title: Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane

abstract: The cytoskeleton of red blood cell (RBC) is anchored to cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ankyrin, whose mutations contribute to numerous human inherited diseases. High-resolution structures of the ankyrin complex have been long sought-after to understand its assembly and disease-causing mutations. Here, we analyzed native complexes on human RBC membrane by stepwise fractionation. Cryo-electron microscopy structures of nine band 3-associated complexes reveal that protein 4.2 stabilizes the cytoplasmic domain of band 3 dimer. In turn, the superhelix-shaped ankyrin binds to this protein 4.2 via ankyrin repeats (ARs) 6-13 and to another band 3 dimer via ARs 17-20, bridging two band 3 dimers in the ankyrin complex. Integration of these structures with both prior and our biochemical data supports a model of ankyrin complex assembly during erythropoiesis and identifies interactions essential for mechanical stability of RBC.

category: biophysics


title: Barrier-free liquid condensates of nanocatalysts as effective concentrators of catalysis

abstract: Molecular confinement of catalysts enhances the catalytic activity significantly. However, physicochemical barriers in traditional confinements restrict the free-passage of substrates/products. To obtain a barrier-free confinement of catalysts, here we explored the liquid-liquid phase separation. Using favourable ionic strength and crowding agents, we recruit the protein-inorganic-composites in phase separated liquid condensates from a solution. The phase separation propensity of these nanocomposites was seen to be independent of the native conformation of the component protein. Using standard catalytic oxidation-reduction reactions, we show that the close-proximity yet diffusive nature of catalysts in solution amplifies the homogeneous catalytic-efficiency of metal particles significantly. Overall, our work demonstrates the roadmap of using inorganic catalysts in homogeneous homogenous solution phase with amplified efficiency and longevity.


title: Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.

abstract: We used single-molecule nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of Superfamily 1 helicase PcrA as it translocates on, or unwinds, several kb-long DNA molecules. We recorded over 2 million enzyme steps under various assisting and opposing forces in diverse ATP and ADP conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA; we show how PcrAs kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base-pairs being unwound. Instead, the sequence of the single stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.


title: Aspirin synergizes with regorafenib to reduce growth of colorectal cancer

abstract:
PurposeRegorafenib is a multi-kinase inhibitor approved for refractory metastatic colorectal cancer. Previous studies have suggested that combining kinase inhibitors with aspirin may improve patient outcomes. We aimed to determine the effects of aspirin and regorafenib combination treatment in preclinical models of colorectal cancer.

Experimental DesignSW480, RKO and LIM1215 colorectal cancer cell lines were treated with aspirin and regorafenib to determine effects on proliferation and cytotoxicity. RNA sequencing and Western blotting were performed to explore underlying molecular effects. Aspirin and regorafenib combination treatment was also tested using organoids derived from three human colorectal cancer tissue specimens. For the in vivo study, SW480-derived tumors were established in athymic mice. Tumor volume was measured during treatment with aspirin and regorafenib, followed by immunohistochemical staining for markers of proliferation and apoptosis.

ResultsAspirin and regorafenib synergistically inhibited proliferation of colorectal cancer cell lines and patient-derived organoids, irrespective of KRAS or BRAF mutation status. This was associated with inhibition of the PI3K-Akt-mTOR pathway and activation of the AMPK pathway. Aspirin and regorafenib effectively inhibited growth of microsatellite stable KRAS-mutant SW480-derived tumors in vivo. Immunohistochemical staining for Ki67 and cleaved caspase 3 showed that combination treatment elicited a synergistic anti-proliferative effect, in addition to a pro-apoptotic effect that was driven by regorafenib.

ConclusionsAspirin and regorafenib demonstrate synergistic anti-proliferative effects in preclinical models of colorectal cancer. This suggests that combining regorafenib with aspirin may be an improved treatment strategy for patients with refractory metastatic colorectal cancer.

category: cancer biology


title: TP53 knockout in MCF7 breast cancer cells induces sensitivity to fluoropyrimidine drugs.

abstract: TP53 mutations are present in all molecular subtypes of breast cancer and often correlate with decreased survival; however, few therapeutic options exist for patients with TP53-mutant breast cancers. To discover therapeutic strategies for these patients, we investigated the sensitivity of 129 FDA-approved chemotherapies to TP53-KO and TP53-WT MCF7 breast adenocarcinoma cells and found p53 loss to confer sensitivity to 5-fluorouracil (5-FU). We then treated the p53-null cells and isogenic controls with F10, a second-generation polymeric fluoropyrimidine, and found this preferential cytotoxicity of TP53-KO cells to be significantly magnified. F10 killing could only minimally be rescued by addition of exogenous uridine, whereas it was completely abrogated by addition of exogenous thymidine, suggesting DNA incorporation to be central to the cytotoxic mechanism of action. Furthermore, F10 killing of p53-null cells was persistent even in heterogeneous cellular mixtures more reflective of natural tumor evolution. Together, our results suggest F10 may widen the therapeutic window for TP53-mutant breast cancer by enhancing genotoxicity in cells resistant to apoptosis.


title: Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis

abstract: The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs) and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time, and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP and NBN clusters, and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.

category: neuroscience


title: Restriction of dietary protein in rats increases progressive-ratio motivation for protein

abstract: Low-protein diets can impact food intake and appetite, but it is not known if motivation for food is changed. In the present study, we used an operant behavioral task - the progressive ratio test - to assess whether motivation for different foods was affected when rats were maintained on a protein-restricted diet (PR, 5% protein diet) compared to non-restricted control rats (NR, 18% protein). Rats were tested either with nutritionally-balanced pellets (18.7% protein, Experiment 1) or protein-rich pellets (35% protein, Experiment 2) as reinforcers. Protein restriction increased breakpoint for protein-rich pellets, relative to non-restricted rats, whereas no difference in breakpoint for nutritionally-balanced pellets was observed between groups. When given free access to either nutritionally-balanced pellets or protein-rich pellets, PR and NR rats did not differ in their intake. We also tested whether a previous history of protein restriction might affect present motivation for different types of food, by assessing breakpoint of previously PR animals that were subsequently put on standard maintenance chow (protein-repleted rats, PRep, Experiment 2). PRep rats did not show increased breakpoint relative to their initial encounter with protein-rich pellets while they were protein-restricted. This study demonstrates that restriction of dietary protein induces a selective increased motivation for protein-rich food, a behavior that rapidly disappears once rats are not in need of protein.


title: Compartmental Neuropeptide Release Measured Using a New Oxytocin Sensor

abstract: As a peptide hormone and neuromodulator, oxytocin (OT) plays a critical role in a variety of physiological and pathophysiological processes in both the central nervous system and the periphery. However, the processes that regulate spatial OT release in the brain remain enigmatic. Here, we developed a genetically encoded GPCR activation-based (GRAB) OT sensor called GRABOT1.0. Using this sensor, we directly visualized stimulation-induced OT release from specific compartments of OT neurons in acute brain slices, and discovered that N-type calcium channels predominantly mediate axonal OT release, while L-type calcium channels mediate somatodendritic OT release. In addition, we found that components in the fusion machinery of OT release differ between axon terminals versus somata and dendrites. Finally, we demonstrated the sensor responses to the activation of OT neurons in various brain regions in vivo and revealed region specific OT release during male courtship behavior. Taken together, these results provide key insights regarding the role of compartmental OT release in the control of physiological and behavioral functions.


title: The First Inhibitor of Meso-Diaminopimelate Biosynthesis with Antibacterial Activity Against Multi-Drug Resistant Bacteria

abstract:
Antibiotic resistance represents one of the biggest threats to global health. While several of our current antibiotics target the peptidoglycan within the bacterial cell wall, only a fraction of its components has been explored for antibiotic development. A component that remains under-exploited is meso-diaminopimelate (meso-DAP), a constituent of the cross-linking peptide in Gram-negative bacteria. In this study, we employed a high throughput chemical screen to identify the first inhibitor of meso-DAP biosynthesis with antibacterial activity. Indeed, the compound was shown to have minimum inhibitory concentration values of 8-16 g/mL against a panel of multi-drug resistant Acinetobacter baumannii strains, including those resistant to the last resort antibiotic carbapenem. Importantly, the compound targets the meso-DAP biosynthesis pathway specifically, with no off-target effects observed in human cell lines, and no resistance exhibited upon continuous treatment, under the conditions tested. Furthermore, we revealed for the first time that meso-DAP biosynthesis inhibition prevents biofilm formation and disrupts established biofilms in A. baumannii. Using a Galleria mellonella model, we showed that this compound improves survival rates against A. baumannii infection by up to 40% relative to the no treatment controls. Lastly, we determined that the inhibitor potentiates the activity of several antibiotic classes, including carbapenems. Thus, this study provides proof-of-concept that meso-DAP biosynthesis represents a promising target for the development of standalone antibacterial agents with a new mode of action as well as adjuvants to be used in combinatorial regimens to rejuvenate our current antibiotic arsenal to combat resistance.

