth.

Translational RelevanceTriple-negative breast cancer (TNBC) is the most treatment-refractory breast cancer subtype and has limited targeted therapy options. TNBC contains a cancer stem cell (CSC) population that underlies growth and therapeutic resistance. We leveraged an aberrant intracellular protein complex in TNBC CSCs containing the gap junction subunit connexin 26 (Cx26), focal adhesion kinase (FAK), and NANOG to develop a peptide-based therapeutic strategy. We show that TNBC can be selectively targeted using this approach. Our findings provide a new strategy for treatment of this patient population by disrupting intracellular Cx26 signaling, which is linked to the CSC and epithelial-to-mesenchymal programs. Our results support the development of therapeutics targeting the Cx26/NANOG/FAK complex via peptide- and small molecule-based approaches, and future efforts will also focus on the development of a combinatorial treatment strategy with FDA-approved chemotherapeutics.

One Sentence SummaryA peptide-based targeting strategy for triple-negative breast cancer was developed by targeting connexin 26-associated signaling partners.


title: Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity

abstract:
Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous.

To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity - and not inverse similarity - between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism.

Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.

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category: systems biology


title: Differential antibody dynamics to SARS-CoV-2 infection and vaccination

abstract: Optimal immune responses furnish long-lasting (durable) antibodies protective across dynamically mutating viral variants (broad). To assess robustness of mRNA vaccine-induced immunity, we compared antibody durability and breadth after SARS-CoV-2 infection and vaccination. While vaccination delivered robust initial virus-specific antibodies with some cross-variant coverage, pre-variant SARS-CoV-2 infection-induced antibodies, while modest in magnitude, showed highly stable long-term antibody dynamics. Vaccination after infection induced maximal antibody magnitudes with enhanced longitudinal stability while infection-naive vaccinee antibodies fell with time to post-infection-alone levels. The composition of antibody neutralizing activity to variant relative to original virus also differed between groups, with infection-induced antibodies demonstrating greater relative breadth. Differential antibody durability trajectories favored COVID-19-recovered subjects with dual memory B cell features of greater early antibody somatic mutation and cross-coronavirus reactivity. By illuminating an infection-mediated antibody breadth advantage and an anti-SARS-CoV-2 antibody durability-enhancing function conferred by recalled immunity, these findings may serve as guides for ongoing vaccine strategy improvement.


title: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

abstract: Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER) and upload onto major histocompatibility type I (MHC-I) protein complexes for cell-surface transport and cross-presentation. Perplexingly, DCs often exhibit activation of the ER-stress sensor IRE1 in the absence of classical ER stress--leaving the underlying mechanism unexplained. Here we show that antigen-derived hydrophobic peptides directly engage ER-resident IRE1 by masquerading as unfolded proteins. Furthermore, IRE1 activation depletes MHC-I heavy-chain mRNAs through regulated IRE1-dependent decay (RIDD), thereby curtailing antigen cross-presentation. In tumor-bearing mice, IRE1 disruption increased MHC-I expression on tumor-infiltrating DCs, and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1 inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings elucidate the mechanism and consequence of antigen-driven IRE1 activation in DCs, yielding a promising combination strategy for cancer immunotherapy.


title: FIBCD1 is a Conserved Receptor for Chondroitin Sulphate Proteoglycans of the Brain Extracellular Matrix and a Candidate Gene for a Complex Neurodevelopmental Disorder

abstract: The brain extracellular matrix (ECM) is enriched in chondroitin sulphate proteoglycans (CSPGs) with variable sulphate modifications that intimately participate in brain maturation and function. Very little is known about how the changing biophysical properties of the CSPGs are signalled to neurons. Here, we report Fibrinogen C Domain Containing 1 (FIBCD1), a known chitin-binding receptor of the innate immune system, to be highly expressed in the hippocampus and to specifically bind CSPGs containing 4-O sulphate modification (CS-4S). Cultured Fibcd1 knockout (KO) neurons lack phenotypic and transcriptomic responses to CSPG stimulation. Further, Fibcd1 KO mice exhibit accumulation of CS-4S, likely resulting in deficits of hippocampal-dependent learning tasks and abrogated synaptic remodelling, a phenotype rescued by enzymatic digestion of CSPGs. Likewise, neuronal specific knockdown of a Fibcd1 orthologue in flies results in neuronal morphological changes at the neuromuscular junctions and behavioural defects. Finally, we report two undiagnosed patients with a complex neurodevelopmental disorder with deleterious variants in FIBCD1, strongly implicating FIBCD1 in the development of the disease. Taken together, our results demonstrate that FIBCD1 is a novel, evolutionarily conserved component of ECM sulphation recognition that is crucial for neuronal development and function.


