title: Loss of β-ketoacyl acyl carrier protein synthase III activity restores multidrug-resistant Escherichia coli sensitivity to previously ineffective antibiotics

abstract:
Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, nine out of 12 are Gram-negative, with four being of  Critical Priority. One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial Type II Fatty Acid Synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic susceptible and antibiotic resistant clinical isolates. The fabH gene encodes the {beta}-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multi-drug resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens.

IMPORTANCEGram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor towards antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids and their synthesis constitutes an attractive antimicrobial target as it follows distinct pathways in prokaryotes and eukaryotes. Herein, we identified a component of fatty acid synthesis, FabH, as a  gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are re-sensitised to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.


title: Alteration in long noncoding RNAs in response to oxidative stress and ladostigil in SH-SY5Y cells

abstract: Microglia activation causes neuroinflammation, which is a hallmark of neurodegenerative disorders, brain injury, and aging. Ladostigil, a bifunctional reagent with antioxidant and anti-inflammatory properties, reduced microglial activation and enhanced brain functioning in elderly rats. In this study, we studied SH-SY5Y, a human neuroblastoma cell line, and tested viability in the presence of hydrogen peroxide and Sin1 (3-morpholinosydnonimine), which generates reactive oxygen and nitrogen species (ROS/RNS). Both stressors caused significant apoptosis and necrotic cell death that was attenuated by ladostigil. Our results from RNA-seq experiments show that long non-coding RNAs (lncRNAs) account for 30% of all transcripts in SH-SY5Y cells treated with Sin1 for 24 hours. Altogether, we identify 94 differently expressed lncRNAs in the presence of Sin1, including MALAT1, a highly expressed lncRNA with anti-inflammatory and anti-apoptotic functions. Additional activities of Sin-1 upregulated lncRNAs include redox homeostasis (e.g., MIAT, GABPB1-AS1), energy metabolism (HAND2-AS1), and neurodegeneration (e.g., MIAT, GABPB1-AS1, NEAT1). Four lncRNAs implicated as enhancers were significantly upregulated in cells exposed to Sin1 and ladostigil. Finally, we show that H2O2 and Sin1 increased the expression of DJ-1, a redox sensor and modulator of Nrf2 (nuclear factor erythroid 2- related factor 2). Nrf2 (NFE2L2 gene) is a major transcription factor regulating antioxidant genes. In the presence of ladostigil, DJ-1 expression is restored to its baseline. The mechanisms governing SH-SY5Y cell survival and homeostasis are highlighted by the beneficial role of ladostigil in the crosstalk involving Nrf2, antioxidant transcription factor DJ-1, and lncRNAs. Stress-dependent induction of lncRNAs represents an underappreciated regulatory level that contributes to cellular homeostasis and the capacity of SH-SY5Y to cope with oxidative stress.

category: cell biology


title: Pre-existing chromosomal polymorphisms in pathogenic E. coli potentiate the evolution of antibiotic resistance by MCR-1 plasmid acquisition

abstract: Bacterial pathogens show high levels of standing genetic diversity, but the influence of this diversity on the evolution of antibiotic resistance remains unclear. Here we address this problem in the context of colistin, a  last line of defense antibiotic. Using experimental evolution, we show that a plasmid carrying the MCR-1 colistin resistance gene dramatically increases the ability of E. coli populations to evolve high-level colistin resistance by acquiring mutations in lpxC, an essential chromosomal gene involved in lipopolysaccharide biosynthesis. Crucially, lpxC mutations increase colistin resistance in the presence of the MCR-1 gene, but decrease the resistance of wild-type cells, revealing positive sign epistasis for antibiotic resistance. Analysis of public genomic datasets shows that lpxC polymorphisms are common in pathogenic E. coli carrying MCR-1, highlighting the clinical relevance of this interaction. Importantly, lpxC diversity is high in pathogenic E. coli from regions with no history of MCR-1 acquisition, suggesting that pre-existing lpxC polymorphisms have potentiated the evolution of high-level colistin resistance by MCR-1 acquisition. More broadly, these findings highlight the importance of standing genetic variation and plasmid/chromosomal interactions in the evolutionary dynamics of antibiotic resistance.

category: evolutionary biology


title: Are profile mixture models over-parameterized?

abstract:
AO_SCPLOWBSTRACTC_SCPLOWSite heterogeneity of the amino acid substitution process accounts for the biochemical constraints on the range of admissible amino acids at specific sites. Phylogenetic models of protein sequence evolution that do not account for site heterogeneity are more prone to long-branch attraction artifacts.

Profile mixture models are used to model site heterogeneity. Even though model, tree, and mixing parameters are statistically consistent, the performance of these models with short alignments is unclear. Here we explore the behavior of tree topology estimates and marginal cumulative distributions with short simulated alignments. We find that over-parameterization is not a problem for complex profile mixture models and that simple models behave poorly. Misspecification of the frequency distributions does not cause a problem if the estimated cumulative distribution function adequately approximates the true one. Also, we find that misspecification of the exchangeabilities can severely affect parameter estimation and that an increase in likelihood does not necessarily reflect better tree estimation. Although the inclusion of more taxa often helps, it can hurt estimation if the exchangeabilities are badly misspecified.

Finally, we explore the effects of including an  F-class with the overall amino acid frequencies of the dataset as an additional class in the profile mixture model. Surprisingly, the F-class does not seem to help parameter estimation significantly, and it can decrease the probability of correct tree estimation, depending on the scenario, despite the fact that it tends to improve likelihood scores. We also investigate this with several empirical data sets.


title: Mitotic bookmarking by CTCF controls selected genes during the fast post-mitotic genome reactivation of ES cells

abstract: Mitosis leads to a global downregulation of transcription that then needs to be efficiently restored. In somatic cells, this is mediated by a transient hyper-active state that first leads to the reactivation of genes necessary to rebuild the interphasic cell and then of those executing specific cell functions. Here, we hypothesized that cells displaying rapid cell cycles may display accelerated gene reactivation dynamics. To test this, we focused on mouse Embryonic Stem (ES) cells, which have a short cell cycle and spend a minor time in G1. Compared to previous studies, we observed a uniquely fast global reactivation, which displays little specificity towards housekeeping versus cell identity genes. Such lack of specificity may enable the restoration of the entirety of regulatory functions before the onset of DNA replication. Genes displaying the fastest reactivation dynamics are associated with binding of CTCF, a transcription factor that largely maintains binding to its targets on DNA during mitosis. Nevertheless, we show that the post-mitotic global burst is robust and largely insensitive to CTCF depletion. There are, however, around 350 genes that respond to CTCF depletion rapidly after mitotic exit. Remarkably, these are characterised by promoter-proximal mitotic bookmarking by CTCF. We propose that the structure of the cell cycle imposes distinct constrains to post-mitotic gene reactivation dynamics in different cell types, via mechanisms that are yet to be identified but that can be modulated by mitotic bookmarking factors.

category: genomics


title: Large-scale comparison of immunoassay- and aptamer-based plasma proteomics through genetics and disease

abstract: The authors have withdrawn this manuscript because this paper was posted prematurely in advance of a UK Biobank Pharma Proteomics Project consortium effort. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author


title: Molecular insights into the recognition of acetylated histone modifications by the BRPF2 bromodomain

abstract:
HBO1 (HAT bound to ORC), a member of the MYST family of histone acetyltransferases (HATs), was initially identified as a binding partner of the origin recognition complex (ORC) that acetylates free histone H3, H4, and nucleosomal H3. It functions as a quaternary complex with the BRPF (BRPF1/2/3) scaffolding protein and two accessory proteins, ING4/5 and Eaf6. BRPF2 interaction with HBO1 has been shown to be important for regulating H3K14 acetylation during embryonic development. However, how the BRPF2 directs the HBO1 HAT complex to chromatin to regulate its HAT activity towards nucleosomal substrates remains unclear. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminus of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K5acK12ac modifications. Moreover, pull-down assays on the endogenous histones and mononucleosomes isolated from human cells confirmed that the BRPF2 bromodomain specifically showed a stronger affinity to H4K5ac marks. Further, mutational analysis of BRPF2 bromodomain coupled with ITC binding and pull-down assays on the histone substrates identified critical residues responsible for acetyllysine binding. Together, our study provides novel insights into how the histone binding function of the BRPF2 bromodomain directs the recruitment of the HBO1 HAT complex to chromatin to regulate gene expression.