ImportanceResistance levels to available antibiotics continues to rise, with a growing number of Gram-negative bacterial infections, in particular A. baumannii infections, becoming life-threatening. Despite this, there have been no new classes of antibiotics against Gram-negative bacteria introduced to the market over the last 40 years. Hence, new targets and therapeutics are urgently required to combat these clinically important pathogens. One such target is meso-DAP, a critical component of the cross-linking peptides in the cell walls of Gram-negative bacteria. Here, we describe the first inhibitor of bacterial meso-DAP biosynthesis, with antibacterial activity against multi-drug resistant Gram-negative bacterial strains, including carbapenem-resistant A. baumannii. We also reveal that meso-DAP biosynthesis inhibition affects biofilm stability and potentiates the activity of several antibiotic classes. This study highlights the need to further explore meso-DAP biosynthesis and other unexploited targets in the search for antibiotics with new modes of action.

category: microbiology


title: HIV, Pathology and epigenetic age acceleration in different human tissues

abstract:
BackgroundEpigenetic clocks based on patterns of DNA methylation have great importance in understanding aging and disease; however, there are basic questions to be resolved in their application. It remains unknown whether epigenetic age acceleration (EAA) within an individual shows strong correlation between different primary tissue sites, the extent to which tissue pathology and clinical illness correlates with EAA in the target organ, and if EAA variability across tissues differs according to sex. Considering the outsized role of age-related illness in Human Immunodeficiency Virus-1 (HIV), these questions were pursued in a sample enriched for tissue from HIV-infected individuals.

MethodsWe used a custom methylation array to generate DNA methylation data from 661 samples representing 11 human tissues (adipose, blood, bone marrow, heart, kidney, liver, lung, lymph node, muscle, spleen, and pituitary gland) from 133 clinically-characterized, deceased individuals, including 75 infected with HIV. We developed a multimorbidity index based on the clinical disease history.

ResultsEpigenetic age was moderately correlated across tissues. Blood had the greatest number and degree of correlation, most notably with spleen and bone marrow. However, blood did not correlate with epigenetic age of liver. EAA in liver was weakly correlated with EAA in kidney, adipose, lung, and bone marrow. Clinically, hypertension was associated with EAA in several tissues, consistent with the multi-organ impacts of this illness. HIV infection was associated with positive age acceleration in kidney and spleen. Male sex was associated with increased epigenetic acceleration in several tissues. Preliminary evidence indicates that Amyotrophic Lateral Sclerosis is associated with positive EAA in muscle tissue. Finally, greater multimorbidity was associated with greater EAA across all tissues.

ConclusionBlood alone will often fail to detect EAA in other tissues. While hypertension is associated with increased EAA in several tissues, many pathologies are associated with organ-specific age acceleration.

category: molecular biology


title: Functional assessment of the ionotropic receptors IR25a and IR93a in predator perception in the freshwater crustacean Daphnia using RNA interference

abstract: Acquiring environmental information is vital for organisms as it informs about the location of resources, mating partners, and predators. The freshwater crustacean Daphnia detects predator specific chemical cues released by its predators and subsequently develops defensive morphological features that reduce the predation risk. The detection of such chemical information is generally processed via distinct chemoreceptors that are located on chemoreceptor cells. Lately an ancestral type of ionotropic receptors (IRs) has been identified in crustaceans. IRs and the putative co-receptors IR25a and IR93a are postulated to be involved in chemoreception However, functional roles have not been assessed. Here, using three Daphnia species as model, we report that the two co-receptors are expressed within the chemosensory antennules and gene expression is increased with predator perception. Importantly, RNA interference mediated knock-down of the two IRs impedes species-specific defense expression in the three Daphnia species. Our results suggest that (albeit not testing the enigmatic receptor protein directly), the reduction of two associated proteins has impaired the functional aggregation of the postulated chemoreceptor complex. This in turn has hampered the perception of environmentally relevant chemical cues resulting in a substantial reduction of defensive morphological features.

category: ecology


title: How nutrition and energy needs affect bumble beepollination services: A mathematical model

abstract: The balance between nutrition and energy needs has an important impact on the spatial distribution of foraging animals. In the present paper, we focus on the case of bumble bees moving around a landscape in search of pollen (to meet nutritional needs) and nectar (to meet energy needs). Depending on the colony demands, bumble bees can concentrate their foraging effort towards either pollen rich flower species or nectar rich flower species. This behaviour allows us to establish a strategy - a spatial landscape design - which can maximize the pollination services of crops that are nutritionally deficient for pollinators by adding nutritionally rich wildflower patches. To do this, we formulate a mathematical partial integro-differential equation model to predict the spatial distribution of foraging bumble bees. We use our model to predict the location, composition and quantity of the wildflower patches adjacent to crop fields that will be most beneficial for crop pollination services. Our results show that relatively small quantities of wildflowers in specific locations with respect to the nest sites and the crop can have a positive impact on pollination services when the composition (i.e., pollen to nectar ratio) of the added wildflowers is significantly different from the composition of the existing crop flowers.


title: Spatial patterns from a dispersal limitation perspective: revealing biotic interactions

abstract: Local spatial distributions of populations are often studied in comparison to Complete Spatial Randomness (CSR) and are found to be ubiquitously aggregated, likely due to dispersal limitation. Here we theoretically examine the advantages of comparing observed distributions to simulated populations subject only to drift and Dispersal Limitation (DL). Compared to this DL null, local competition produces overdispersion out to surprisingly large scales--much larger than the scale of competitive interactions. Furthermore, strong overdispersion provides a hallmark that a key requirement of stable coexistence is met, as it can only be observed if intraspecific competition is substantially stronger than interspecific competition. Dispersion compared to CSR is insensitive to competition and as a result unreflective of its presence. Hence, we suggest DL as a complement to CSR since the former focuses on biologically relevant spatial scales and has the potential to detect biotic interactions and habitat specificity.


title: Seeing the future: a better way to model and test for adaptive developmental plasticity

abstract: Early life conditions can have profound effects on individual health, longevity, and biological fitness. Two classes of hypotheses are used to explain the evolutionary origins of these effects: developmental constraints (DC) hypotheses, which focus on the deleterious effects of low-quality early-life environments, and predictive adaptive response (PAR) models, which focus on organisms predictions about their adult environment, phenotypic adaptations based on that prediction, and the deleterious consequences of incorrect predictions. Despite their popularity, these ideas remain poorly defined. To remedy this, we provide mathematical definitions for DC, PARs, and related concepts, and develop statistical tests derived from these definitions. We use simulations to demonstrate that PARs are more readily detected by tests based on quadratic regressions than by tests based on more commonly used interaction regression models. Specifically, quadratic regression-based tests on simulated data yield 90.7% sensitivity and 71.5% specificity in detecting PARs, while interaction-based tests yield sensitivity and specificity roughly equal to chance. We demonstrate that the poor performance of interaction models stems from two problems: first, they are mathematically incapable of detecting a central prediction of PAR, and second, they conceptually conflate PARs with DC. Our results emphasize the value of formal statistical modeling to reveal the theoretical underpinnings of verbal and visual models, and their importance for helping resolve conflicting and ambiguous results in this field of research. We conclude by providing recommendations for how researchers can make use of explicit definitions and properly-aligned visualizations and statistical tests to make progress in this important research area.