title: AgRP neurons coordinate the mitigation of activity-based anorexia

abstract: Anorexia nervosa (AN) is a debilitating and deadly disease characterized by low body mass index due to diminished food intake, and oftentimes concurrent hyperactivity. A high percentage of AN behavioral and metabolic phenotypes can be replicated in rodents given access to a voluntary running wheel and subject to food restriction, termed activity-based anorexia (ABA). Despite the well-documented body weight loss observed in AN human patients and ABA rodents, much less is understood regarding the neurobiological underpinnings of these maladaptive behaviors. Moreover, while exercise has been shown to diminish the activity of hunger-promoting hypothalamic agouti-related peptide (AgRP) neurons, much less is known regarding their activity and function in the mediation of food intake during ABA. Here, feeding microstructure analysis revealed ABA mice decreased food intake due to increased interpellet interval retrieval and diminished meal number. Longitudinal activity recordings of AgRP neurons in ABA animals revealed a maladaptive inhibitory response to food. We then demonstrated that ABA development or progression can be mitigated by chemogenetic AgRP activation through the reprioritization of food intake (increased meal number) over hyperactivity. These results elucidate a potential neural target for the amelioration of behavioral maladaptations present in AN patients.


title: The architecture and operating mechanism of a cnidarian stinging organelle

abstract: The stingers of jellyfish, sea anemones and other cnidarians, known as nematocysts, are remarkable cellular weapons used for both predation and defense1. Nematocysts are specialized organelles which consist of a pressurized capsule containing a coiled harpoon-like thread2. These structures are in turn built within specialized cells known as nematocytes3. When triggered4, the capsule explosively discharges, ejecting the coiled thread which punctures the target and rapidly elongates by turning inside out in a process called eversion5,6. Due to the structural complexity of the thread and the extreme speed of discharge, the precise mechanics of nematocyst firing have remained elusive7. Here, using a combination of live and super-resolution imaging, 3D electron microscopy and genetic perturbations, we define the step-by-step sequence of nematocyst operation in the model sea anemone Nematostella vectensis. This analysis reveals the complex biomechanical transformations underpinning the operating mechanism of nematocysts, one of the natures most exquisite biological micro-machines. Further, this study will provide insight into the form and function of related cnidarian organelles and serve as a template for the design of bioinspired microdevices.


title: Not so local: the population genetics of convergent adaptation in maize and teosinte.

abstract: What is the genetic architecture of local adaptation and what is the geographic scale that it operates over? We investigated patterns of local and convergent adaptation in five sympatric population pairs of traditionally cultivated maize and its wild relative teosinte (Zea mays subsp. parviglumis). We found that signatures of local adaptation based on the inference of adaptive fixations and selective sweeps are frequently exclusive to individual populations, more so in teosinte compared to maize. However, for both maize and teosinte, selective sweeps are frequently shared by several populations, and often between the subspecies. We were further able to infer that selective sweeps were shared among populations most often via migration, though sharing via standing variation was also common. Our analyses suggest that teosinte has been a continued source of beneficial alleles for maize, post domestication, and that maize populations have facilitated adaptation in teosinte by moving beneficial alleles across the landscape. Taken together, out results suggest local adaptation in maize and teosinte has an intermediate geographic scale, one that is larger than individual populations, but smaller than the species range.

category: evolutionary biology


title: Intranasal administration of a VLP-based vaccine against COVID-19 induces neutralizing antibodies against SARS-CoV-2 and Variants of Concerns

abstract:
BackgroundThe highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of COVID-19 vaccines.

MethodsIn the current study, we tested in murine model the immunogenicity of a conventional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal vaccination. The candidate vaccine, CuMVTT-RBD, has been immunologically optimized to incorporate tetanus-toxin and is self-adjuvanted with TLR7/8 ligands.

ResultsCuMVTT-RBD elicited strong RBD- and spike- specific systemic IgG and IgA antibody responses of high avidity. Local immune responses were assessed and results demonstrate strong mucosal antibody and plasma cell production in lung tissue. The induced systemic antibodies could efficiently recognize and neutralize different Variants of Concerns of mutated SARS-CoV-2 RBDs.