Table of Content Artwork

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category: biochemistry


title: Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila Melanogaster (Pupal Retina)

abstract: A novel pair of protein Tyrosine Phosphatases in Drosophila Melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized having fundamental role in cellular processes including effects on metabolism, cell proliferation and differentiation. This study presents comparative homology modeling of low molecular weight phosphotyrosine protein phosphatase (PTPs) from Drosophila melanogaster (Dr-PTPs) and their complexation with potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homology is approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel {beta} sheet with flanking  helices on both sides, showing two right-handed {beta}--{beta} motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs activation. The structure guided studies of both proteins clearly reveal a common mechanism of action, inhibitor binding at the active site and will expected to contribute towards the basic understanding of functional association of this enzyme with other molecules.


title: An assessment of bioinformatics tools for the detection of human endogenous retroviral insertions in short-read genome sequencing data

abstract: There is a growing interest in the study of human endogenous retroviruses (HERVs) given the substantial body of evidence that implicates them in many human diseases. Although their genomic characterization presents numerous technical challenges, next-generation sequencing (NGS) has shown potential to detect HERV insertions and their polymorphisms in humans, and a number of computational tools to detect them in short-read NGS data exist. In order to design optimal analysis pipelines, an independent evaluation of the currently available tools is required. We evaluated the performance of a set of such tools using a variety of experimental designs and types of NGS datasets. These included 50 human short read whole-genome sequencing samples, matching long and short read NGS data, and simulated short-read NGS data. Our results highlight the performance variability of the tools across the datasets and suggest that different tools might be suitable for different study designs. Using multiple tools and a consensus approach is advisable if computationally feasible and wet-lab validation via PCR is advisable where biological samples are available.

category: bioinformatics


title: Zn-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

abstract:
The toxic gain of function of Cu/Zn superoxide dismutase (SOD1) associated with the neurodegenerative disease - Amyotrophic lateral sclerosis (ALS), is believed to occur via misfolding and/or aggregation. SOD1 is also associated with stress granules (SGs) which are a type of membraneless organelle believed to form via liquid-liquid phase separation (LLPS) of several proteins containing low-complexity, disordered regions. Using a combination of experiments and computer simulations, we report here that structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The phase-separated droplets give rise to aggregates which eventually form toxic amyloids upon prolonged incubation. The addition of exogenous Zn to immature, metal-free SOD1 and the severe ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. In contrast, the Zn-induced inhibition of LLPS and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In conclusion, our work establishes a role for Zn-dependent modulation of SOD1 disorder and LLPS as a precursor phenomenon which may lead to the formation of toxic amyloids associated with ALS.

Significance StatementThe formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. SOD1 is a metalloenzyme which is stabilized by the metal co-factor - Zn. In this work, we utilize experimental and simulation techniques to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.

category: biophysics


title: Resistance to EGFR inhibitors in lung cancer occurs through horizontal transfer and is associated with increased caveolins expression

abstract:
Resistance to treatment is a major clinical problem and a major cause of cancer-related deaths. Understanding the biological basis of resistance acquisition is of utmost importance to improve the clinical management of cancer patients. NGS analysis of human lung cancer (LC) tumors from patients that relapsed after treatment with EGFR-tyrosine kinase inhibitors (TKI), revealed that the p.T790M resistance mutation is not present in all the relapsing tumor cells, suggesting that LC cells can become resistant even if not carrying the p.T790M mutation. Using in vitro treatments with conditioned medium (CM) and in vivo co-inoculation experiments, we show that LC cells sensitive to EGFR-TKIs (S cells) acquire resistance faster when treated with CM from LC cells resistant to EGFR-TKIs (R cells) or when co-inoculated with R cells in opposite flanks of the same animal. Importantly, we show that acquisition of resistance is not due to the emergence of subpopulations of cancer cells with new resistance mutations. Using transcriptomics, we show that acquisition of resistance is associated with upregulation of genes involved in endocytosis, namely caveolins CAV1 and CAV2. These findings were validated in human clinical samples, where an increase in CAV1 and CAV2 expression was associated with tumor relapse after treatment with EGFR-TKIs. Our results suggest that acquisition of resistance to targeted therapies results from the combined effect of selection of cells harboring specific resistance mutations and horizontal transfer of the resistance phenotype. These findings may pave the way to bring intercellular communication into the realm of cancer treatment.

One Sentence SummaryResistance to EGFR inhibitors is transferred horizontally between lung cancer cells and is associated with gain of expression of caveolins.

category: cancer biology


title: The effects of brief heat during early booting on reproductive, developmental and physiological performance in common wheat (Triticum aestivum L.)

abstract: Rising temperatures due to climate change threaten agricultural crop productivity. As a cool-season crop wheat is heat sensitive, but often exposed to high temperatures during cultivation. In the current study, a bread wheat panel of spring wheat genotypes, including putatively heat-tolerant Australian and CIMMYT genotypes, was exposed to a 5-day mild (34{degrees}C/28{degrees}C, day/night) or extreme (37{degrees}C/27{degrees}C) heat stress during the sensitive pollen developmental stage. Worsening effects on anther morphology were observed as heat stress increased from mild to extreme. Even under mild heat a significant decrease in pollen viability and grain number per spike from primary spike was observed compared with the control (21{degrees}C/15{degrees}C), with Sunstar and two CIMMYT breeding lines performing well. A heat-specific positive correlation between the two traits indicates the important role of pollen fertility for grain setting. Interestingly, both mild and extreme heat induced development of new tillers after the heat stress, providing an alternative sink for accumulated photosynthates and significantly contributing to the final yield. Measurements of flag leaf maximum potential quantum efficiency of Photosystem II (Fv/Fm) showed an initial inhibition after the heat treatment, followed by a full recovery within a few days. Despite this, model fitting using chlorophyll SPAD measurements showed an earlier onset or faster senescence rate under heat stress. The data presented here provide interesting entry points for further research into pollen fertility, tillering dynamics and leaf senescence under heat. The identified tolerant wheat genotypes can be used to dissect the underlying mechanisms and breed climate-resilient wheat.

category: plant biology


title: Use of sugarcane mosaic virus for virus-induced gene silencing in maize

abstract: Previously, sugarcane mosaic virus (SCMV) was developed as a vector for transient expression of heterologous genes in Zea mays (maize). Here we show that SCMV can also be applied for virus-induced gene silencing (VIGS) of endogenous maize genes. Comparison of sense and antisense VIGS constructs targeting maize PDS (phytoene desaturase) showed that antisense constructs resulted in a greater reduction in gene expression. In a time course of gene expression after infection with VIGS constructs targeting PDS, Les22 (Lesion mimic 22), and Ij1 (Iodent japonica1), efficient expression silencing was observed two, three, and four weeks after infection with SCMV. However, at week five, expression of Les22 and Ij1 was no longer significantly reduced compared to control plants. The defense signaling molecule jasmonate-isoleucine (JA-Ile) can be inactivated by 12C-hydroxylation and hydrolysis, and knockout of these genes leads to increased herbivore resistance. JA-Ile hydroxylases and hydrolases have been investigated in Arabidopsis, rice, and Nicotiana attenuata. To determine whether the maize homologs of these genes function in plant defense, we silenced expression of ZmCYP94B1 (predicted JA-Ile hydroxylase) and ZmJIH1 (predicted JA-Ile hydrolase) by VIGS with SCMV. Although ZmCYP94B1 and ZmJIH1gene expression silencing increased resistance to Spodoptera frugiperda (fall armyworm), Schistocerca americana (American birdwing grasshopper), and Rhopalosiphum maidis (corn leaf aphid), there was no additive effect from silencing the expression of both genes. Further work will be required to determine the more precise functions of these enzymes in regulating maize defenses.


title: Regulation of Hedgehog Signaling Through Arih2-Mediated Smoothened Ubiquitination and Endoplasmic Reticulum-Associated Degradation

abstract: During Hedgehog signaling, the ciliary levels of Ptch1 and Smo are regulated by pathway activity. At the basal state, Ptch1 localizes to cilia and prevents the ciliary accumulation and activation of Smo. Upon stimulation with Hedgehog ligand, Ptch1 exits cilia and relieves the inhibition of Smo. Uninhibited Smo concentrates in cilia, becomes activated, and activates the downstream steps of the pathway. Loss of the ubiquitin E3 ligase Arih2 elevates the cellular level of Smo, causes Smo to inappropriately localize to cilia at the at the basal state, and elevates basal expression of Hedgehog responsive genes. Mice express two isoforms of Arih2 with different N-termini, but neither isoform localizes to cilia. Instead, Arih2 is found in the nucleus and Arih2{beta} is found on the cytoplasmic face of the endoplasmic reticulum. Re-expression of endoplasmic reticulum-localized Arih2{beta}, but not nuclear-localized Arih2 returns the cellular Smo levels back to normal and rescues the ciliary Smo accumulation phenotype. When Arih2{beta} is missing, protein aggregates accumulate in the endoplasmic reticulum and the unfolded protein response is activated. Inhibitor studies suggest that Arih2{beta} functions to mark excess or misfolded Smo for degradation by endoplasmic reticulum-associated degradation. When Arih2{beta} is defective, excess Smo, possibly misfolded, is delivered to the cell surface and cilium where it interferes with pathway regulation. These findings add another level of complexity to the Hedgehog pathway.