category: evolutionary biology


title: Multi-ancestry fine-mapping improves precision to identify causal genes in transcriptome-wide association studies

abstract: Transcriptome-wide association studies (TWAS) are a powerful approach to identify genes whose expression associates with complex disease risk. However, non-causal genes can exhibit association signals due to confounding by linkage disequilibrium patterns (LD) and eQTL pleiotropy at genomic risk regions which necessitates fine-mapping of TWAS signals. Here, we present MA-FOCUS, a multi-ancestry framework for the improved identification of genes underlying traits of interest. We demonstrate that by leveraging differences in ancestry-specific patterns of LD and eQTL signals, MA-FOCUS consistently outperforms single-ancestry fine-mapping approaches with equivalent total sample size across multiple metrics. We perform 15 blood trait TWAS using genome-wide summary statistics (average NEA=511k, NAA=13k) and lymphoblastoid cell line eQTL data from cohorts of primarily European and African continental ancestries. We recapitulate evidence demonstrating shared genetic architectures for eQTL and blood traits between the two ancestry groups and observe that gene-level effects correlate 20% more strongly across ancestries compared with SNP-level effects. We perform fine-mapping using MA-FOCUS and find evidence that genes at TWAS risk regions are more likely to be shared across ancestries rather than ancestry-specific. Using multiple lines of evidence to validate our findings, we find gene sets produced by MA-FOCUS are more enriched in hematopoietic categories compared to alternative approaches (P = 1.73 x 10-16). Our work demonstrates that including, and appropriately accounting for, genetic diversity can drive deeper insights into the genetic architecture of complex traits.

category: genetics


title: First whole-genome sequence of Cervid atadenovirus A outside of the United States from an Adenoviral hemorrhagic disease epizootic of black-tailed deer in Canada

abstract: A complete 30,616 nucleotide Cervid atadenovirus A genome was determined from tissues of a black-tailed deer that died in 2020 in British Columbia, Canada. Unique, non-synonymous SNPs in the E1B, Iva2 and E4.3 coding regions, and deletions totalling 74 nucleotides not observed in moose and red deer isolates were present.

category: genomics


title: A network-based drug repurposing approach identifies new treatment opportunities for the systemic chronic inflammation underlying multiple complex diseases

abstract: Complex diseases are associated with a wide range of cellular, physiological, and clinical phenotypes. To advance our understanding of disease mechanisms and our ability to treat these diseases, it is critical to delineate the molecular basis and therapeutic avenues of specific disease phenotypes, especially those that are associated with multiple diseases. Inflammatory processes constitute one such prominent phenotype, being involved in a wide range of health problems including ischemic heart disease, stroke, cancer, diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, and autoimmune and neurodegenerative conditions. While hundreds of genes might play a role in the etiology of each of these diseases, isolating the genes involved in the specific phenotype (e.g. inflammation "component") could help us understand the genes and pathways underlying this phenotype across diseases and predict potential drugs to target the phenotype. Here, we present a computational approach that integrates gene interaction networks, disease-/trait-gene associations, and drug-target information to accomplish this goal. We apply this approach to isolate gene signatures of complex diseases that correspond to chronic inflammation and prioritize drugs to reveal new therapeutic opportunities.

category: bioinformatics


title: Sequence- and chemical specificity define the functional landscape of intrinsically disordered regions

abstract:
Intrinsically disordered protein regions (IDRs) pervasively engage in essential molecular functions, yet they are often poorly conserved as assessed by sequence alignment. To understand the seeming paradox of how sequence variability is compatible with function, we examined the functional determinants for a poorly conserved but essential IDR. We show that IDR function depends on two distinct but related properties: sequence- and chemical specificity. While sequence-specificity works via linear binding motifs, chemical-specificity reflects the sequence-encoded chemistry of multivalent interactions through amino acids across an IDR. Unexpectedly, an apparently essential binding motif can be removed if compensatory changes to the sequence chemistry are made, highlighting the orthogonality and interoperability of both properties. Our results provide a general framework to understand the functional constraints on IDR sequence evolution.

One-Sentence SummaryInteractions driven by intrinsically disordered regions can be understood using a two-dimensional landscape that defines binding via motif-dependent and motif-independent contributions.


title: Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

abstract:
Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons, and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside virus-like particles can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here we present a set of acceptor vectors that provide a quick and efficient way to build, test and optimise cargo loading inside P22 virus-like particles. We prototyped the system using yellow fluorescent protein then applied it to mevalonate kinases, a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different mevalonate kinases required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimise cargo enzyme behaviour in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behaviour.

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category: synthetic biology


title: Optimal routing to cerebellum-like structures

abstract: The vast expansion from mossy fibers to cerebellar granule cells produces a neural representation that supports functions including associative and internal model learning. This motif is shared by other cerebellum-like structures, including the insect mushroom body, electrosensory lobe of electric fish, and mammalian dorsal cochlear nucleus, and has inspired numerous theoretical models of its functional role. Less attention has been paid to structures immediately presynaptic to granule cell layers, whose architecture can be described as a "bottleneck" and whose functional role is not understood. We therefore develop a general theory of cerebellum-like structures in conjunction with their afferent pathways. This theory predicts the role of the pontine relay to cerebellar cortex and the glomerular organization of the insect antennal lobe. It also reconciles theories of nonlinear mixing with recent observations of correlated granule cell activity. More generally, it shows that structured compression followed by random expansion is an efficient architecture for flexible computation.


title: Low-cost sample preservation methods for high-throughput processing of rumen microbiomes

abstract:
BackgroundThe use of rumen microbial community (RMC) profiles to predict methane emissions has driven interest in ruminal DNA preservation and extraction protocols that can be processed cheaply while also maintaining or improving DNA quality for RMC profiling. Our standard approach for preserving rumen samples, as defined in the Global Rumen Census (GRC), requires time-consuming pre-processing steps of freeze drying and grinding prior to international transportation and DNA extraction. This impedes researchers unable to access sufficient funding or infrastructure. To circumvent these pre-processing steps, we investigated three methods of preserving rumen samples for subsequent DNA extraction, based on existing lysis buffers Tris-NaCl-EDTA-SDS (TNx2) and guanidine hydrochloride (GHx2), or 100% ethanol.

ResultsRumen samples were collected via stomach intubation from 151 sheep at two time-points two weeks apart. Each sample was separated into four subsamples and preserved using the three preservation methods and the GRC method (n = 4x302). DNA was extracted and sequenced using Restriction Enzyme-Reduced Representation Sequencing to generate RMC profiles. Differences in DNA yield, quality and integrity, and sequencing metrics were observed across the methods (p < 0.0001). Ethanol exhibited poorer quality DNA (A260/A230 < 2) and more failed samples compared to the other methods. Samples preserved using the GRC method had smaller relative abundances in gram-negative genera Anaerovibrio, Bacteroides, Prevotella, Selenomonas, and Succiniclasticum, but larger relative abundances in the majority of 56 additional genera compared to TNx2 and GHx2. However, log10 relative abundances across all genera and time-points for TNx2 and GHx2 were on average consistent (R2 > 0.99) but slightly more variable compared to the GRC method. Relative abundances were moderately to highly correlated (0.68 {+/-} 0.13) between methods for samples collected within a time-point, which was greater than the average correlation (0.17 {+/-} 0.11) between time-points within a preservation method.

ConclusionsThe two modified lysis buffers solutions (TNx2 and GHx2) proposed in this study were shown to be viable alternatives to the GRC method for RMC profiling in sheep. Use of these preservative solutions reduces cost and improves throughput associated with processing and sequencing ruminal samples. This development could significantly advance implementation of RMC profiles as a tool for breeding ruminant livestock.


title: Having the 'right' microbiome matters for host trait expression and the strength of mutualism between duckweeds and microbes

abstract: An organisms phenotypes and fitness often depend on interactive effects of its genome (Ghost), microbiome (Gmicrobe), and environment (E). These G xG, G xE, and G xG xE effects fundamentally shape host-microbiome (co)evolution and may be widespread, but are rarely compared within a single experiment. We collected and cultured Lemna minor (duckweed) and its associated microbiome from 10 sites across an urban-to-rural ecotone. We factorially manipulated host genotype and microbiome in two environments (low and high zinc, an urban aquatic stressor) in an experiment with 200 treatments: 10 host genotypes x 10 microbiomes x 2 environments. Host genotype explained the most variation in L. minor fitness and traits, while microbiome effects depended on host genotype (G xG) or environment (G xE). Hosts had higher fitness and microbes grew fastest when tested microbiomes more closely matched field microbiomes, suggesting some local adaptation between hosts and their microbiota. High microbiome similarity also led to more predictable host trait expression. In contrast, although zinc decreased host fitness, we observed no local adaptation of urban duckweed or microbes to high-zinc conditions. Thus, we found that host fitness and trait expression are contingent on microbiome composition, with implications for microbiome engineering and host-microbiome evolution.