ConclusionIn summary, intranasal vaccination with CuMVTT-RBD shows high immunogenicity and induces protective systemic and local specific antibody response against SARS-CoV-2 and its variants.

One sentence summaryEvaluation of an intransal administrated conventional VLP-based vaccine against COVID-19 in a murine model.


title: Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

abstract:
Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant ({Delta}phz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the {Delta}phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions.

ImportanceDue to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.


title: Kv1.3 induced hyperpolarisation and Cav3.2-mediated calcium entry are required for efficientKaposi's sarcoma-associated herpesvirus lytic replication

abstract: Understanding the host factors critical for Kaposis sarcoma-associated herpesvirus (KSHV) lytic replication can identify new targets for therapeutic intervention. Using pharmacological and genetic silencing approaches, we reveal for the first time that KSHV requires a B cell expressed voltage-gated K+ channel, Kv1.3, to enhance lytic replication. We show that the KSHV replication and transcription activator (RTA) protein upregulates Kv1.3 expression, leading to enhanced K+ channel activity and hyperpolarisation of the B cell membrane. Enhanced Kv1.3 activity then promotes intracellular Ca2+ influx through Cav3.2, a T-type Ca2+ channel, leading to the Ca2+ driven nuclear localisation of NFAT and the subsequent NFAT1-responsive gene expression. Importantly, KSHV lytic replication and infectious virion production could be inhibited by both Kv1.3 and Cav3.2 blockers or through Kv1.3 silencing. These findings provide new mechanistic insight into the essential role of host ion channels during KSHV infection and highlight Kv1.3 and Cav3.2 as new druggable host factors that are key to the successful completion of KSHV lytic replication.


title: Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

abstract: The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an AAC(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.


title: An inactivated multivalent influenza A virus vaccine is broadly protective in mice and ferrets

abstract:
Influenza A viruses (IAVs) present major public health threats from annual seasonal epidemics, from pandemics caused by novel virus subtypes, and from viruses adapted to a variety of animals including poultry, pigs and horses. Vaccines that broadly protect against all such IAVs, so-called "universal" influenza vaccines, do not currently exist, but are urgently needed. This study demonstrates that an inactivated, multivalent whole virus vaccine, delivered intramuscularly or intranasally, is broadly protective against challenges with multiple IAV HA/NA subtypes in both mice and ferrets, including challenges with IAV subtypes not contained in the vaccine. This vaccine approach indicates the feasibility of eliciting broad "universal" IAV protection, and identifies a promising candidate for influenza vaccine clinical development.

One-Sentence SummaryAn inactivated, whole avian influenza virus vaccine delivered intramuscularly or intranasally provides extremely broad protection against antigenically divergent viral challenge and is a promising candidate for a "universal" influenza virus vaccine.


title: Comparative genomic analysis of skin and soft tissue Streptococcus pyogenes isolates from low- and high-income settings

abstract: Streptococcus pyogenes is a leading cause of human morbidity and mortality, especially in resource limited settings. The World Health Organisation has recently made a vaccine for S. pyogenes a global health priority to reduce the burden of the post-infection rheumatic heart disease. For a vaccine to be active against all relevant strains in each region, molecular characterisation of circulating S. pyogenes isolates is needed. We performed extensive comparative whole genome analyses of S. pyogenes isolates from skin and soft tissue infections in The Gambia, West Africa, where there is a high burden of such infections. To act as a comparator to this low-income country (LIC) collection of isolates, we performed genome sequencing of isolates from skin infections in Sheffield, UK, as representative high-income country (HIC) isolates. LIC isolates from The Gambia were genetically more diverse (46 emm-types in 107 isolates) compared to HIC isolates from Sheffield (23 emm-types in 142 isolates), with only 7 overlapping emm-types and with diverse genetic backgrounds. Characterisation of other molecular markers indicated some shared features, including a high prevalence of the skin infection-associated emm-pattern D and the variable fibronectin-collagen-T antigen (FCT) types FCT-3 and FCT-4. A previously unidentified FCT (FCT-10) was identified in the LIC isolates, belonging to two different emm-types. A high proportion (79/107; 73.8%) of LIC isolates carried genes for tetracycline resistance, compared to 53/142 (37.3%) HIC isolates. There was also evidence of different circulating prophages, as very few prophage-associated DNases and lower numbers of superantigens were detected in LIC isolates. Our study provides much needed insight into the genetics of circulating isolates in a LIC (The Gambia), and how they differ from those circulating in HICs (Sheffield, UK). Common molecular features may act as bacterial drivers for specific infection types, regardless of the diverse genetic background.