title: Differential roles of neural crest- and endothelial-derived FOXC2 in trabecular meshwork and Schlemms Canal in glaucomatous pathology

abstract: Impaired development and maintenance of the Schlemms Canal (SC) is associated with perturbed aqueous humor outflow regulation and glaucoma progression. Key molecular mechanisms, such as ANGPT/TIE2, PROX1, and VEGF-C/VEGFR-3 regulate SC development and maintenance, but mechanisms of paracrine signaling from neighboring tissues, including the trabecular meshwork (TM) are poorly understood. Here, we show Foxc2 is critical within the neural crest (NC)-derived TM and SC endothelium for development of the aqueous humor outflow pathway. In mice, NC- specific deletion of Foxc2 results in abnormal anterior eye segment development, including impaired SC morphogenesis and functional maintenance, loss of SC identity, and impaired maintenance of intraocular pressure (IOP). Visible light optical coherence tomography angiography analysis also demonstrated functional impairment of the SC in response to changes in IOP in NC-Foxc2-/- mice, suggesting increased TM stiffness. Utilization of single-cell RNA-sequencing (scRNA-seq) analysis then identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM-associated cell clusters, including increased matrix metalloproteinase (MMP) expression, which can generate soluble TIE2 that acts as an ANGPT trap. As FOXC2 is also critically involved in development of the lymphatic vasculature in other tissues, we also show that endothelial-specific deletion of Foxc2 resulted in impaired SC morphogenesis due to loss of TIE2 expression, which was rescued by deletion of the TIE2 phosphatase VE-PTP. Thus, NC-Foxc2 is critical for development of the TM, and both NC- and endothelial-Foxc2 are key for maintenance of SC identity and its morphogenesis.

category: developmental biology


title: Using landscape genomics to infer genomic regions involved in environmental adaptation of soybean genebank accessions

abstract: Understanding how crops adapt to specific environmental conditions is becoming increasingly important in the face of accelerating climate change, but the genetics of local adaptation remain little understood for many crops. Landscape genomics can reveal patterns of genetic variation that indicate adaptive diversification during crop evolution and dispersal. Here, we examine genetic differentiation and association signatures with environmental gradients in soybean (Glycine max) germplasm groups from China that were inferred from the USDA Soybean Germplasm Collection (N = 17,019 accessions) based on population structure and passport information. We recover genes previously known to be involved in soybean environmental adaptation and report numerous new candidate genes in selection signatures implicated by genomic resources such as the genome annotation and gene expression datasets to function in flowering regulation, photoperiodism and stress reaction cascades. Linkage disequilibrium network analysis suggested functional relationships between genomic regions with selection signatures, consistent with the polygenic nature of environmental adaptation. We tested whether haplotypes associated with environmental adaptation in China were present in 843 North American and 160 European soybean cultivars and found that haplotypes in major genes for early maturity have been selected during breeding, but also that a large number of haplotypes exhibiting putative adaptive variation for cold regions at high latitudes are underrepresented in modern cultivars. Our results demonstrate the value of landscape genomics analysis in genebank germplasm as a starting point for the study of crop environmental adaptation and have the potential to inform future research efforts focused on improved soybean adaptation. Functional validation of candidate genes will support understanding of their adaptive roles and likely enable the transfer of beneficial adaptive variation into modern breeding germplasm.


title: When Vibrios Take Flight: A Meta-analysis of Pathogenic Vibrios Species in Wild and Domestic Birds

abstract: Of the over 100 species in the genus Vibrio, approximately twelve are associated with clinical disease, such as cholera and vibriosis. Crucially, eleven of those twelve--V. alginolyticus, V. cholerae, V. cincinnatiensis, V. hollinsae, e.g., Grimontia hollinsae, V. furnissii, V. mimicus, V. parahaemolyticus, V. vulnificus, V. harveyi, V. scophthalmi and V. metschnikovi--have also been isolated from birds. Since 1965, pathogenic Vibrio species have been consistently isolated from aquatic and ground-foraging bird species, which has implications for public health, as well as the One Health paradigm defined as an ecology-inspired, integrative framework for the study of health and disease, inclusive of environmental, human, and animal health. In this meta-analysis, we identified 76 studies from the primary literature which report on or examine birds as hosts for pathogenic Vibrio species. We found that the burden of disease in birds was most commonly associated with V. cholerae, followed by V. metschnikovi and V. parahaemolyticus. Meta-analysis wide prevalences of the Vibrio pathogens varied from 19% for V. parahaemolyticus to 1% for V. mimicus. Wild and domestic birds were both affected, which may have implications for conservation, as well as agriculturally associated avian species. As pathogenic Vibrios become more abundant throughout the world as a result of warming estuaries and oceans, susceptible avian species should be continually monitored as potential reservoirs for these pathogens.

category: ecology


title: Keratinocyte-Associated Protein 3 is a novel gene for adiposity with differential effects in males and females

abstract:
ObjectiveDespite the obesity crisis in the United States, the underlying genetics are poorly understood. Our lab previously identified Keratinocyte-associated protein 3, Krtcap3, as a candidate gene for adiposity where increased expression of Krtcap3 correlated with decreased fat mass. Here we seek to confirm that Krtcap3 expression affects adiposity traits.

MethodsWe developed an in vivo whole-body Krtcap3 knock-out (KO) rat model. Wild-type (WT) and KO rats were placed onto a high-fat or low-fat diet at six weeks of age and were maintained on diet for 13 weeks, followed by assessments of metabolic health. We hypothesized that Krtcap3-KO rats will have increased adiposity and a worsened metabolic phenotype relative to WT.

ResultsWe found that KO male and female rats have significantly increased body weight versus WT. KO females ate more, had more fat mass, but were also more insulin sensitive than WT. Alternatively, KO males weighed more and were more insulin resistant than WT, with no differences in eating or fat mass.

ConclusionsThis study validates Krtcap3 in body weight regulation and demonstrates sex-specific effects on food intake, adiposity, and insulin sensitivity. Future studies will investigate how Krtcap3 is acting and seek to better understand these sex differences.

Study Importance QuestionsWhat is already known about this subject? O_LIOver 900 low-risk, common genetic variants for BMI have been identified, but these still only explain a fraction of the heritability and many of the underlying causal genes remain unknown
C_LIO_LIKrtcap3 has been identified as a candidate gene for obesity in both rats and humans, but no verification or functional studies have been done
C_LI

What are the new findings in your manuscript? O_LIIdentified Krtcap3 as a novel gene that impacts feeding behavior and adiposity in female rats
C_LIO_LIDetermined that Krtcap3 impacts insulin sensitivity differentially in male and female rats
C_LI

How might your results change the direction of research or the focus of clinical practice? O_LIThis work may lead to identification of new pathways that contribute to obesity without metabolic complications, which will advance understanding of the biology of obesity and potentially identify novel drug targets
C_LIO_LIThis work highlights the need to investigate sex differences in the genetics of obesity
C_LI

category: genetics


title: Inverted genomic regions between reference genome builds in humans impact imputation accuracy and decrease the power of association testing

abstract: Over the last two decades, the human reference genome has undergone multiple updates as we complete a linear representation of our genome. There are two versions of human references currently used in the biomedical literature, GRCh37/hg19 and GRCh38, and conversions between these versions are critical for quality control, imputation, and association analysis. In the present study we show that genomic coordinates for single nucleotide variants (SNVs) in regions inverted between different builds of the reference genome are erroneously converted by the TOPMed imputation server. Depending on the array type, we estimate the inappropriate conversions of variant coordinates would occur in about 2-5 Mb of the genome. Independent of the imputation server, however, in-house conversions of variant coordinates are routine yet impact of mapped variants to these inverted regions and its downstream analytical consequences are underrecognized. We observed that errors for palindromic variants in these inverted regions cannot be detected by standard quality control procedures and destabilize the local haplotype structure, leading to loss of imputation accuracy and power in association analyses. Though only a small proportion of the genome is affected, we show that these regions include important disease susceptibility variants that would be lost due to poor imputation. For example, we show that a known locus associated with prostate cancer on chr10 would have its association P-value drop from 2.86x10-7 to 0.0011 in a case-control analysis of 20,286 Africans and African Americans (10,643 cases and 9,643 controls). We devise a straight-forward heuristic based on the popular tool liftOver that can easily detect and correct these variants in the inverted regions between genome builds to locally improve imputation accuracy.


title: scAnnotate: an automated cell type annotation tool for single-cell RNA-sequencing data

abstract:
MotivationSingle-cell RNA-sequencing (scRNA-seq) technology enables researchers to investigate a genome at the cellular level with unprecedented resolution. An organism consists of a heterogeneous collection of cell types, each of which plays a distinct role in various biological processes. Hence, the first step of scRNA-seq data analysis often is to distinguish cell types so that they can be investigated separately. Researchers have recently developed several automated cell type annotation tools based on supervised machine learning algorithms, requiring neither biological knowledge nor subjective human decisions. Dropout is a crucial characteristic of scRNA-seq data widely used in differential expression analysis. However, existing supervised learning methods for cell annotation do not use the dropout information explicitly. This motivated us to build a novel cell annotation tool that fully utilizes this information.