title: Antisense oligonucleotides targeting exon 11 are able to partially rescue the Neurofibromatosis Type 2 phenotype in vitro

abstract: Neurofibromatosis type 2 (NF2) is an autosomal dominant condition characterized by the development of multiple tumours of the nervous system caused by loss of function variants in the NF2 gene. The clinical presentation of the disease is variable and related to the type of the inherited constitutional mutation. Here, we evaluated the use of antisense oligonucleotides, specifically phosphorodiamidate morpholino oligomers (PMOs), to reduce the severity of the effects of NF2 truncating variants by converting them to milder hypomorphic forms in vitro. Our results showed that the PMOs designed for variants located at +/- 13 from the intron-exon boundary region interfered with the correct splicing signalling of both NF2 wild-type and mutated alleles. On the other hand, we were able to specifically induce the skipping of exons 4, 8 and 11 of both the mutated and the WT allele maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11) able to partially rescue the phenotype observed in primary fibroblast cultures from NF2 patients harbouring a germline loss of function variant. This encouraging result is an in vitro proof of concept of the use of antisense therapy to develop personalized therapies for NF2.


title: Predictive link between systemic metabolism and immune signaling in the brain of APOE4 mice

abstract: The {varepsilon}4 variant of apolipoprotein E (APOE) is the strongest and most common genetic risk factor for Alzheimers disease (AD). While the mechanism of conveyed risk is incompletely understood, promotion of inflammation, dysregulated metabolism, and protein misfolding and aggregation have been implicated as potential contributions to accelerating disease. Here we determined the concurrent effects of systemic metabolic changes and brain inflammation in young (3-month-old) and aged (18-month-old) female mice carrying the human APOE4 gene. Using functional metabolic assays alongside multivariate modeling of hippocampal cytokine signaling, we found that immune signaling patterns in the brain are predictive of systemic metabolic outcomes, independent of AD proteinopathies. The relationship between cytokine signaling networks in the hippocampus and systemic glucose tolerance and adiposity differ by APOE genotype, where APOE4 mice produce lower levels of neuroprotective cytokines as they age and as their metabolic phenotype declines. To our knowledge, ours is the first study to identify a quantitative and predictive link between systemic metabolism and specific pathological immune signaling signatures in the brain. Our results highlight the AD-relevant effects of APOE4 beyond the brain and highlight the potential for bidirectional systemic influences of risk factors in the brain and periphery.

category: physiology


title: Vagal involvement in non-dipping phenotype of Hsd11b2 knockout rats

abstract: The Syndrome of Apparent Mineralocorticoid Excess (SAME) is a hypertensive disorder caused by deficiency of 11b-hydroxysteroid dehydrogenase type 2. Blood pressure is directly influenced by dietary salt intake, but the causes of salt-sensitivity are not fully resolved. We modelled SAME in Fischer 344 rats, using zinc finger nuclease targeting of the Hsd11b2 gene. The F344 genetic background showed modest salt sensitivity: blood pressure increased by [~]6mmHg when diet was switched from control (0.3% Na) to high salt (3% Na) diet. Homozygous knockout (Hsd2-/-) rats exhibited severe hypertension on control diet (mean arterial blood pressure of [~]180 mmHg compared to [~]115 mmHg in wild-types) and displayed no dipping in blood pressure in the inactive/sleep phase. They also displayed reduced heart rate (339 bpm versus 384 bpm in F344 controls). Low salt diet (0.03% Na) caused a dramatic fall in Hsd2-/- blood pressure (to [~]141mmHg), restoration of robust circadian variation in blood pressure, and an increase in heart rate (to 364bpm). This was mirrored by a restoration of circadian variation in the Poincare plot descriptor, SD1, suggesting involvement of parasympathetic dysfunction in the non-dipping phenotype. Alpha adrenoceptor blockade with prazosin treatment resulted in a further decrease in blood pressure (to [~]124mmHg), which blunted circadian rhythm, together with an increase in heart rate (to [~]394bpm). This rat model of human hypertension reveals clear links between dietary salt, autonomic nervous system dysfunction, and the non-dipping blood pressure phenotype.


title: Two bridgehead invasions of Xylella fastidiosa subsp. multiplex in France

abstract:
Of American origin, a wide diversity of Xylella fastidiosa strains belonging to different subspecies have been reported in Europe since 2013 and its discovery in Italian olive groves. Strains from the subspecies multiplex (ST6 and ST7) were first identified in France in 2015 in urban and natural areas. To trace back the most probable introduction scenario, the molecular evolution rate of this subspecies was estimated at 3.2165 x 10-7 substitutions per site per year, based on heterochronous genome sequences collected worldwide. This rate allowed the dating of the divergence between French and American strains in 1987 for ST6 and in 1971 for ST7. The development of a new VNTR-13 scheme allowed tracing the spread of the bacterium in France, hypothesizing an American origin. Our results suggest that both sequence types were initially introduced and spread in PACA; then they were introduced in Corsica in two waves from the PACA bridgehead populations.

TeaserTipdating and ABC analyses on genomic and microsatellite data were combined to provide scenarios for X. fastidiosa introduction in France.


title: Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life

abstract: The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life1, 2. It is involved in multiple branches of proteostasis2, 3, and as a molecular capacitor in morphological evolution4. Although reduced Hsp90 levels cause phenotypic variations5, 6 and correlate with aging7, whether eukaryotic cells and organisms can tune the basal Hsp90 protein levels to alleviate physiologically accumulated stress is unknown. To begin to explore this question, we investigated whether and how mice adapt to the deletion of three out of four alleles encoding cytosolic Hsp90, one Hsp90{beta} allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90{beta} protein to at least wild-type levels. Further mechanistic studies revealed an internal ribosome entry site in the 5UTR of the Hsp90{beta} mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We found that the minimum amount of total Hsp90 that is required to support viability of mammalian cells and organisms is 50-70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced [≥]100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have been markedly increased during eukaryotic evolution. The incompressible part of the steady-state levels of Hsp90 may have increased to accommodate the ever-growing complexity of the proteome8 on the path towards mammals.


title: Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions

abstract: In Parkinsons disease and other synucleinopathies, the intrinsically disordered, presynaptic protein alpha-synuclein misfolds and aggregates. We hypothesise that the exposure of alpha-synuclein to different cellular environments, with different chemical compositions, pH and binding partners, alters its biological and pathological function by inducing changes in molecular conformation. Our custom instrumentation and software enable measurement of the amide hydrogen exchange rates of wild-type alpha-synuclein at amino acid resolution under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal compartments of cells. We characterised the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Our findings reveal that the C-terminal residues of alpha-synuclein are driving its nucleation and thus its aggregation. Furthermore, the entire NAC region and specific other residues strongly promoted elongation of fibrils. This provides new detail on our current understanding of the relationship between the local chemical environment and monomeric conformations of alpha-synuclein.


title: RpoN is required for a functional type III secretion system in Yersinia pseudotuberculosis

abstract: Pathogenic bacteria use a broad range of virulence factors to thrive within their host. Yersinia pseudotuberculosis, a gram-negative enteropathogen in humans, utilises a Type III secretion system to overcome the hosts innate immune response. A global regulator previously shown to be essential for virulence in Y. pseudotuberculosis is RpoN. We show here that a strain lacking RpoN has a severely reduced capacity to secrete Yop effectors. This strain has a substantially reduced expression of genes encoding structural components of the secretion apparatus, the ysc operons, while expression of genes encoding effectors and translocators is less affected. We show that RpoN regulates a complex network, where one part is suggested to contribute to inducing Type III secretion via the enhancer-binding protein GlrR/RpoN regulon. By analogy, during inducement of Type III secretion, RpoN has a positive effect on expression of the sigma factor RpoE, which also is known to act downstream of GlrR. Interestingly, we found putative RpoE binding sites upstream of the ysc operons, and also a partial rescue of Yop secretion in the rpoN mutant strain by overexpression of RpoE. Our findings suggest that the RpoN-mediated effect on expression of type III genes involves a sigma factor hierarchy, where RpoN via RpoE contributes to inducing Type III secretion.


title: Tumor elimination by clustered microRNAs miR-306 and miR-79 via non-canonical activation of JNK signaling

abstract: JNK signaling plays a critical role in both tumor promotion and tumor suppression. Here, we identified clustered microRNAs (miRNAs) miR-306 and miR-79 as novel tumor-suppressor miRNAs that specifically eliminate JNK-activated tumors in Drosophila. While showing no significant effect on normal tissue growth, miR-306 and miR-79 strongly suppressed growth of multiple tumor models including malignant tumors caused by Ras activation and cell polarity defects. Mechanistically, these miRNAs commonly target the mRNA of an E3 ubiquitin ligase Drosophila ring finger protein 146 (dRNF146). We found that DRNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a non-canonical manner. Thus, downregulation of dRNF146 by miR-306 and miR-79 leads to hyper-enhancement of JNK activation. Our data show that, while JNK activity is essential for tumor growth, elevation of miR-306 or miR-79 overactivate JNK signaling to the lethal level via non-canonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer.