title: Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy

abstract: SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.


title: Spexin modulates molecular thermogenic profile of adipose tissue and thermoregulatory behaviors

abstract:
Thermoregulation is a physiological process by which a mammal regulates body temperature in response to its environment. Within the human body, thermoregulatory behaviors and metabolism are modulated by circulating metabolic factors. In our study, we tested the ability of the neuropeptide spexin, which shares sequence homology to galanin, to regulate these functions in female mice. Supraphysiological levels of spexin in C57BL/6 mice were insufficient to protect against diet-induced obesity after 50 days of treatment. Behavioral analysis of long-term spexin treatment appeared to modulate anxiety-like behaviors by promoting exploratory behaviors and thermoregulatory behaviors of nest building that ceased when animals were housed at thermoneutral temperatures. Upon examination of the molecular profile of brown and white adipose tissue, treatment disrupted the thermogenic profile of white adipose tissue, in which {beta}3-adrenergic receptor expression was downregulated. Our results reveal novel functions for spexin as a modulator of thermoregulatory behaviors and adipose tissue metabolism.

HighlightsO_LISpexin treatment did not protect against diet-induced obesity in female mice.
C_LIO_LISpexin-treatment promoted thermoregulatory behaviors of nest building.
C_LIO_LIBehaviors normalized when animals were housed in thermoneutral temperatures.
C_LI

Funding SourcesNot applicable

Disclosure SummaryNothing to disclose

category: molecular biology


title: Characterization of aquaporin1b (AQP1b) mRNA in mud loach (Misgurnus mizolepis) in response to heavy metal and immunostimulant stimuli

abstract:
Aquaporins (AQPs) facilitate the transport of water or other small solutes into cells in the presence of osmotic gradients. However, the current understanding of piscine AQP gene with cellular stress responses has been still limitedly exemplified. In present study, we characterized the mud loach AQP1b gene at the nucleotide and amino acid levels. We identified three AQP 1b transcript variants (mmAQP1b_tv1, mmAQP1b_tv2, and mmAQP1b_tv3). Then, we examined the AQP1b promoter region and observed several transcription factor binding sites (TFBS) for nuclear factor of activated T-cells (NFAT), SRY-box, c-AMP responsive element binding protein (CREB), GATA binding factor, and hepatic nuclear factor-1. Interestingly, mmAQP1b transcription was differentially modulated by heavy metal or immunostimulant challenge. Further studies to deepen the knowledge of fish AQP-mediated adaptation response potentially relevant to molecular pathogenesis are warranted.

Summary statementWe identified mud loach AQP1b transcript variants and consensus sequences involved in stress or innate immunity in promotor region. AQP1b transcription was differentially modulated by heavy metal or immunostimulant challenge.


title: Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants

abstract: The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.


title: SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells

abstract: Chimeric antigen receptor (CAR) is a single-pass transmembrane receptor designed to specifically target and eliminate cancers. While CARs prove highly efficacious against B cell malignancies, the intracellular signaling events which promote CAR T cell activity remain elusive. To gain further insight into both CAR T cell signaling and the potential signaling response of cells targeted by CAR, we analyzed phosphopeptides captured by two separate phopshoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we report that CD19-CAR T cells upregulated several key phosphorylation events also observed in canonical T cell receptor (TCR) signaling while Raji B cells exhibited a significant decrease in B cell receptor-signaling related phosphorylation events in response to coculture. Our data suggest that CD19-CAR stimulation activates a mixture of unique CD19-CAR-specific signaling pathways and canonical TCR signaling while global phosphorylation in Raji B cells is reduced after association with the CD19-CAR T cells.


title: Tetragonisca angustula resin gathering behaviour of Schizolobium parahyba

abstract: Tetragonisca angustula is a very important stingless bees species. This study aimed to report the resin gathering behaviour of this species of a possible new resin source: Schizolobium parahyba. Evaluations of temperature, number of bees in gathering activity, time and season were used to characterize bees behaviour. Resin gathering activities were concentered between 10 and 14 hours, with a moderate linear positive correlation index with the temperature. No bee activity was observed when temperatures were below 16.69{degrees}C. Gathering suffered extreme reduction during winter and greater activity in summer. Bees exploration were concentered on the younger leafs and apical portion of S. parahyba. Older leafs were mainly ignored, and gradually the exploring activity was constantly migrating to most young parts, while apical exploration was consistent all the studied period.