ResultsWe present scAnnotate, an automated cell annotation tool based on supervised machine learning algorithms. We use a marginal mixture model to describe both the dropout proportion and the non-dropout expression level distribution of a gene. We develop a marginal model based ensemble learning approach to avoid having to specify and estimate a high-dimensional joint distribution for all genes. First, we build a  weak classifier using the mixture model for each gene. Then, we combine  weak classifiers of all genes into a single  strong classifier to annotate cells. Using 11 real scRNA-seq data, we demonstrate that scAnnotate is competitive against 9 other methods, and that it accurately annotates cells when training and test data are (1) similar, (2) cross-platform, and (3) cross-species.

AvailabilityAn R package, scAnnotate, is in preparation and will be publicly available by March 2022.

ContactXuekui Zhang, xuekui@uvic.ca


title: Quantitative sequence basis for the E. coli transcriptional regulatory network

abstract: The transcriptional regulatory network (TRN) of E. coli consists of thousands of interactions between regulators and DNA sequences. Inherently the DNA sequence is the primary determinant of the TRN; however, it is well established that the presence of a DNA binding motif does not guarantee a functional regulatory protein binding site. Thus, the extent to which the TRN architecture can be predicted by the genome DNA sequence alone remains unclear. Here, we developed machine learning models that predict the TRN structure of E. coli based on genome sequence. Models were constructed successfully (cross-validation AUROC >= 0.8) for 84% (57/68) of valid E. coli regulons identified from top-down analysis of RNA-seq data. We found that: 1) While regulatory motif strength is the most important sequence feature for determining regulon membership, additional features such as DNA shape substantially influence membership; 2) complex regulons involving multiple interacting regulators could be unraveled by machine learning; 3) investigating regulons where initial ML models failed revealed new regulator-specific sequence features that improved model accuracy. Finally, while regulon structure can appear to be variable across estimation methods and strains, we found that strong regulatory sequence features underlie both the genes that appear most consistently in regulons across estimation methods as well as the core regulon genes in the Fur pan-regulon. This work develops a quantitative understanding of the sequence basis of the TRN and suggests a path towards computationally-guided control of transcriptional regulation for synthetic biology applications.


title: Statistical Mechanical theory for spatio-temporal evolution of Intra-tumor heterogeneity in cancers: Analysis of Multiregion sequencing data

abstract: Variations in characteristics from one region (sub-population) to another is commonly observed in complex systems, such as glasses and a collection of cells. Such variations are manifestations of heterogeneity, whose spatial and temporal behavior is hard to describe theoretically. In the context of cancer, intra-tumor heterogeneity (ITH), characterized by cells with genetic and phenotypic variability that co-exist within a single tumor, is often the cause of ineffective therapy and recurrence of cancer. Next-generation sequencing, obtained by sampling multiple regions of a single tumor (multi-region sequencing, M-Seq), has vividly demonstrated the pervasive nature of ITH, raising the need for a theory that accounts for evolution of tumor heterogeneity. Here, we develop a statistical mechanical theory to quantify ITH, using the Hamming distance, between genetic mutations in distinct regions within a single tumor. An analytic expression for ITH, expressed in terms of cell division probability () and mutation probability (p), is validated using cellular-automaton type simulations. Application of the theory successfully captures ITH extracted from M-seq data in patients with exogenous cancers (melanoma and lung). The theory, based on punctuated evolution at the early stages of the tumor followed by neutral evolution, is accurate provided the spatial variation in the tumor mutation burden is not large. We show that there are substantial variations in ITH in distinct regions of a single solid tumor, which supports the notion that distinct subclones could co-exist. The simulations show that there are substantial variations in the sub-populations, with the ITH increasing as the distance between the regions increases. The analytical and simulation framework developed here could be used in the quantitative analyses of the experimental (M-Seq) data. More broadly, our theory is likely to be useful in analyzing dynamic heterogeneity in complex systems such as super-cooled liquids.


title: Conformational Ensemble of Monomeric α-Synuclein in Aqueous and Crowded Environments as revealed by Markov State Model

abstract:
140-residue intrinsically disordered protein -synuclein (S) is known to be susceptible to environmental cues/crowders and adopts conformations that are vastly variable in the extent of secondary structure and tertiary interactions. Depending upon the nature of these interactions, some of the conformations may be suitable for its physiological functions while some may be predisposed to aggregate with other partners into higher ordered species or to phase separate. However, the inherently heterogenous and dynamic nature of S has precluded a clear demarcation of its monomeric precursor between aggregation-prone and functionally relevant aggregation-resistant states. Here, we optimally characterise a set of metastable conformations of S by developing a comprehensive Markov state model (MSM) using cumulative 108 {micro}s-long all-atom MD simulation trajectories of monomeric S. Notably, the dimension of the most populated metastable (85%) state (Rg [~] 2.59 ({+/-}0.45) nm) corroborates PRENMR studies of S monomer and undergoes kinetic transition at 0.1-150 {micro}s time-scale with weakly populated (0.06%) random-coil like ensemble (Rg [~] 5.85 ({+/-}0.43) nm) and globular protein-like state (14%) (Rg [~] 1.95 ({+/-}0.08) nm). The inter-residue contact maps identify a set of mutually interconverting aggregation-prone {beta}-sheet networks in the NAC region and aggregation-resistant long-range interactions between N- and C-terminus or helical conformations. The presence of crowding agents compacts the MSM-derived metastable conformations in a non-monotonic fashion and skews the ensemble by either introducing new tertiary contacts or reinforcing the innate contacts to adjust to the excluded-volume effects of such environments. These observations of crucial monomeric states would serve as important steps towards rationalising routes that trigger S-associated pathologies.

Significance statement-synuclein, a neuronal protein, is often associated with neurogenerative diseases due to its tendency to self-assemble into higher ordered aggregates. While the monomeric precursor of this protein is intrinsically disordered, it is also known to be susceptible to biological environmental cues and adopts a wide range of conformations that are either primed for aggregation or remain in auto-inhibitory states. However, the inherently heterogenous nature of the monomeric form has prevented a clear dissection of aggregation-prone and functionally relevant aggregation-resistant states. Here, we resolve this via an atomistic characterisation of an optimal set of crucial metastable monomeric conformations via statistical modelling of computer simulated data. The investigation also sheds light on crowding-induced modulation of the ensemble and eventual fibrillation pathways.


title: Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

abstract:
Cancer genomes harbor a broad spectrum of structural variants (SV) driving tumorigenesis, a relevant subset of which are likely to escape discovery in short reads. We employed Oxford Nanopore Technologies (ONT) sequencing in a paired diagnostic and post-therapy medulloblastoma to unravel the haplotype-resolved somatic genetic and epigenetic landscape. We assemble complex rearrangements and such associated with telomeric sequences, including a 1.55 Megabasepair chromothripsis event. We uncover a complex SV pattern termed  templated insertion thread, characterized by short (mostly <1kb) insertions showing prevalent self-concatenation into highly amplified structures of up to 50kbp in size. Templated insertion threads occur in 3% of cancers, with a prevalence ranging to 74% in liposarcoma, and frequent colocalization with chromothripsis. We also perform long-read based methylome profiling and discover allele-specific methylation (ASM) effects, complex rearrangements exhibiting differential methylation, and differential promoter methylation in seven cancer-driver genes. Our study shows the potential of long-read sequencing in cancer.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY I) We investigate a single patient with chromothriptic sonic hedgehog medulloblastoma (Li-Fraumeni syndrome), with tissue samples taken from blood, the primary tumor at diagnosis, and a post-treatment (relapse) tumor. II) Data on the three samples has been collected from four sources, 1) Illumina whole-genome, 2) Illumina transcriptome sequencing, 3) Illumina Infinium HumanMethylation450k, as well as 4) long-read whole-genome sequencing using Oxford Nanopore Technologies (ONT) sequencing. III) An integrative analysis combines genomic, epigenomic as well as transcriptomic data to provide a comprehensive analysis of this heavily rearranged tumor sample. Long and short read sequencing data is used to inform the analysis of complex structural genomic variants and methylation called from haplotyped ONT reads and validated through the methylation array data allows for a haplotype-resolved study of genomic and epigenomic variation, which can then be examined for transcriptional effect. IV) This integrative analysis allows us to identify a large number of inter- and intra-chromosomal genomic rearrangements (A) including a complex rearrangement pattern we term templated insertion threads (B), as well as sample-specific and haplotype specific methylation patterns of known cancer genes (C).