category: cell biology


title: Mitotically heritable, RNA polymerase II-independent H3K4 dimethylation stimulates INO1 transcriptional memory

abstract: For some inducible genes, the rate and molecular mechanism of transcriptional activation depends on the prior experiences of the cell. This phenomenon, called epigenetic transcriptional memory, accelerates reactivation and requires both changes in chromatin structure and recruitment of poised RNA Polymerase II (RNAPII) to the promoter. Memory of inositol starvation in budding yeast involves a positive feedback loop between transcription factor-dependent interaction with the nuclear pore complex and histone H3 lysine 4 dimethylation (H3K4me2). While H3K4me2 is essential for recruitment of RNAPII and faster reactivation, RNAPII is not required for H3K4me2. Unlike RNAPII-dependent H3K4me2 associated with transcription, RNAPII-independent H3K4me2 requires Nup100, SET3C, the Leo1 subunit of the Paf1 complex and, upon degradation of an essential transcription factor, is inherited through multiple cell cycles. The writer of this mark (COMPASS) physically interacts with the potential reader (SET3C), suggesting a molecular mechanism for the spreading and re-incorporation of H3K4me2 following DNA replication.


title: The nature and chromosomal landscape of endogenous retroviruses (ERVs) integrated in the sheep nuclear genome

abstract: Endogenous retroviruses (ERVs) represent genomic components of retroviral origin that are found integrated in the genomes of various species of vertebrates. These genomic elements have been widely characterized in model organisms and humans. However, composition and abundances of ERVs have not been categorized fully in all domestic animals. The advent of next generation sequencing technologies, development of bioinformatics tools, availability of genomic databases and molecular cytogenetic techniques have revolutionized the exploration of the genome structure. Here, we investigated the nature, abundance, organization and assembly of ERVs and complete genomes of Jaagsiekte sheep retrovirus (JSRV) from high-throughput sequencing (HTS) data from two Iraqi domestic sheep breeds. We used graph-based read clustering (RepeatExplorer), frequency analysis of short motifs (k-mers), alignment to reference genome assemblies and fluorescent in situ hybridization (FISH). Three classes of ERVs were identified with the total genomic proportions of 0.55% from all analyzed whole genome sequencing raw reads, while FISH to ovine metaphase chromosomes exhibited abundant centromeric to dispersed distribution of these ERVs. Furthermore, the complete genomes of JSRV of two Iraqi sheep breeds were assembled and phylogenetically clustered with the known enJSRV proviruses in sheep worldwide. Characterization of partial and complete sequences of mammalian ERVs is valuable to provide insights into the genome landscape, to help with future genome assemblies and to identify potential sources of disease when ERVs become active.


title: Whole-Genome Sequencing and Comparative Genomic Analysis of Potential Biotechnological Strains from Trichoderma harzianum, Trichoderma atroviride, and Trichoderma reesei

abstract: Fungi of the genus Trichoderma exhibit high genetic diversity and can thus be utilized in a large range of biotechnological applications. While Trichoderma reesei is the primary source of industrial enzymatic cocktails, Trichoderma atroviride and Trichoderma harzianum are widely used as commercial biocontrol agents against plant diseases. Recently, T. harzianum IOC-3844 (Th3844) and T. harzianum CBMAI-0179 (Th0179) demonstrated great potential in the enzymatic conversion of lignocellulose into fermentable sugars. Despite such potential, the genomes of both hydrolytic strains remain unclear. Herein, we performed whole-genome sequencing and assembly of Th3844 and Th0179 strains. To assess the genetic diversity within the genus Trichoderma, the results of both strains were compared with those from T. atroviride CBMAI-00020 (Ta0020) and T. reesei CBMAI-0711 (Tr0711). The resulting assembly revealed a total length of 40 Mb, (Th3844), 39 Mb (Th0179), 36 Mb (Ta0020), and 32 Mb (Tr0711), in which 10,786 (Th3844), 11,322 (Th0179), 10,082 (Ta0020), and 8,796 (Tr0711) genes were predicted. Then, the annotation of the predicted CDS sequences from the evaluated strain genes revealed 413 (Th3844), 413 (Th0179), 377 (Ta0020), and 329 (Tr0711) CAZymes. The orthology analysis revealed 18,349 orthogroups, which encompassed 95% of the total genes, 3,378 orthogroups with all species present, and 408 species-specific orthogroups. A genome-wide phylogenetic analysis modeled from the 1,864 single-copy orthogroups provided details on the relationship of the newly sequenced species with other Trichoderma and with more evolutionarily distant genera. Structural variants revealed genomic rearrangements between Th3844, Th0179, Ta0020, and Tr0711 with the T. reesei QM6a reference genome as well as the functional effects of such variants on the evaluated strains. In conclusion, the findings presented herein allow the genetic diversity of the evaluated strains, including those from the same species, to be viewed, offering opportunities to further explore such fungal genomes in future biotechnological and industrial applications.


title: Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay

abstract: Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an modified massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required for silencing (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization of transcription factors binding sites.


title: Structure of the vasopressin hormone-V2 receptor-β-arrestin1 ternary complex

abstract: Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with {beta}-arrestin1. It reveals an atypical position of {beta}-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/{beta}-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the {beta}-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.

category: biochemistry


title: Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

abstract: Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode 8-9 kinesins in their genomes. Of these, two - kinesin-8B and kinesin-8X -are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8Bs essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.


title: Extensive in vitro and in vivo protein translation via in situ circularized RNAs

abstract: RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.

category: bioengineering


title: Delayed viral vector mediated delivery of neurotrophin-3 improves skilled hindlimb function and stability after thoracic contusion in rats

abstract:
It has been reported that intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 hours after a severe T9 contusion in rats induced lumbar spinal neuroplasticity, partially restored locomotive function and reduced spasms during swimming. Here we investigated whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 hours following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-hours following trauma, persistently elevating serum levels of the neurotrophin. NT3 improved trunk stability, accuracy of stepping during skilled locomotive tasks, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotion function in the open field. The number of vGlut1+ (likely proprioceptive afferent) boutons on gastrocnemius -motor neurons was increased after injury but normalised following NT3 treatment suggestive of a mechanism in which the functional effects may be mediated through proprioceptive feedback. Ex vivo MRI revealed substantial loss of grey and white matter at the lesion epicentre but no effect of delayed NT3 treatment to induce neuroprotection or prevent secondary damage. Spasms and hyperreflexia were not reliably induced in this severe injury model suggesting a more complex anatomical or physiological cause to their induction. We have shown that delayed intramuscular AAV-NT3 treatment can promote recovery in skilled stepping and coordinated swimming supporting a role for NT3 as a therapeutic strategy for spinal injuries potentially through modulation of somatosensory feedback.