category: ecology


title: Bacterial supergroup specific "Cost" of Wolbachia infections in Nasonia vitripennis

abstract: The maternally-inherited endosymbiont, Wolbachia, is known to alter the reproductive biology of its arthropod hosts for its benefit and can induce both positive and negative fitness effects in many hosts. Here we describe the effects of the maintenance of two distinct Wolbachia infections, one each from supergroups A and B, on the parasitoid host Nasonia vitripennis. We compare the effect of Wolbachia infections on various traits between the uninfected, single A infected, single B infected, and the double infected strains with their cured versions. Contrary to the previous reports, our results suggest that there is a significant cost associated with the maintenance of Wolbachia infections where traits like family size, fecundity, longevity, and rates of male copulation are compromised in Wolbachia infected strains. The double infected and supergroup B infection strains show higher Wolbachia titer than supergroup A. The double infected Wolbachia strain has the most detrimental impact on the host as compared to single infections. Moreover, there is a supergroup-specific negative impact on these wasps as the supergroup B infections elicit the most pronounced negative effects. These findings raise important questions on the mechanism of survival and maintenance of these reproductive parasites in arthropod hosts.


title: Strong experimental support for the hologenome hypothesis revealed from Drosophila melanogaster selection lines

abstract: Recently it has been proposed, that the holobiont, i.e., the host and its associated microbiome, constitute a distinct biological entity, on which selection operates. This is a fascinating idea that so far has limited empirical justification. Here Drosophila melanogaster lines from a large-scale artificial selection experiment, where we selected for stress resistance traits and for longevity, were used to test the hologenome hypothesis. We raised flies from all selection regimes, including a regime where flies were kept at benign standard laboratory condition (control regime) throughout the duration of the experiment, under common garden conditions and sequenced the microbiome of the flies. We found abundant differences in microbial communities between control and selection regimes, but not between replicate lines within the regimes, and microbial diversity was higher in selected relative to control lines. Several major core Drosophila bacterial species were differentially abundant in the different selection regimes despite flies being exposed to similar nutritional and general environmental conditions. Our results support the idea that the host and microbiome genomes have evolved in concert and provide experimental support for the hologenome theory of evolution.


title: The transitivity of the Hardy-Weinberg law

abstract: The reduction of multi-allelic polymorphisms to variants with fewer alleles, two in the limit, is addressed. The Hardy-Weinberg law is shown to be transitive in the sense that a multi-allelic polymorphism that is in equilibrium will retain its equilibrium status if any allele together with its corresponding genotypes is deleted from the population. Similarly, the transitivity principle also applies if alleles are joined, which leads to the summation of allele frequencies and their corresponding genotype frequencies. These basic polymorphism properties are intuitive, but they have apparently not been formalized or investigated. This article provides a straightforward proof of the transitivity principle, and its usefulness in practical genetic data analysis with multi-allelic markers is explored. In general, results of statistical tests for Hardy-Weinberg equilibrium obtained with polymorphisms that are reduced by deletion or joining of alleles are seen to be consistent with the formulated transitivity principle. We also show how the transitivity principle allows one to identify equilibrium-offending alleles, and how it can provide clues to genotyping problems and evolutionary changes. For microsatellites, which are widely used in forensics, the transitivity principle implies one expects similar results for statistical tests that use length-based and sequence-based alleles. High-quality autosomal microsatellite databases of the US National Institute of Standards and Technology are used to illustrate the use of the transitivity principle in testing both length-based and sequence-based microsatellites for Hardy-Weinberg proportions. Test results for Hardy-Weinberg proportions for the two types of microsatellites are seen to be largely consistent and can detect allele imbalance.


