title: Harnessing cellular perception apparatus for smart metabolic reprogramming

abstract: Metabolic reprogramming (MRP) is a fundamental approach in synthetic biology that involves redirecting metabolic flux and remodeling metabolic networks. However, only few approaches have been made in effective metabolic operations, especially at global level of metabolic networks. Naturally existing cellular perception apparatuses (CPAs), such as histidine kinases (HKs), are considered to sit on sensitive nodes of the metabolic network, which can trigger natural MRP upon perceiving environmental fluctuations. We develop a plateform for global MRP by natural environmental stimulation based on the combinational interference of CPAs. The plateform consists of a CRISPRi-mediated dual-gene combinational knockdown (CDCK) strategy and survivorship-based metabolic interaction analysis (SMIA). A total of 35 histidine kinase (HK) genes and 24 glycine metabolism genes were selected as targets to determine effectiveness of our approach for fast-growing chassis Vibrio FA2. Combined knockdown of several genes of HKs and glycine metabolism increased the glycine production. Other other hand, effects of CDCK on bacterial antibiotic resistance were assessed by targeting HKs. Many HKs were identified to be associated with antibiotic resistance in Vibrio FA2, of which combinational knockdown of two HK genes sasA_8 and 04288 reduced the ampicillin resistance. This MRP strategy is powerful and cost-effective, and can be considered as a smart strategy capable of operating a broad range of metabolic networks in microorganisms.


title: VMHdm/cSF-1 Neuronal Circuits Regulate Skeletal Muscle PGC1-α via the Sympathoadrenal Drive

abstract: To adapt to metabolically challenging environments, the central nervous system (CNS) orchestrates metabolism of peripheral organs including skeletal muscle. The organ-communication between the CNS and skeletal muscle has been investigated, yet our understanding of the neuronal pathway from the CNS to skeletal muscle is still limited. Neurons in the dorsomedial and central parts of the ventromedial hypothalamic nucleus (VMHdm/c) expressing steroidogenic factor-1 (VMHdm/cSF-1 neurons) are key for metabolic adaptations to exercise, including increased basal metabolic rate and skeletal muscle mass in mice. However, the mechanisms by which VMHdm/cSF-1 neurons regulate skeletal muscle function remain unclear. Here, we show that VMHdm/cSF-1 neurons increase the sympathoadrenal activity and regulate skeletal muscle peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1) in mice via multiple downstream nodes. Optogenetic activation of VMHdm/cSF-1 neurons dramatically elevates mRNA levels of skeletal muscle Pgc-1, which regulates a spectrum of skeletal muscle function including protein synthesis and metabolism. Mechanistically, the sympathoadrenal drive coupled with {beta}2 adrenergic receptor ({beta}2AdR) is essential for VMHdm/cSF-1 neurons-mediated increases in skeletal muscle PGC1-. Specifically, adrenalectomy and knockout of {beta}2AdR block augmented skeletal muscle PGC1- by VMHdm/cSF-1 neuronal activation. Optogenetic functional mapping reveals that downstream nodes of VMHdm/cSF-1 neurons are functionally redundant to increase circulating epinephrine and skeletal muscle PGC1-. Collectively, we propose that VMHdm/cSF-1 neurons-skeletal muscle pathway, VMHdm/cSF-1 neurons[->]multiple downstream nodes[->]the adrenal gland[->]skeletal muscle {beta}2AdR, underlies augmented skeletal muscle function for metabolic adaptations.

category: neuroscience


title: M2e-specific monoclonal antibody cocktails against influenza A virus are superior to individual monoclonal antibody treatments, universally effective, and viral escape mutant resistant

abstract: Influenza virus has pandemic potential, seasonal epidemics burden the human population, and viral resistance has developed to all available treatment options. A universally effective, escape mutant resistant therapeutic agent is desperately needed. We previously described extensive analyses of seven Matrix Protein 2 ectodomain-specific monoclonal antibodies (M2e-mAbs) as the basis for an effective and universal treatment for influenza A virus (IAV) infection. On the strength of these findings, we developed an effective prophylactic cocktail agent using three M2e-mAbs distinct in their M2e epitopes. This cocktail protected mice challenged with laboratory and pandemic IAV strains at a low dose significantly better than single M2e-mAb treatments. Notably, no viral escape mutants developed in immunocompetent and immunodeficient mice after viral passaging in the presence of single, cocktail, or alternating M2e-mAb treatments. Our study reveals the superiority of M2e-mAb cocktails, their universal effectiveness, and resistance to viral escape. It will critically shape future influenza-therapeutic development.