Key PointsO_LITargeted delivery of NT3 to hindlimb and trunk muscles at a clinically relevant 48h following a severe thoracic contusion aids fine locomotor control and synchronised movement.
C_LIO_LINT3 mediated improvements in trunk stability, accuracy of stepping during skilled locomotive tasks, and alternation of the hindlimbs during swimming through the normalisation of vGlut1+ boutons on presumptive proprioceptive afferents innervating these muscles.
C_LIO_LI250kDyn thoracic contusion does not reliably result in measurable signs of spasticity.
C_LI


title: Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling

abstract: Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, we show that the protein AuTophaGy 5 (ATG5) functions in neurons to regulate the cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA R1, which sequesters the PKA catalytic subunit and diminishes the cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins mediating AMPAR trafficking. Glutamatergic neurons-confined ATG5 deletion augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.


title: Rearing environment persistently modulates the phenotype of mice

abstract: The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions 1-4. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment 5. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between rearing facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multi-center study, where inbred mice from the same breeding stock were reared in five different facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that common variation among rearing facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organization. We detected changes in chromatin organization in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity and regulation of behavior. Our findings demonstrate that common environmental differences between rearing facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. We expect our findings to stimulate further research into the mechanisms and drivers of these epigenetic changes mediated by the laboratory environment. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and a need to account for the animals environmental background in study design to produce robust and replicable findings.


title: A single-cell resolution gene expression atlas of the larval zebrafish brain

abstract: The advent of multimodal brain atlases promises to accelerate discoveries in neuroscience by offering in silico queries of cell types, connectivity and gene expression in regions of interest. We employed multiplexed fluorescent in situ RNA hybridization chain reaction (HCR) to generate expression maps for an initial set of 200 marker genes across the larval zebrafish brain. The data were registered to the Max Planck Zebrafish Brain (mapzebrain) atlas, thus allowing co-visualization of gene expression patterns, single-neuron tracings, transgenic lines and anatomical segmentations. Additionally, brain activity maps of freely swimming larvae were generated at single-cell resolution using HCR labeling of the immediate-early gene cfos and integrated into the atlas. As a proof of concept, we identified a novel class of cerebellar eurydendroid cells that express calb2a, project to the hypothalamus and are activated in animals that have recently ingested food. Thus, a cellular-resolution gene expression atlas will not only help with rapid identification of marker genes for neuronal populations of interest, but also bridge the molecular and circuit levels by anchoring genetic information to functional activity maps and synaptic connectivity.


title: STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological dysregulation of NKG2Dhi CD8 killer T cells

abstract:
The association between cancer and autoimmune disease is unexplained, exemplified by T-cell large granular lymphocytic leukemia (T-LGL) where gain-of-function somatic mutations in STAT3 correlate with co-existing autoimmunity. To resolve whether these mutations are the cause or consequence of CD8 clonal expansions and autoimmunity, here we analyse patients with germline STAT3 GOF syndrome and mice with the T-LGL mutation STAT3K658Nor the most common germline mutation, STAT3T716M. STAT3 GOF mutations drove accumulation of effector CD8 T cell clones highly expressing the NKG2D receptor for MHC-I-related molecules expressed on stressed cells, and the genes for inflammatory/cytotoxic granzymes, perforin, interferon-{gamma} and Ccl5/Rantes. CD8 cells were essential to lethal disease in Stat3K658Nmice and their accumulation required NKG2D and the receptor for IL-15 and IL-2, IL2RB. These results demonstrate that STAT3 GOF mutations cause effector CD8 T cell oligoclonal accumulation and that these rogue T cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

IN BRIEFLeukemia and autoimmune-associated STAT3 gain-of-function mutations dysregulate CD8 T cells to cause autoimmune pathology and oligoclonal expansion of cytotoxic killer CD8 T cells, that depend upon NKG2D and IL2RB receptors for signals displayed on stressed, damaged, infected, or mutated tissues.

category: immunology


title: Exponential magnetophoretic gradient for the direct isolation of basophils from whole blood in a microfluidic system

abstract: Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 L of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9%{+/-}3.6% and recovery of 95.6%{+/-}3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment.


title: Comparative genomics of Staphylococcus capitis reveals determinants of speciation

abstract: Staphylococcus capitis is primarily described as a human skin commensal but is now emergent as an opportunistic pathogen isolated from bloodstream and prosthetic joint infections, and neonatal intensive care unit (NICU) associated sepsis. We used comparative genomic analyses of S. capitis to provide new insights of commensal scalp isolates from varying skin states, and to expand our current knowledge of the species populations (scalp isolates, n = 59, contextual isolates, n = 127). A highly recombinogenic population structure was revealed, with genomes including the presence of a range of previously described staphylococcal virulence factors, cell wall-associated proteins, and two-component systems. Genomic differences between the two described S. capitis subspecies were explored and reveal determinants associated exclusively with each. The subspecies ureolyticus was distinguished from subspecies capitis by differences in antimicrobial resistance genes and gene clusters linked to survival on the skin. This study will aid further research into classification of S. capitis and virulence linked phylogroups that is important to monitor the spread and evolution of S. capitis.


title: Ancestral reconstruction of duplicated signaling proteins reveals the evolution of signaling specificity

abstract: Gene duplication is crucial to generating novel signaling pathways during evolution. However, it remains unclear how the redundant proteins produced by gene duplication ultimately acquire new interaction specificities to establish insulated paralogous signaling pathways. Here, we used ancestral sequence reconstruction to resurrect and characterize a bacterial two-component signaling system that duplicated in -proteobacteria. We determined the interaction specificities of the signaling proteins that existed before and immediately after this duplication event and then identified key mutations responsible for establishing specificity in the two systems. Just three mutations, in only two of the four interacting proteins, were sufficient to establish specificity of the extant systems. Some of these mutations weakened interactions between paralogous systems to limit crosstalk. However, others strengthened interactions within a system, indicating that the ancestral interaction, although functional, had the potential to be strengthened. Our work suggests that protein-protein interactions with such latent potential may be highly amenable to duplication and divergence.


title: Cellular forces during early spreading of T lymphocytes on ultra-soft substrates

abstract:
T cells use forces to read out and act on the mechanical parameters of their microenvironment, which includes antigen presenting cells (APCs). Here we explore the early interaction of T cells with an APC-mimicking ultra-soft polymer gel exhibiting physiologically relevant stiffness in the range of 350-450 Pa. We quantify the dependence of cell spreading and stiffness on gel elasticity, and measure early time traction forces. We find that coating the surface with an antibody against the CD3 region of the TCR-complex elicits small but measurable gel deformation in the early recognition phase, which we quantify in terms of stress or energy. We show that the time evolution of the energy follows one of three distinct patterns: active fluctuation, intermittent, or sigmoidal signal. Addition of either anti-CD28 or anti-LFA1 has little impact on the total integrated energy or the maximum stress. However, the relative distribution of the energy patterns does depend on the additional ligands. Remarkably, the forces are centrifugal at very early times, and only later turn into classical in-ward pointing centripetal traction.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: ATAC-Seq and RNA-Seq combined analysis elucidates the impact of BORIS/CTCFL on melanoma progression, mediated by chromatin accessibility and gene expression alterations

abstract: Melanoma accounts for most deaths form skin cancer, since it metastasizes early, and it usually diagnosed at an advance stage. Brother of Regulator of Imprinted Sites (BORIS), also known as CCCTC binding factor-Like (CTCFL), is a transcriptional modulator that becomes aberrantly expressed in melanoma. Yet, the role of BORIS in melanoma remains elusive, in our latest study we sought to uncover its effect on the biological processes and pathways in melanoma cells lines, by using a doxycycline inducible system expressing ectopic BORIS, we investigated its effect on chromatin accessibility at genes promoters and transcriptomics changes, utilizing Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) and RNA-Seq respectively. A differential analysis revealed BORIS target genes and transcription factors (TFs) it governs, in its role as a tumor driver factor. It targets include an over representation of oncogenes, specially upregulated tumor progressors associated with an invasive phenotype such as PDL-1, CTLA4 and GDF6, a known metastatic driver gene. In addition, activators TFs include TEAD4, STAT6 and SP1, while repressed TFs are known tumor suppressors, such as P53,P73 and SOX10. Together, these findings situate BORIS as a potential factor that can lead to an invasiveness phenotype, which drive melanoma progression towards metastatic dissemination.


title: Evolutionary trade-offs in osmotic and ionic regulation and the expression of gill ion transporter genes in high latitude, Neotropical cold clime crabs from the 'end of the world'

abstract:
Seeking to identify consequences of evolution at low temperature, we examine hyper/hypo-osmotic and ionic regulation and gill ion transporter gene expression in two sub-Antarctic Eubrachyura from the Beagle Channel, Tierra del Fuego. Despite sharing the same osmotic niche, Acanthocyclus albatrossis tolerates a wider salinity range (2-65 {per thousand}S) than Halicarcinus planatus (5-60 {per thousand}S); respective lower and upper critical salinities are 4 and 12 {per thousand}S, and 63 and 50 {per thousand}S. Acanthocyclus albatrossis is a weak hyperosmotic regulator, while H. planatus hyper-osmoconforms; isosmotic points are 1,380 and {approx}1,340 mOsm kg-1 H2O. Both crabs hyper/hypo-regulate [Cl-] well with iso-chloride points at 452 and 316 mmol L-1 Cl-, respectively. [Na+] is hyper-regulated at all salinities. mRNA expression of gill Na+/K+-ATPase is salinity-sensitive in A. albatrossis, increasing {approx}1.9-fold at 5 {per thousand}S compared to 30 {per thousand}S, decreasing at 40 to 60 {per thousand}S. Expression in H. planatus is very low salinity-sensitive, increasing {approx}4.7-fold over 30 {per thousand}S, but decreasing at 50 {per thousand}S. V(H+)-ATPase expression decreases in A. albatrossis at low and high salinities as in H. planatus. Na+-K+-2Cl- symporter expression in A. albatrossis increases 2.6-fold at 5 {per thousand}S, but decreases at 60 {per thousand}S compared to 30 {per thousand}S. Chloride uptake may be mediated by increased Na+-K+-2Cl- expression but Cl- secretion is independent of symporter expression. These unrelated eubrachyurans exhibit similar systemic osmoregulatory characteristics and are better adapted to dilute media; however, the gene expressions underlying ion uptake and secretion show marked interspecific divergences. Cold clime crabs may have limited energy expenditure by regulating hemolymph [Cl-] alone, apportioning resources for other metabolic processes.