title: Whole-genome sequencing analysis reveals the population history of Mus musculus in Madagascar

abstract: In Madagascar, the house mouse (Mus musculus) is thought to have colonized with human activities and is now one of the most abundant rodents on the island. In this study, we determined the whole-genome sequences of five Madagascar house mice captured from the wild. We examined the evolutionary history of the population by analyzing the mitochondrial and autosomal genomes. We confirmed that the mitochondrial genome lineages of Madagascar house mice formed a monophyletic clade placed at one of the most basal positions in the species. An analysis of autosomal genomic sequences indicates that the Madagascar house mice are genetically members of M. m. castaneus (CAS), but also contain genetic elements of M. m. domesticus (DOM) resulting from hybridization between subspecies. The signature of a strong population bottleneck 1000-3000 years ago was observed in both mitochondrial and autosomal genomic data. All samples showed strong genetic affinity to many CAS samples across a wide range of Indian Ocean coastal and island regions, with divergence time estimated around 4000 years ago. These findings support that the Madagascar house mice started to colonize the island with human agricultural activity, and experienced complex history for the establishment.


title: Whole-genome sequencing analysis of wild-caught house mice Mus musculus from Madagascar


title: Cytosolic aspartate aminotransferase moonlights as a ribosome binding modulator of Gcn2 activity during oxidative stress

abstract: Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2 phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or  moonlighting function that modulates the ISR independent of its aspartate aminotransferase activity.


title: Mechanistic insight into light-dependent recognition of Timeless by Drosophila cryptochrome

abstract: Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light and triggering its degradation. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM binding. A generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay enables quantification of CRY binding to TIM in dark and light. The assay is utilized to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bind TIM constitutively, whereas those incapable of photoreduction bind TIM weakly. In response to flavin redox state, two conserved histidine residues contribute to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify protein-protein interactions and photoreceptor targeting.


title: Rapid, multianalyte detection of opioid metabolites in wastewater

abstract: By monitoring opioid metabolites, wastewater-based epidemiology (WBE) could be an excellent tool for real-time information on consumption of illicit drugs. A key limitation of WBE is the reliance on costly laboratory-based techniques that require substantial infrastructure and trained personnel, resulting in long turnaround times. Here, we present an aptamer-based graphene field effect transistor (AptG-FET) platform for simultaneous detection of three different opioid metabolites. This platform provides a reliable, rapid, and inexpensive method for quantitative analysis of opioid metabolites in wastewater (WW). The platform delivers a limit of detection (LOD) 2-3 orders of magnitude lower than previous reports, but in line with the concentrations range (pg/ml to ng/ml) of these opioid metabolites present in real samples. To enable multianalyte detection we developed a facile, reproducible, and high yield fabrication process producing twenty G-FETs with integrated side gate platinum (Pt) electrodes on a single chip. Our devices achieved the simultaneous and selective multianalyte detection of three different metabolites: Noroxycodone (NX), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and Norfentanyl (NF) in wastewater.


title: Subclonal evolution and expansion of THY1-positive cells is associated with recurrence in glioblastoma

abstract:
PurposeGlioblastoma (GBM) is a lethal disease characterized by inevitable recurrence. Here we investigate the molecular pathways mediating resistance, with the goal of identifying therapeutic opportunities to target this tumor.

Experimental DesignWe developed a longitudinal in vivo recurrence model utilizing patient-derived explants to produce paired specimens (pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR). These specimens were evaluated for treatment response and to identify gene expression pathways driving treatment resistance. Findings were clinically validated using spatial transcriptomics of human GBMs.

ResultsThese studies reveal in replicate cohorts, a gene expression profile characterized by upregulation of mesenchymal and stem-like genes at recurrence. Analyses of clinical databases revealed increased expression of this transcriptional profile to be significantly associated with worse median overall survival (248 days vs 430 days, p=0.0004), and upregulation of this profile at recurrence. Most notably, we identified upregulation of TGF{beta} signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Utilizing cell sorting, we observed that THY1-positive cells represented <10% of cells in the treatment-naive tumors and 75-96% in the recurrent tumors. We then isolated THY1-positive cells from treatment-naive patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naive human GBM, we conducted spatial transcriptomic analyses. This revealed rare THY1+ regions characterized by mesenchymal/stem-like gene expression, analogous to our recurrent mouse model samples, which co-localized with macrophages within the perivascular niche. Since TGF{beta} signaling contributes to a mesenchymal/stem-like phenotype, we inhibited TGF{beta}RI activity in vivo which resulted in decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors.