C_FIG_DISPLAY


title: Increased Relative Biological Effectiveness of Orthovoltage X-rays Compared to γ-rays in Preclinical Irradiation

abstract:
PurposeRadionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. There are institutional initiatives to replace radionuclide sources with lower-energy X-ray sources amidst concerns of radiological terrorism. As researchers transition, there are questions whether the biological effects of {gamma}-rays may be recapitulated with orthovoltage X-rays, since different energy may cause different biological effects. We, therefore, sought to compare the effects of orthovoltage X-rays and 137Cs {gamma}-rays using mouse models of acute radiation syndrome.

Experimental Design137Cs {gamma}-rays were compared with Orthovoltage X-rays, generated at 300 kVp, 10 mA with 1 mm Cu or Thoraeus filtration. We assessed 30-day overall survival following whole-body irradiation and calculated LD50 by logistic regression. Comparing equivalent doses delivered with different average energies ([E]), we assessed bone marrow, spleen, and intestinal histology and flow cytometry.

ResultsThe LD50 doses are 6.7 Gy, 7.4 Gy and 8.1 Gy with 1 mm Cu filtered ([E]=120 keV), and Thoraeus filtered X-rays ([E]=160 keV), and 137Cs (E=662 keV), respectively. At constant dose, hematopoietic injury was most severe with 1 mm Cu filtered X-rays with the greatest reduction in bone marrow cellularity, stem and progenitor populations, and intestinal crypts and OLFM4+ intestinal stem cells. Thoraeus filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage.

ConclusionsOur study reveals a dichotomy between physical dose and biological effect relevant as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose-reduction to achieve comparable biological effects.

Statement of Translational RelevanceRadiation is used in translational studies in fields ranging from hematopoiesis and stem cell biology to cancer radiotherapy, with 137Cs and 60Co radionuclide sources serving as the most common irradiators. Due to the threat of radiological terrorism using stolen radionuclides, there are institutional initiatives to replace these sources with orthovoltage X-ray irradiators. Yet, as shown in this study, the biological effects of radiation are highly dependent on radiation energy. Lower energy orthovoltage X-rays are absorbed differently than higher energy radionuclide {gamma}-rays, provoking more severe hematopoietic, immunologic, and gastrointestinal radiation injury. Thus, an identical physical dose delivered with beams of differing energy does not produce the same biologic effect. As researchers transition between these sources, it is critical that we appreciate that radiation doses are not interchangeable between them. Understanding the significance of physical dose delivered using different methods will allow us to contextualize past results with future studies.

category: pathology


title: A pipeline for natural small molecule inhibitors of endoplasmic reticulum stress

abstract:
The homeostasis of eukaryotic cells is inseverable of that of the endoplasmic reticulum (ER). The main function of this organelle is the synthesis and folding of a significant portion of cellular proteins, while also being the major calcium reservoir of the cell. Upon unresolved ER stress, a set of stress response signaling pathways that are collectively labeled as the unfolded protein response (UPR) is activated. Prolonged or intense activation of this molecular machinery may be deleterious. It is known that compromised ER homeostasis, and consequent UPR activation, characterize the pathogenesis of neurodegenerative disease.

In an effort to discover new small molecules capable of countering ER stress, we subjected a panel of over 100 natural molecules to a battery of assays designed to evaluate several hallmarks of ER stress. The effect of the compounds on calcium homeostasis, key gene and protein expression, and levels of protein aggregation were evaluated in fibroblasts, and subsequently in neuronal cells. This framework resulted in the identification of several bioactive molecules capable of countering ER stress and deleterious events associated to it, among which delphinidin stands out as the most promising candidate against neurodegeneration.

category: pharmacology and toxicology


title: Generation of human nociceptor-enriched sensory neurons for the study of pain-related dysfunctions

abstract: In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically-useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly-enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9,000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.


title: Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response

abstract: Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, each Brachypodium AFP gene encodes two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models suggesting that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins that form ice at high sub-zero temperatures, consistent with decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Strikingly, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22), with structural modelling suggesting this was due to affinity of the LRR domains to flg22. Thus, these genes play distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins ability to adsorb to ice as well as attenuating bacterial ice nucleation and the host immune response.


title: Kir6.2-containing KATP channels are necessary for glucose dependent increases in amyloid-beta and Alzheimer's-related pathology

abstract: Increased neuronal excitability contributes to amyloid-{beta} (A{beta}) production and aggregation in the Alzheimers disease (AD) brain. Previous work from our lab demonstrated that hyperglycemia, or elevated blood glucose levels, increased brain excitability and A{beta} release potentially through inward rectifying, ATP-sensitive potassium (KATP) channels. KATP channels are present on several different cell types and help to maintain excitatory thresholds throughout the brain. KATP channels are sensitive to changes in the metabolic environment, which are coupled to changes in cellular excitability. Therefore, we hypothesized that neuronal KATP channels are necessary for the hyperglycemic-dependent increases in extracellular A{beta} and eliminating KATP channel activity will uncouple the relationship between metabolism, excitability, and A{beta} pathology. First, we demonstrate that Kir6.2/KCNJ11, the pore forming subunits, and SUR1/ABCC8, the sulfonylurea receptors, are predominantly expressed on excitatory and inhibitory neurons in the human brain and that cortical expression of KCNJ11 and ABCC8 change with AD pathology in humans and rodent models. Next, we crossed APP/PS1 mice with Kir6.2 -/- mice, which lack neuronal KATP channel activity, to define the relationship between KATP channels, A{beta}, and hyperglycemia. Using in vivo microdialysis and hyperglycemic clamps, we explored how acute elevations in peripheral blood glucose levels impacted hippocampal interstitial fluid (ISF) glucose, lactate, and A{beta} levels in APP/PS1 mice with or without KATP channels. Kir6.2+/+, APP/PS1 mice and Kir6.2-/-, APP/PS1 mice were exposed to a high sucrose diet for 6 months to determine the effects of chronic hyperglycemia on A{beta} deposition. We found that elevations in blood glucose levels correlate with increased ISF A{beta}, amyloidogenic processing of amyloid precursor protein (APP), and amyloid plaque pathology in APP/PS mice with intact KATP channels. However, neither acute hyperglycemia nor chronic sucrose overconsumption raised ISF A{beta} or increased A{beta} plaque burden in APP/PS1 mice lacking Kir6.2-KATP channel activity. Mechanistic studies demonstrate ISF glucose not only correlates with ISF A{beta} but also ISF lactate. Without KATP channel activity, ISF lactate does not increase during hyperglycemia, which correlates with decreased monocarboxylate transporter 4 (MCT4) expression, a lactate transporter responsible for astrocytic lactate release. This suggests that KATP channel activity regulates ISF lactate during hyperglycemia, which is important for A{beta} release and aggregation. These studies identify a new role for Kir6.2-KATP channels in Alzheimers disease pathology and suggest that pharmacological antagonism of Kir6.2-KATP channels holds therapeutic promise in reducing A{beta} pathology, especially in diabetic and prediabetic patients.

category: neuroscience


title: Synaptic Location Is a Determinant of the Detrimental Effects of α-Synuclein Pathology to Glutamatergic Transmission in the Basolateral Amygdala

abstract: -Synuclein (Syn) is a soluble protein enriched at presynaptic boutons and its abnormal aggregation is a hallmark of Parkinsons disease (PD). The amygdala shows selective vulnerability to the formation of insoluble Syn aggregates, and its circuit dysfunction may contribute to prevalent psychiatric deficits in PD. Yet, how Syn aggregates affect the amygdala function remains elusive. In the present study, we examined the presence of Syn in glutamatergic axon terminals and how its aggregation affects glutamatergic transmission in the basolateral amygdala (BLA). Using confocal microscopy, we showed that Syn is primarily present in the axon terminals expressing vesicular glutamate transporter 1 (vGluT1), but not in those expressing vGluT2. Using electrophysiology and optogenetics, we found that glutamatergic neurotransmission from vGluT1+ axon terminals are functionally more vulnerable than vGluT2+ axon terminals to the toxic effects of pathological Syn aggregation triggered by Syn preformed fibrils (PFFs). Using the PFFs models and mouse genetics, we showed that loss of Syn promotes short-term depression of cortico-BLA synapses responding to repetitive stimuli, leading to an input-selective filtering of cortical inputs to the BLA. Together, we demonstrate that Syn expresses specifically in a subset of synapses in mouse amygdala, which causes an input-specific decrease of cortical inputs to the BLA as Syn aggregation forms. These results might be relevant to the reduced cortical control of amygdala function that has been associated with psychiatric deficits in PD.