Summary statementSub-Antarctic crabs may skimp on osmoregulatory capabilities to apportion energy for metabolic processes. They regulate chloride but not sodium or osmolality. Transporter gene expressions diverge markedly. Adaptive, differential ion regulation may characterize cold clime crabs.


title: An in vivo model of echovirus-induced meningitis in neonates

abstract:
Echoviruses are amongst the most common causes of aseptic meningitis worldwide, which can cause long-term sequelae and death, particularly in neonates. However, the mechanisms by which these viruses induce meningeal inflammation is poorly understood, owing at least in part to the lack of in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we developed an in vivo neonatal mouse model that recapitulates key aspects of echovirus-induced meningitis. We found that expression of the human homologue of the neonatal Fc receptor (FcRn), the primary echovirus receptor, in neonatal mice was not sufficient for infection of the brain. However, ablation of type I, but not III, IFN signaling in mice expressing human FcRn permitted high levels of echovirus replication in the brain, with corresponding clinical symptoms including delayed motor skills and hind limb weakness. We also defined the immunological response of the brain to echovirus infections and identified key cytokines induced by this infection. Lastly, we found that echoviruses robustly replicate in the leptomeninges, where they induce profound inflammation and cell death. Together, this work establishes an in vivo model of aseptic meningitis associated with echovirus infections and defines the specificity of echoviral infections within the meninges.

Significance StatementEchoviruses are amongst the most common causes of aseptic meningitis worldwide, which can cause long-term sequelae or even death. The mechanisms by which echoviruses infect the brain are poorly understood, largely owing to the lack of robust in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we establish a neonatal mouse model of echovirus-induced aseptic meningitis and show that expression of the human homologue of the neonatal Fc receptor (FcRn), the primary receptor for echoviruses, and ablation of type I interferon (IFN) signaling are required to recapitulate echovirus-induced meningitis and clinical disease. These findings provide key insights into the host factors that control echovirus-induced meningitis and a model that could be used to test anti-echovirus therapeutics.


title: Cell cycle and developmental control of cortical excitability in Xenopus laevis

abstract: Interest in cortical excitability - the ability of the cell cortex to generate traveling waves of protein activity - has grown considerably over the past twenty years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in X. laevis eggs and embryos, and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties - such as wave frequency and amplitude - and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to non-furrow cortical excitability and showed that these wave regimes are mutually exclusive.


title: Population genetic structure of the maize weevil, Sitophilus zeamais, in southern Mexico

abstract: The maize weevil, Sitophilus zeamais, is a ubiquitous pest of maize and other cereal crops worldwide and remains a threat to food security in subsistence communities. Few population genetic studies have been conducted on the maize weevil, but those that exist have shown that there is very little genetic differentiation between geographically dispersed populations and that it is likely the species has experienced a recent range expansion within the last few hundred years. While the previous studies found little genetic structure, they relied primarily on mitochondrial and nuclear microsatellite markers for their analyses. It is possible that more fine-scaled population genetic structure exists due to local population dynamics, the biological limits of natural species dispersal, and the isolated nature of subsistence farming communities. In contrast to previous studies, here, we utilized genome-wide single nucleotide polymorphism data to evaluate the genetic population structure of the maize weevil from the southern and coastal Mexican states of Oaxaca and Chiapas. Our study is the first to find significant genetic population structure in the maize weevil. Here, we show that although there continues to be gene flow between populations of maize weevil, that fine-scale genetic structure exists. It is possible that this structure is shaped by the movement and trade of maize by humans in the region, by geographic barriers to gene flow, or a combination of factors.


title: Interaction of Fungal lipase with potential phytotherapeutics

abstract:
Interaction of thymol, carvacrol and linalool with fungal lipase and Human Serum Albumin (HSA) have been investigated employing UV-Vis, Fluorescence and Circular dichroism spectroscopy along with docking studies. Thymol, carvacrol and linalool displayed approximately 50% inhibition at 1.5 mmol/litre concentrations using para-nitrophenyl palmitate. UV-Vis spectroscopy give evidence of the formation of lipase-linalool, lipasecarvacrol and lipase-thymol complex at the ground state. Three molecules also showed complex formation with HSA at the ground state. Fluorescence spectroscopy shows strong binding of lipase to thymol (Ka of 2.6 x 109 M-1) as compared to carvacrol (4.66 x 107 M-1) and linalool (5.3 x 103 M-1). Number of binding sites showing stoichiometry of association process on lipase is found to be 2.52 (thymol) compared to 2.04 (carvacrol) and 1.12 (linalool). Secondary structure analysis by CD spectra results, following 24 hours incubation at 25{degrees}C, with thymol, carvacrol and linalool revealed decrease in negative ellipticity for lipase indicating loss in helical structure as compared with the native protein. The lowering in negative ellipticity was in the order of thymol > carvacrol > linalool.

Results of Fluorescence and CD spectroscopy taken together suggests that thymol and carvacrol are profound disrupter of lipase structure.

Fluorescence spectra following binding of all three molecules with HSA caused blue shift which suggests the compaction of the HSA structure. Association constant of thymol and HSA is 9.6 x 108 M-1 which along with  n value of 2.41 suggests strong association and stable complex formation, association constant for carvacrol and linalool was in range of 107 and 103 respectively.

Docking results give further insight into strong binding of thymol, carvacrol and linalool with lipase having free energy of binding as -7.1 kcal/mol, -5.0 kcal/mol and -5.2 kcal/mol respectively.

To conclude, fungal lipases can be attractive target for controlling their growth and pathogenicity. Employing UV-Vis, Fluorescence and Circular dichroism spectroscopy we have shown that thymol, carvacrol and linalool strongly bind and disrupt structure of fungal lipase, these three phytochemicals also bind well with HSA. Best anti-lipase molecules based on disruption of lipase structure and HSA structure conservation is thymol.


title: Induction of senescence upon loss of the Ash2l core subunit of H3K4 methyltransferase complexes

abstract: Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence.


title: Allele Dispersion Score: Quantifying the range of allele frequencies across populations, based on UMAP

abstract: Genomic variation plays a crucial role in biology, serving as a base for evolution - allowing for adaptation on a species or population level. At the individual level, however, specific alleles can be implicated in diseases. To interpret genetic variants identified in an individual potentially affected with a rare genetic disease, it is fundamental to know the population frequency of each allele, ideally in an ancestry matched cohort. Equity in human genomics remains a challenge for the field, and there are not yet cohorts representing most populations. Currently, when ancestry matched cohorts are not available, pooled variant libraries are used, such as gnomAD, the Human Genome Diversity Project (HGDP) or the 1,000 Genomes Project (now known as IGSR: International Genome Sample Resource). When working with a pooled collection of variant frequencies, one of the challenges is to determine efficiently if a variant is broadly spread across populations or appears selectively in one or more populations. While this can be accomplished by reviewing tables of population frequencies, it can be advantageous to have a single score that summarizes the observed dispersion. This score would not require classifying individuals into populations, which can be complicated if it is a homogenous population, or can leave individuals excluded from all the predefined population groups. Moreover, a score would not display fine-scaled population information, which could have privacy implications and consequently be inappropriate to release. Therefore, we sought to develop a scoring method based on a Uniform Manifold Approximation and Projection (UMAP) where, for each allele, the score can range from 0 (the variant is limited to a subset of close individuals within the whole cohort) to 1 (the variant is spread among the individuals represented in the cohort). We call this score the Allele Dispersion Score (ADS). The scoring system was implemented on the IGSR dataset, and compared to the current method consisting in displaying variant frequencies for several populations in a table. The ADS correlates with the population frequencies, without requiring grouping of individuals.