ConclusionsThese findings reveal that GBM recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive, mesenchymal/stem-like cells within the perivascular niche. Furthermore, our data demonstrate the promise of targeting upregulated pathways in resistant subclones as a novel mechanism to achieve therapeutic response, and specifically that THY1 expression may represent a biomarker of response to TGF{beta} inhibition.


title: INTEGUMENT-SPECIFIC TRANSCRIPTIONAL REGULATION IN THE MID- STAGE OF FLAX SEED DEVELOPMENT INFLUENCES THE RELEASE OF MUCILAGE AND THE SEED OIL CONTENT

abstract: Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of these integrative biology approach, suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.


title: Large-scale miRNA-Target Data Analysis to Discover miRNA Co-regulation Network of Abiotic Stress Tolerance in Soybeans

abstract: Although growing evidence shows that microRNA (miRNA) regulates plant growth and development, miRNA regulatory networks in plants are not well understood. Current experimental studies cannot characterize miRNA regulatory networks on a large scale. This information gap provides a good opportunity to employ computational methods for global analysis and to generate useful models and hypotheses. To address this opportunity, we collected miRNA-target interactions (MTIs) and used MTIs from Arabidopsis thaliana and Medicago truncatula to predict homologous MTIs in soybeans, resulting in 80,235 soybean MTIs in total. A multi-level iterative bi-clustering method was developed to identify 483 soybean miRNA-target regulatory modules (MTRMs). Furthermore, we collected soybean miRNA expression data and corresponding gene expression data in response to abiotic stresses. By clustering these data, 37 MTRMs related to abiotic stresses were identified including stress-specific MTRMs and shared MTRMs. These MTRMs have gene ontology (GO) enrichment in resistance response, iron transport, positive growth regulation, etc. Our study predicts soybean miRNA-target regulatory modules with high confidence under different stresses, constructs miRNA-GO regulatory networks for MTRMs under different stresses and provides miRNA targeting hypotheses for experimental study. The method can be applied to other biological processes and other plants to elucidate miRNA co-regulation mechanisms.


title: Multiplex knockout of trichome-regulating MYBs in poplar affects light sensitivity and triterpene accumulation

abstract:
As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula x P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar.

ONE SENTENCE SUMMARYTargeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.


title: Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA


title: Molecular basis for the R-type anion channel QUAC1 activity in guard cells

abstract: The rapid (R)-type anion channel plays a central role in controlling stomatal closure in plant guard cells, thus regulating the exchange of water and photosynthetic gas (CO2) in response to environmental stimuli. The activity of the R- type anion channel is regulated by malate. However, the molecular basis of the R-type anion channel activity remains elusive. Here, we describe the first cryo-EM structure of the R-type anion channel QUAC1 at 3.5 [A] resolution in the presence of malate. The structure reveals that the QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore for passing anions across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted bi-layer architecture, with the associated dimeric interfaces nearly perpendicular. Our structural and functional analyses reveal that QUAC1 functions as an inward rectifying anion channel and suggests a mechanism for malate-mediated channel activation. Altogether, our study uncovers the molecular basis for a novel class of anion channels and provides insights into the gating and modulation of the R-type anion channel.


title: Microtubule-based perception of mechanical conflicts controls plant organ morphogenesis

abstract: Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana, the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite wall of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling, and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.


title: Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

abstract: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages from livestock. Porcine and bovine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages. Pig PSCdMs were productively infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and African Swine Fever Virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, Pig PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied in parental stem cells, or directly by lentiviral vector transduction. PSCs and differentiated derivatives therefore provide a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in livestock.


title: Role of mitotic diffusion barriers in regulating the asymmetric division of activated CD8 T cells

abstract: Upon their activation, naive CD8 T cells divide and differentiate into short-lived effector cells, relevant for exerting immune control, and long-lived memory cells, relevant for long-term immunity. The proportion of memory cells generated depends highly on the context of activation and whether the activated cell divides symmetrically or asymmetrically. However, how T cells control the extent of their asymmetry during their first division in response to contextual signals is not known. Using fluorescence loss in photo-bleaching (FLIP) experiments, we show that the metabolic and plasma membrane asymmetry of mitotic T cells depend on the regulated assembly of a lateral diffusion barrier in their endoplasmic reticulum (ER-) membrane. In asymmetrically dividing T cells, the degrees of asymmetry correlated tightly to barrier strength, whereas symmetrically dividing T cells did not establish such a barrier. Direct positive or negative interference with barrier assembly enhanced or abrogated metabolic and plasma membrane asymmetry, respectively, indicating that barrier strength is a direct and decisive determinant of mitotic asymmetry. Thus, together our data identify diffusion barrier-mediated compartmentalization as a mechanism for how asymmetric T cell regulate their long-term response as a function of the activatory context.