title: Urocortin-3 neurons in the perifornical area mediate the impact of chronic stress on female infant-directed behavior

abstract:
Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons but does not affect maternal behavior in mothers. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

Significance statementWhile a large body of research has studied the impact of maternal stress on offspring, few studies have focused on the neural circuitry underlying reduced maternal behavior in stressed mothers. In this study, we examine the neural substrates involved in reduced infant-directed behavior caused by chronic stress. We find that perifornical area neurons expressing the neuropeptide urocortin-3 are critical mediators of the impact of stress on infant-directed behavior in females.


title: Combined single-cell RNA-seq profiling and enhancer editing reveals critical spatiotemporal controls over thalamic nuclei formation in the murine embryo

abstract: The thalamus is the principal information hub of the vertebrate brain, with essential roles in sensory and motor information processing, attention, and memory. The molecular mechanisms regulating the formation of thalamic nuclei are unclear. We apply longitudinal single-cell RNA-sequencing, regional abrogation of Sonic-hedgehog (Shh), and spatial profiling of gene expression to map the developmental trajectories of thalamic progenitors, intermediate progenitors, and post-mitotic neurons as they coalesce into distinct thalamic nuclei. These data reveal that the complex architecture of the thalamus is established early during embryonic brain development through the coordinated action of four cell differentiation lineages derived from Shh-dependent and independent progenitors. We systematically characterize the gene expression programs that define these lineages across time and demonstrate how their disruption upon Shh depletion causes pronounced locomotor impairment resembling infantile Parkinsons disease. These results reveal key principles of thalamic development and provide mechanistic insights into neurodevelopmental disorders resulting from thalamic dysfunction.


title: Coupling of metataxonomics and culturing improves description of bacterial communities from a multi-contaminated waste sediment and identifies a novel Rhizorhapis sp. with metal resistance potential

abstract: Long-term contaminated environments have been recognized as potential hotspots for bacterial discovery in taxonomic and functional terms for bioremediation purposes. Here, bacterial communities from waste sediment collected from a former industrial dumpsite and contaminated with petroleum hydrocarbon and heavy metals were investigated through the parallel application of culture-independent (16S rRNA gene amplicon sequencing) and -dependent (plate culturing followed by colony picking and isolates identification by 16S Sanger sequencing) approaches. The bacterial diversities retrieved by both approaches greatly differed. Bacteroidetes and Proteobacteria were dominant in the culture-independent community, while Firmicutes and Actinobacteria were the main culturable groups. Only 2.7% of OTUs (operational taxonomic units) in the culture-independent dataset were cultured. Most of the culturable OTUs were absent or in very low abundances in the culture-independent dataset, revealing that cultivation is a useful tool to study the rare bacterial biosphere. One culturable OTUs (comprising only the isolate SPR117) was identified as a potential new species in the genus Rhizorhapis (class Alphaproteobacteria) and was selected for further characterization. Phytopathogenicity tests showed that Rhizorhapis sp. strain SPR117 (ATCC TSD-228) is not pathogenic for lettuce, despite the only described species within this genus, Rhizorhapis suberifaciens, is causal agent of the lettuce corky root disease. The genome of the strain SPR117 was sequenced, assembled in 256 contigs with a length of 4,419,522 bp and a GC content of 59.9% and its further annotation revealed the presence of genes related to the resistance to arsenic, copper and iron, among other metals. Therefore, the coupling of metataxonomic and culturing is a useful tool to obtain not only an improved description of bacterial communities in contaminated environments, but also to isolate microorganisms with bioremediation potential.

category: microbiology


title: High retention of genomic variation and fitness-related traits in the effective population of reintroduced wolves in Yellowstone National Park

abstract: Cooperatively breeding species exhibit numerous strategies to avoid mating with close relatives, inherently reducing effective population size. For species of management concern, accurate estimates of inbreeding and trait depression are crucial for the species future. We utilized genomic and pedigree data for Yellowstone National Park gray wolves to investigate the contributions of foundation stock lineages, genetic architecture of the effective population, and putative fitness consequences of inbreeding. Our dataset spans 25 years and seven generations since reintroduction, encompassing 152 nuclear families and 329 litters. We found over 87% of the pedigree foundation genomes persisted and report influxes of allelic diversity from two translocated wolves from a divergent source in Montana. As expected for group-living species, mean kinship significantly increased over time, although we found high retention of genetic variation. Strikingly, the effective population carried a significantly lower level of genome-wide inbreeding coefficients and autozygosity with shorter decays for linkage disequilibrium relative to the non-breeding population. Lifespan and heterozygosity were higher in the effective population, although individuals who had their first litter at an older age also had higher inbreeding coefficients. Our findings highlight genetic contributions to fitness, and the importance of effective population size and gene flow to counteract loss of genetic variation in a wild, free-ranging social carnivore. It is crucial for managers to mitigate factors that significantly reduce effective population size and genetic connectivity, which supports the dispersion of genetic variation that aids in rapid evolutionary responses to environmental challenges.


title: Weighting by Gene Tree Uncertainty Improves Accuracy of Quartet-based Species Trees

abstract: Phylogenomic analyses routinely estimate species trees using methods that account for gene tree discordance. However, the most scalable species tree inference methods, which summarize independently inferred gene trees to obtain a species tree, are sensitive to hard-to-avoid errors introduced in the gene tree estimation step. This dilemma has created much debate on the merits of concatenation versus summary methods and practical obstacles to using summary methods more widely and to the exclusion of concatenation. The most successful attempt at making summary methods resilient to noisy gene trees has been contracting low support branches from the gene trees. Unfortunately, this approach requires arbitrary thresholds and poses new challenges. Here, we introduce threshold-free weighting schemes for the quartet-based species tree inference, the metric used in the popular method ASTRAL. By reducing the impact of quartets with low support or long terminal branches (or both), weighting provides stronger theoretical guarantees and better empirical performance than vanilla ASTRAL. Significantly, weighting by support makes ASTRAL a statistically consistent estimator with estimated (not true) gene trees as input, assuming support values satisfy certain conditions. More consequentially, weighting dramatically improves accuracy in a wide range of simulations and reduces the gap with concatenation in conditions with low gene tree discordance and high noise. On empirical data, weighting improves congruence with concatenation and increases support. Together, our results show that weighting, enabled by a new optimization algorithm we introduce, dramatically improves the utility of summary methods and can reduce the incongruence often observed across analytical pipelines.


title: Genome assembly and isoform analysis of a highly heterozygous New Zealand fisheries species, the tarakihi (Nemadactylus macropterus)

abstract: Although being some of the most valuable and heavily exploited wild organisms, few fisheries species have been studied at the whole-genome level. This is especially the case in New Zealand, where genomics resources are urgently needed to assist fisheries management attains its sustainability goals. Here we generated 55 Gb of short Illumina reads (92x coverage) and 73 Gb of long Nanopore reads (122x) to produce the first genome assembly of the marine teleost tarakihi (Nemadactylus macropterus), a highly valuable fisheries species in New Zealand. An additional 300 Mb of Iso-Seq RNA reads were obtained from four tissue types of another specimen to assist in gene annotation. The final genome assembly was 568 Mb long and consisted of 1,214 scaffolds with an N50 of 3.37 Mb. The genome completeness was high, with 97.8% of complete Actinopterygii BUSCOs. Heterozygosity values estimated through k-mer counting (1.00%) and bi-allelic SNPs (0.64%) were high compared to the same values reported for other fishes. Repetitive elements covered 30.45% of the genome and 20,169 protein-coding genes were annotated. Iso-Seq analysis recovered 91,313 unique transcripts (isoforms) from 15,515 genes (mean ratio of 5.89 transcripts per gene), and the most common alternative splicing event was intron retention. This highly contiguous genome assembly along with the isoform-resolved transcriptome will provide a useful resource to assist the study of population genomics, as well as comparative eco-evolutionary studies in other teleost and related organisms.


title: Vasor: Accurate prediction of variant effects for amino acid substitutions in MDR3

abstract:
Background / RationaleThe phosphatidylcholine floppase MDR3 is an essential hepatobiliary transport protein. MDR3 dysfunction is associated with various liver diseases, ranging from severe progressive familial intrahepatic cholestasis to transient forms of intrahepatic cholestasis of pregnancy and familial gallstone disease. Single amino acid substitutions are often found as causative of dysfunction, but identifying the substitution effect in in vitro studies is time- and cost-intensive.