title: ClonoCluster: a method for using clonal origin to inform transcriptome clustering

abstract: Clustering cells based on their high dimensional profiles is an important data reduction process by which researchers infer distinct categories of cellular state. The advent of cellular barcoding, however, provides an alternative means by which to group cells: by their clonal origin. We developed ClonoCluster, a computational method that combines both clone and transcriptome information to create hybrid clusters that weight both kinds of data with a tunable parameter. We generated hybrid clusters across six independent datasets and found that ClonoCluster generated qualitatively different clusters in all cases. The markers of these hybrid clusters were different but had equivalent fidelity to transcriptome-only clusters. The genes most strongly associated with the rearrangements in hybrid clusters were ribosomal function and extracellular matrix genes. We also developed the complementary tool Warp Factor that incorporates clone information in popular 2D visualization techniques like UMAP. Integrating ClonoCluster and Warp Factor revealed biologically relevant markers of cell identity.


title: Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections

abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.


title: Ecological Observations Based on Functional Gene Sequencing Are Sensitive to the Amplicon Processing Method

abstract:
Until recently, the de-facto method for short read-based amplicons reconstruction is a sequence similarity threshold approach (Operational taxonomic Units OTUs). This assumption was relaxed by shifting to Amplicon Sequencing Variants (ASVs) where distributions are fitted to abundance profiles of individual genes using a noise-error model. Whilst OTUs-based approach is still useful for 16SrRNA/18S rRNA regions, where typically 97-99% thresholds are used, their utility to functional genes is still debatable as there is no consensus on how to cluster the sequences together. Here, we compare OTUs- and ASVs-based reconstruction approaches as well as taxonomy assignment methods, Naive Bayesian Classifier (NBC) and Bayesian Lowest Common Ancestor Algorithm (BLCA), using functional genes dataset from the microbial nitrogen-cycling community in the Brouage mudflat (France). A range of OTU similarity thresholds and ASV were used to compare amoA (AOA and AOB), nxrB, nirS, nirK and nrfA communities between differing sedimentary structures. We show that for AOA-amoA and nrfA, the use of ASV led to differences in the communities between sedimentary structures whereas the use of OTUs didnt. Conversely, significant differences were detected when using OTU (97%) for AOB-amoA but not with ASV or OTUs at other similarity thresholds. Interestingly, conclusions drawn from the other three functional genes were consistent between amplicon reconstruction methods. We also show that, when the sequences in the reference-database are related to the environment in question, BLCA leads to more phylogenetically relevant classifications. However, when the reference database contains sequences more dissimilar to the ones retrieved, NBC helps obtain more information.

ImportanceSeveral analysis pipelines are available to microbial ecologists to process amplicon sequencing data yet to-date, there is no consensus as to the most appropriate method, and it becomes more difficult for genes that encode a specific function (functional genes). Standardised approaches need to be adopted to increase reliability and reproducibility of environmental amplicon sequencing-based datasets. In this paper, we argue that the recently developed ASV approach offers a better opportunity to achieve such standardisation compared to OTUs for functional genes. We also propose a comprehensive framework for quality filtering of the sequencing reads based on protein sequence verification and merging.


title: Microbial turnover and dispersal events occur in sync with plant phenology in the perennial evergreen tree crop, Citrus sinensis

abstract: Emerging research indicates that plant-associated microbes can alter plant developmental timing. However, it is unclear if host phenology impacts microbial community assembly. Microbiome studies in annuals or deciduous perennial plants face challenges in separating effects of tissue age from phenological driven effects on the microbiome. In contrast, evergreen perennial trees, like Citrus sinensis, retain leaves for years allowing for uniform sampling of similarly aged leaves from the same developmental cohort. This aids in separating phenological effects on the microbiome from impacts due to annual leaf maturation/senescence. Here we used this system to test the hypothesis that host phenology acts as a driver of microbiome composition. Citrus sinensis leaves and roots were sampled during seven phenological stages. Using amplicon-based sequencing, followed by diversity, phylogenetic, differential abundance, and network analyses we examined changes in bacterial and fungal communities. Host phenological stage is the main determinant of microbiome composition, particularly within the foliar bacteriome. Microbial enrichment/depletion patterns suggest that microbial turnover and dispersal were driving these shifts. Moreover, a subset of community shifts were phylogenetically conserved across bacterial clades suggesting that inherited traits contribute to microbe-microbe and/or plant-microbe interactions during specific phenophases. Plant phenology influences microbial community composition. These findings enhance understanding of microbiome assembly and identify microbes that potentially influence plant development and reproduction.


title: In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance.

abstract: In vitro selection of remdesivir-resistant SARS-CoV-2 revealed the emergence of a V166L substitution, located outside of the polymerase active site of the nsp12 protein, after 9 passages. V166L remained the only nsp12 substitution after 17 passages at a final concentration of 10 {micro}M RDV, conferring a 2.3-fold increase in EC50. When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to RDV resistance.


title: Micro and Nanoplastics Interactions with Plant Species Trends, Meta-Analysis, and Perspectives

abstract:
The ubiquitous presence of nano plastics (NPx) and micro plastics (MPx) in the environment has been demonstrated, and as such, the exposure scenarios, mechanisms of plant response, and ultimate risk must be determined. However, the current literature reports ambiguous outcomes and provides limited mechanistic insight into critical governing processes. Here, we performed a meta-analysis of the most recent literature investigating the effect of MPx/NPx on plant species under laboratory and field conditions so as to evaluate the current state of knowledge. Toxic effects of MPx/NPx exposure in plants varies as a function of plant species and interestingly, generally non-significant responses are reported in staple crops. NPx (<100 nm) more negatively affected plant development parameters (n=341) (n is total number of observations), photosynthetic pigments (n=80), and biochemical indicators (n=91) than did MPx (>100 nm). Surprisingly, NPx exposure yielded negligible effects on germination rate (n=17), although root morphology (n=45) was negatively affected. Alternatively, MPx negatively affected on germination (n= 27) and generally non-significant affect with regard to root morphology (n=64). The effect of MPx/NPx on plant health decreases with increasing exposure time. No specific trends were evident for the production of biochemical enzymes as related to MPx/NPx concentration or size. Future work should include crop full life cycle studies to highlight the accumulation of MPx/NPx in edible tissues and also to investigate potential trophic transfer of MPx/NPx. Furthermore, we provide a framework for additional investigative work to address these and other knowledge gaps and to enable accurate assessment of the fate and risk of these materials to environmental and human health.

Environmental significanceAccumulation of plastic (MPx and NPx) particles is increasing in environmental compartments, and this might be threatened to agricultural plants.

Graphical Abstract

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category: plant biology


title: Plcg2M28L interacts with high fat-high sugar diet to accelerate Alzheimers disease-relevant phenotypes in mice

abstract: Obesity is recognized as a significant risk factor for Alzheimers disease (AD). Studies have supported the notion that obesity accelerates AD-related pathophysiology in mouse models of AD. The majority of studies to date have focused on the use of early-onset AD models. Here we evaluate the impact of genetic risk factors on late-onset AD (LOAD) in mice fed a high fat/high sugar diet. We focused on three mouse models created through the IU/JAX/Pitt MODEL-AD Center, LOAD1, LOAD1.Plcg2M28L and LOAD1.Mthfr677C>T. At 2 months of age, animals were placed on a high fat/high sugar diet (HFD) that induces obesity, or a control diet (CD) that does not, until 12 months of age. Throughout the study, blood was collected to assess cholesterol and glucose. Positron emission tomography/computed tomography (PET/CT) was completed prior to sacrifice to image for glucose utilization and brain perfusion. At the completion of the study, blood and brains were collected for analysis. As expected, animals fed the HFD, regardless of genotype or sex, showed a significant increase in body weight compared to those fed the CD. Glucose and cholesterol increased as a function of HFD as well. Interestingly, LOAD1.Plcg2M28L demonstrated an increase in microglia density as well as alterations in regional brain glucose and perfusion when on a HFD. These changes were not observed in LOAD1 or LOAD1.Mthfr677C>T animals when fed a HFD. Furthermore, LOAD1.Plcg2M28L but not LOAD1.Mthfr677C>T or LOAD1 animals showed transcriptomics correlations to human AD modules. Our results show HFD affects brain health in a genotype-specific manner. Further insight into this process may have significant implications in the development of lifestyle interventions for treatment of AD.