title: Effect of dietary zinc supplementation on the gastrointestinal microbiota and host gene expression in the Shank3B-/- mouse model of autism spectrum disorder

abstract:
Shank genes are implicated in ~1% of people with autism and mice with Shank3 knock out mutations exhibit autism-like behaviours. Zinc deficiency and gastrointestinal problems can be common among people with autism, and zinc is a key element required for SHANK protein function and gut development. In Shank3B-/- mice, a supplementary zinc diet reverses autism behaviours. We hypothesise that dietary zinc may alter the gut microbiome, potentially affecting the gut-microbiome-brain axis, which may contribute to changes in autism-like behaviours. To test this, four types of gastrointestinal samples (ileum, caecum, colon, faecal) were collected from wild-type and knock-out Shank3B-/- mice on either control or supplemented-zinc diets. Cage, genotype and zinc diet each contributed significantly to bacterial community variation (accounting for 12.8%, 3.9% and 2.3% of the variation, respectively). Fungal diversity differed significantly between wild-type and knock-out Shank3B-/- mice on the control zinc diet, and the fungal biota differed among gut locations. RNA-seq analysis of host (mouse) transcripts revealed differential expression of genes involved in host metabolism that may be regulated by the gut microbiota and genes involved in anti-microbial interactions. By utilising the Shank3B-/- knock-out mouse model we were able to examine the influence of - and interactions between - dietary zinc and ASD-linked host genotype. These data broaden understanding of the gut microbiome in autism and pave the way towards potential microbial therapeutics for gastrointestinal problems in people with autism.

ImportancePreviously, supplemental dietary zinc in the Shank3B-/- mouse model of autism spectrum disorder resulted in observations of ASD behaviours reversal; in this study we also used the Shank3B-/- mouse model to examine the influence of - and interaction between - dietary zinc and ASD-linked host genotype. Sample location along the gastrointestinal tract, genotype and zinc diet explained some of the variation in the microbiota data, with notable bacterial differences between treatment groups. Differential expression of host genes between treatment groups, including antimicrobial interaction genes and gut microbiota-regulated host metabolism genes, suggests that the interplay between gut microbes, the gastrointestinal tract and the brain may play a major role towards the observed amelioration of ASD behaviours seen previously with supplemented dietary zinc. These results widen the scope towards manipulating both dietary zinc and the microbiota itself to ameliorate ASD-related behaviours and associated gastrointestinal issues.


title: Tumor Suppressor p53 Restrains Cancer Cell Dissemination by Modulating Mitochondrial Dynamics

abstract: Tumor suppressor p53 plays a central role in preventing tumorigenesis. Here, we unravel how p53 modulates mitochondrial dynamics to restrain the metastatic properties of cancer cells. p53 inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling to attenuate the protein level of mitochondrial fission process 1 (MTFP1), which fosters the pro-fission dynamin-related protein 1 (Drp1) phosphorylation. This regulatory mechanism allows p53 to restrict cell migration and invasion governed by Drp1-mediated mitochondrial fission. Downregulating p53 expression or elevating the molecular signature of mitochondrial fission correlates with aggressive tumor phenotypes and poor prognosis in cancer patients. Upon p53 loss, exaggerated mitochondrial fragmentation stimulates the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling resulting in epithelial-to-mesenchymal transition (EMT)-like changes in cell morphology, accompanied by accelerated matrix metalloproteinase-9 (MMP9) expression and invasive cell migration. Notably, blocking the activation of mTORC1/MTFP1/Drp1/ERK1/2 axis completely abolishes the p53 deficiency-driven cellular morphological switch, MMP9 expression, and cancer cell dissemination. Our findings unveil a hitherto unrecognized mitochondria-dependent molecular mechanism underlying the metastatic phenotypes of p53-compromised cancers.


title: N-myc Downstream Regulated Gene 1 (NDRG1) functions as a molecular switch for cellular adaptation to hypoxia

abstract: Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation. Consequently, ndrg1a mutants that fail to downregulate NKA, have reduced ATP levels compared to WT embryos. Lastly, we show that sodium azide treatment, which increased lactate levels, was sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a functions as a molecular switch for long term adaptation to hypoxia via metabolic suppression.

category: developmental biology