Main resultsWe developed Vasor (Variant assessor of MDR3), a machine learning-based model to classify novel MDR3 missense variants into the categories benign or pathogenic. Vasor was trained on the, to date, largest dataset specific for MDR3 of benign and pathogenic variants and uses general predictors, namely EVE, EVmutation, PolyPhen-2, I-Mutant2.0, MUpro, MAESTRO, PON-P2, and other variant properties such as half-sphere exposure, PTM site, and secondary structure disruption as input. Vasor consistently outperformed the integrated general predictors and the external prediction tool MutPred2, leading to the current best prediction performance for MDR3 single-site missense variants (on an external test set: F1-score: 0.90, MCC: 0.80). Furthermore, Vasor predictions cover the entire sequence space of MDR3. Vasor is accessible as a webserver at https://cpclab.uni-duesseldorf.de/mdr3_predictor/ for users to rapidly obtain prediction results and a visualization of the substitution site within the MDR3 structure.

ConclusionThe MDR3-specific prediction tool Vasor can provide reliable predictions of single site amino acid substitutions, giving users a fast way to assess initially whether a variant is benign or pathogenic.


title: A conditional survival distribution-based method for censored data imputation: overcoming the hurdle in machine learning-based survival analysis

abstract: Data analyses by machine learning (ML) algorithms are gaining popularity in biomedical research. When time-to-event data are of interest, censoring is common and needs to be properly addressed. Most ML methods cannot conveniently and appropriately take the censoring information into consideration, potentially leading to inaccurate or biased results. We aim to develop a general-purpose method for imputing censored survival data, facilitating downstream ML analysis. In this study, we propose a novel method of imputing the survival times for censored observations. The proposal is based on their conditional survival distributions (CondiS) derived from Kaplan-Meier estimators. CondiS can replace censored observations with their best approximations from the statistical model, allowing for direct application of ML methods. When covariates are available, we extend CondiS by incorporating the covariate information through ML modeling (CondiS-X), which further improves the accuracy of the imputed survival time. Compared with existing methods with similar purposes, the proposed methods achieved smaller prediction errors and higher concordance with the underlying true survival times in extensive simulation studies. We also demonstrated the usage and advantages of the proposed methods through two real-world cancer datasets. The major advantage of CondiS is that it allows for the direct application of standard ML techniques for analysis once the censored survival times are imputed. We present a user-friendly R package to implement our method, which is a useful tool for ML-based biomedical research in this era of big data.


title: Active muscular hydraulics

abstract: What limits the rate of contraction in muscle? The molecular cycling of actomyosin crossbridges has typically been assumed to be rate limiting, but muscle is a soft, wet and active tissue that is spatially heterogeneous and heirarchically organized across multiple scales. We propose a minimal multiscale model that describes muscle as an active sponge and show how contractions generically induce intracellular fluid flow and power active hydraulic oscillations which dictate the fastest rate of muscle contraction. A key feature is the presence of spatially heterogeneous strains and local volumetric deformations, which we validate by reanalyzing data from existing experiments across different species and muscle types. We further demonstrate that the viscoelastic response of muscle is naturally nonreciprocal (or  odd) due to its active and anisotropic nature and highlight a novel consequence, whereby periodic cycles in strain alone can produce work. Our work suggests a revised view of muscle dynamics that emphasizes the multiscale spatial aspects in addition to temporal aspects, with implications for physiology, biomechanics and locomotion.


title: Cardiac myosin filaments are directly regulated by calcium

abstract: Contraction of cardiac and skeletal muscle is initiated by calcium (Ca2+) binding to regulatory proteins on actin-containing thin filaments. During muscle contraction, structural changes in Ca2+-dependent thin filament allow the binding of myosin motors to drive muscle contraction1. The dynamic switching between the resting off states and the active on states of myosin is also critical in regulating muscle contractility2-4. However, the molecular switch on the myosin-containing thick filament that drives this process is not understood. Here we show that cardiac thick filaments are directly Ca2+-regulated. We find that Ca2+ progressively moves the myosin heads from ordered off states close to the thick filament backbone to disordered on states closer to the thin filaments. This Ca2+-dependent structural shift of myosin is accompanied by a biochemically defined transition from the inactive super-relaxed state(s) to the active disordered relaxed state(s)3. Furthermore, we find that this Ca2+-mediated molecular switching is an intrinsic property of cardiac myosin but only when assembled into thick filaments. This novel concept of Ca2+ as a regulatory modulator of the thick filament provides a fresh perspective on cardiac muscle regulation, which may be particularly valuable for devising restorative treatments for pathologies altering the Ca2+ sensitivity of the sarcomere.


title: Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

abstract: The biological processes underlying ischemic stroke, although complex, are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1h of OGD and, with longer OGD duration and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.


title: Oral ingestion of the environmental toxicant trichlorethylene in rats induces alterations in the gut microbiome: relevance to idiopathic Parkinson's disease

abstract:
Microbial alterations within the gut microbiome appear to be a common feature of individuals with Parkinsons disease (PD), providing further evidence for the role of the gut-brain axis in PD development. As a major site of contact with the environment, questions have emerged surrounding the cause and effect of alterations to the gut microbiome by environmental contaminants associated with PD risk, such as pesticides, metals, and organic solvents. Recent data from our lab shows that ingestion of the industrial byproduct and environmental pollutant trichloroethylene (TCE) induces key Parkinsonian pathology within aged rats, including the degeneration of dopaminergic neurons, -synuclein accumulation, neuroinflammation, and endolysosomal deficits. As TCE is the most common organic contaminant within drinking water, we postulated that ingestion of TCE associated with PD-related neurodegeneration may alter the gut microbiome to a similar extent as observed in persons with PD. To assess this, we collected fecal samples from adult rats treated with 200 mg/kg TCE over 6 weeks via oral gavage and analyzed the gut microbiome via whole genome shotgun sequencing. Our results showed changes in gut microorganisms reflective of the microbial signatures observed in individuals with idiopathic PD, such as decreased abundance of short-chain fatty acid producing Blautia and elevated lactic-acid producing Bifidobacteria, as well as genera who contain species previously reported as opportunistic pathogens such as Clostridium. From these experimental data, we postulate that TCE exposure within contaminated drinking water could induce alterations of the gut microbiome that contributes to chronic disease risk, including idiopathic PD.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: A conditional null allele of Dync1h1 enables targeted analyses of dynein roles in neuronal length sensing and neurological disorders

abstract: Size homeostasis is one of the most fundamental aspects of biology and it is particularly important for large cells as neurons. We have previously proposed a motor-dependent length-sensing and growth-regulating mechanism wherein a partial reduction in the levels of microtubule motor proteins should lead to accelerated neuronal growth. This prediction was originally validated in sensory neurons heterozygous for the Loa point mutation in dynein heavy chain 1 (Dync1h1Loa). Here we describe a new mouse model with a conditional allele allowing deletion of exons 24-25 in Dync1h1. Homozygous Islet1-Cre deletion of Dync1h1 is embryonic lethal, but heterozygous animals (Isl1-Dync1h1+/-) survive to adulthood with approximately 50% dynein expression in targeted cell types. Isl1-Dync1h1+/- adult sensory neurons reveal an accelerated growth phenotype, similar to that previously reported in Dync1h1Loa neurons. Moreover, Isl1-Dync1h1+/- mice show mild impairments in gait, proprioception and tactile sensation; and slightly impaired recovery from peripheral nerve injury. Thus, conditional deletion of Dync1h1 exons 24-25 enables targeted studies of the role of dynein in neuronal growth and neurological disorders.


title: Latitudinal cline does not predict variation in the microbiome of wild Drosophila melanogaster

abstract: Microbiomes affect many aspects of host biology, but the eco-evolutionary forces that shape their diversity in natural populations remain poorly understood. Geographic gradients, like latitudinal clines, generate predictable patterns in biodiversity at macroecological scales, but whether these macro-scale processes apply to host-microbiome interactions is an open question. To address this question, we sampled the microbiomes of 13 natural populations of Drosophila melanogaster along a latitudinal cline in eastern United States. The microbiomes were surprisingly consistent across the cline-latitude did not predict either alpha or beta diversity. Only a narrow taxonomic range of bacteria were present in all microbiomes, indicating that strict taxonomic filtering by the host and neutral ecological dynamics are the primary factors shaping the fly microbiome. Additional temporal sampling across two independent sites revealed significant differences in microbial communities over time, suggesting that local environmental differences that vary at fine spatiotemporal scales are more likely to shape the microbiome. Our findings reveal the complexity of eco-evolutionary interactions shaping microbial variation in D. melanogaster and highlight the need for additional sampling of the microbiomes in natural populations along environmental gradients.


title: Statistical curve models for inferring 3D chromatin architecture

abstract: Reconstructing three dimensional (3D) chromatin structure from conformation capture assays (such as Hi-C) is a critical task in computational biology, since chromatin spatial architecture plays a vital role in numerous cellular processes and direct imaging is challenging. We previously introduced Poisson metric scaling (PoisMS), a technique that models chromatin by a smooth curve, which yielded promising results. In this paper, we advance several ways for improving PoisMS. In particular, we address initialization issues by using a smoothing spline basis. The resulting SPoisMS method produces a sequence of reconstructions re-using previous solutions as warm starts. Importantly, this approach permits smoothing degree to be determined via cross-validation which was problematic using our prior B-spline basis. In addition, motivated by the sparsity of Hi-C contact data, especially when obtained from single-cell assays, we appreciably extend the class of distributions used to model contact counts. We build a general distribution-based metric scaling (DBMS) framework, from which we develop zero-inflated and Hurdle Poisson models as well as negative binomial applications. Illustrative applications make recourse to bulk Hi-C data from IMR90 cells and single-cell Hi-C data from mouse embryonic stem cells.


title: Functional Connections Among Neurons within Single Columns of Macaque V1

abstract: Recent developments in high-density neurophysiological tools now make it possible to record from hundreds of single neurons within local, highly interconnected neural networks. Among the many advantages of such recordings is that they dramatically increase the quantity of identifiable, functional connections between neurons thereby providing an unprecedented view of local circuit interactions. Using high-density, Neuropixels recordings from single neocortical columns of primary visual cortex in nonhuman primates, we identified 1000s of functionally connected neuronal pairs using established crosscorrelation approaches. Our results reveal clear and systematic variations in the strength and synchrony of functional connections across the cortical column. Despite neurons residing within the same column, both measures of functional connectivity depended heavily on the vertical distance separating neuronal pairs, as well as on the similarity of stimulus tuning. In addition, we leveraged the statistical power afforded by the large numbers of connected pairs to categorize functional connections between neurons based on their crosscorrelation functions. These analyses identified distinct, putative classes of functional connections within the full population. These classes of functional connections were corroborated by their unique distributions across defined laminar compartments and were consistent with known properties of V1 cortical circuitry, such as the lead-lag relationship between simple and complex cells. Our results provide a clear proof-of-principle for the use of high-density neurophysiological recordings to assess circuit-level interactions within local neuronal networks.


title: Spontaneous behavioural recovery following stroke relates to the integrity of sensory and association cortices

abstract: Stroke is a devastating disease that results in neurological deficits and represents a leading cause of death and disability worldwide. Following a stroke, there is a degree of spontaneous recovery of function, the neural basis of which is of great interest among clinicians in their efforts to reduce disability following stroke and enhance rehabilitation. Conventionally, work on spontaneous recovery has tended to focus on the neural reorganization of motor cortical regions, with comparably little attention being paid to changes in non-motor regions and how these relate to recovery. Here we show, using structural neuroimaging in a macaque stroke model and by exploiting individual differences in spontaneous behavioural recovery, that the preservation of regions in the sensory and association cortices predict animal recovery. To characterize recovery, we performed a clustering analysis using Non-Human Primate Stroke Scale (NHPSS) scores and identified a good versus poor recovery group. By comparing the preservation of brain volumes in the two groups, we found that brain areas in integrity of brain areas in parietal, temporal and somatosensory cortex were associated with better recovery. In addition, a decoding approach performed across all subjects revealed that the preservation of specific brain regions in the parietal, somatosensory and medial frontal cortex predicted recovery. Together, these findings highlight the importance of the sensory and association regions in spontaneous behavioural recovery, supporting the notion that the  sensory retraining of impairments following stroke is critical to recovery and motor function.


title: Enabling interpretable machine learning for biological data with reliability scores

abstract: Machine learning has become an important tool across biological disciplines, allowing researchers to draw conclusions from large datasets, and opening up new opportunities for interpreting complex and heterogeneous biological data. Alongside the rapid growth of machine learning, there have also been growing pains: some models that appear to perform well have later been revealed to rely on features of the data that are artifactual or biased; this feeds into the general criticism that machine learning models are designed to optimize model performance over the creation of new biological insights. A natural question thus arises: how do we develop machine learning models that are inherently interpretable or explainable? In this manuscript, we describe reliability scores, a new concept for scientific machine learning studies that assesses the ability of a classifier to produce a reliable classification for a given instance. We develop a specific implementation of a reliability score, based on our work in Sugden et al. 2018 in which we introduced SWIF(r), a generative classifier for detecting selection in genomic data. We call our implementation the SWIF(r) Reliability Score (SRS), and demonstrate the utility of the SRS when faced with common challenges in machine learning including: 1) an unknown class present in testing data that was not present in training data, 2) systemic mismatch between training and testing data, and 3) instances of testing data that are missing values for some attributes. We explore these applications of the SRS using a range of biological datasets, from agricultural data on seed morphology, to 22 quantitative traits in the UK Biobank, and population genetic simulations and 1000 Genomes Project data. With each of these examples, we demonstrate how interpretability tools for machine learning like the SRS can allow researchers to interrogate their data thoroughly, and to pair their domain-specific knowledge with powerful machine-learning frameworks. We hope that this tool, and the surrounding discussion, will aid researchers in the biological machine learning space as they seek to harness the power of machine learning without sacrificing rigor and biological understanding.


title: On the Mental Status of Stargazer Mice

abstract: In many childhood-onset genetic epilepsies, seizures are accompanied by neurobehavioral impairments and motor disability. In the Stargazer mutant mouse, genetic disruptions of Cacng2 result in absence-like spike-wave seizures, cerebellar gait ataxia and vestibular dysfunction. Here, we combine videotracking and instrumented home-cage monitoring to resolve the neurobehavioral facets of the murine Stargazer syndrome. We find that despite their gait ataxia, stargazer mutants display horizontal hyperactivity and variable rates of repetitive circling behavior. While feeding rhythms, circadian or ultradian oscillations in activity are unchanged, mutants exhibit fragmented sleep bouts, atypical licking dynamics and lowered sucrose preference. Mutants also display an attenuated response to visual and auditory home-cage perturbations, together with profound reductions in voluntary wheel-running. Our results reveal that the seizures and ataxia of Stargazer mutants occur in the context of a more pervasive behavioral syndrome with elements of encephalopathy, repetitive behavior and anhedonia. These findings expand our understanding of the function of Cacng2/CACNG2, variants in which have been identified in patients with bipolar disorder and schizophrenia.


title: Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections

abstract: Bacillus Calmette-Guerin (BCG) is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces trained immunity. Here, we found that intravesical BCG does induce trained immunity based on an increased production of TNF and IL-1{beta} after heterologous ex-vivo stimulation of circulating monocytes 6- 12 weeks after intravesical BCG treatment; and a 37% decreased risk (OR 0.63 (95% CI 0.40- 1.01)) for respiratory infections in BCG-treated versus non-BCG-treated NMIBC patients. An epigenomics approach combining ChIP-sequencing and RNA-sequencing with in-vitro trained immunity experiments identified enhanced inflammasome activity in BCG-treated individuals. Finally, germline variation in genes that affect trained immunity was associated with recurrence and progression after BCG therapy in NMIBC, suggesting a link between trained immunity and oncological outcome.

category: immunology


title: Shifting a Cellular Metabolic Landscape Identifies a Refractory Environment for Flavivirus Replication

abstract:
Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect.

Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.


title: Microbial electrochemical systems for enhanced degradation of glyphosate: Electrochemical performance, degradation efficiency, and analysis of the anodic microbial community

abstract: Glyphosate, one of the most used herbicides worldwide, is known as an aquatic contaminant of concern, and can present adverse impacts in agroecosystems. In this study, we investigated the degradation of glyphosate in microbial electrochemical systems (MESs), and analysed the microbial composition of enriched anodic biofilms, and comparing them with microbial communities of non-MESs enriched cultures. MESs supported higher glyphosate degradation (68.41 {+/-} 1.21 % to 73.90 {+/-} 0.79 %) compared to non-MESs cultures (48.88 {+/-} 0.51 %). The Linear Sweep Voltammetry (LSV) analysis showed that MESs operated at +300 mV, produced a maximum current of 611.95 A, which was the highest among all the applied voltages. 16S amplicon sequencing revealed a significant difference in microbial community composition of MESs anodic biofilms and non-MESs enriched communities. The anodic biofilms were dominated by Rhodococcus (51.26 %), Pseudomonas (10.77 %), and Geobacter (8.67 %) while in non-MESs cultures, methanogens including Methanobrevibacter (51.18 %), and Methanobacterium (10.32 %), were the dominant genera. The present study suggested that MESs could be considered as a promising system for glyphosate degradation.


title: Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of IP6K1 and repression of myo-inositol-3-P synthase

abstract: Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts (MEFs) demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. The current study was carried out to elucidate the role of PA in IP6K1 repression. The results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of MIPS protein. This effect was specific to PA synthesized in the plasma membrane, as ER-derived PA did not induce IP6K1 translocation. PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5 AMP-activated protein kinase (AMPK). Activation of AMPK by glucose deprivation or by treatment with the mood stabilizing drugs valproate (VPA) or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.

category: molecular biology