category: immunology


title: Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature upon SARS-CoV-2 sensing by monocytes in COVID-19

abstract: Alterations in the myeloid immune compartment have been observed in COVID-19, but the specific mechanisms underlying these impairments are not completely understood. Here we examined the functionality of classical CD14+ monocytes as a main myeloid cell component in well-defined cohorts of patients with mild and moderate COVID-19 during the acute phase of infection and compared them to that of healthy individuals. We found that ex vivo isolated CD14+ monocytes from mild and moderate COVID-19 patients display specific patterns of costimulatory and inhibitory receptors that clearly distinguish them from healthy monocytes, as well as altered expression of histone marks and a dysfunctional metabolic profile. Decreased NF{kappa}B activation in COVID-19 monocytes ex vivo is accompanied by an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness characterized by a defect in pro-inflammatory cytokine expression, NF{kappa}B-driven cytokine responses and defective type I IFN response in moderate COVID-19 monocytes. Transcriptionally, COVID-19 monocytes switched their gene expression signature from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in hemostasis and immunothrombosis. In response to SARS-CoV-2 or other viral or bacterial components, monocytes displayed defects in the epigenetic remodelling and metabolic reprogramming that usually occurs upon pathogen sensing in innate immune cells. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 may contribute to disease pathology.


title: CD169+ macrophages orchestrate plasmacytoid dendritic cell arrest and retention for optimal priming in the bone marrow of malaria-infected mice

abstract: Plasmacytoid dendritic cells (pDC) are the most potent producer of type I interferon (IFN), but how pDC are primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MP), pDC initiate type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7-sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional clustering with CD169+ MP in the BM. Lastly, we demonstrate that STING-sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and egress from the BM. Collectively, these results link pDC acquisition of type I IFN secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.


title: Dynamics of drug response in single mycobacterial cells by microfluidic dose-response assay

abstract: Preclinical analysis of drug efficacy is critical for drug development. However, conventional bulk-cell assays statically assess the mean population behavior, lacking resolution on drugescaping cells. Inaccurate estimation of efficacy can lead to overestimation of compounds, whose efficacy will not be confirmed in the clinic, or lead to rejection of valuable candidates. Time-lapse microfluidic microscopy is a powerful approach to characterize drugs at high spatiotemporal resolution, but hard to apply on a large scale. Here we report the development of a microfluidic platform based on a pneumatic operating principle, which is scalable and compatible with long-term live-cell imaging and with simultaneous analysis of different drug concentrations. We tested the platform with mycobacterial cells, including the tubercular pathogen, providing the first proof of concept of a single-cell dose-response assay. This dynamic in-vitro model will prove useful to probe the fate of drug-stressed cells, providing improved predictions of drug efficacy in the clinic.

category: microbiology


title: Melanization is an important antifungal defense mechanism in Galleria mellonella hosts

abstract: A key component of insect immunity is melanin encapsulation of microbes. Melanization is also a part of an immune process known as nodulation, which occurs when insect hemocytes surround microbes and produce melanin. Insect nodules are analogous to mammalian immune granulomas. Melanin is believed to kill microbes through the production of toxic intermediates and oxidative damage. However, it is unclear to what extent immune melanin is directly fungicidal during infections of insect hosts. We reported previously that C. neoformans cells are encapsulated with host-derived melanin within hemocyte nodules. Here we report an association between melanin-based immune responses by Galleria mellonella wax moth larvae and fungal cell death of C. neoformans during infection. To monitor melanization in situ, we applied a tissue-clearing technique to G. mellonella larvae, revealing that nodulation occurs throughout the organism. Further, we developed a protocol for time-lapse microscopy of extracted hemolymph following exposure to fungal cells, which allowed us to visualize and quantify the kinetics of the melanin-based immune response. Using this technique, we found evidence that cryptococcal melanins and laccase enhance immune melanization in hemolymph. We used these techniques to also study the fungal pathogen Candida albicans infections of G. mellonella. We find that the yeast form of C. albicans was the primary targets of host melanization, while filamentous structures were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive and break through the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in insect hosts.


title: Single-cell profiling of lncRNAs in human germ cells and molecular analysis reveals transcriptional regulation of LNC1845 on LHX8

abstract:
Non-coding RNAs exert diverse functions in many cell types. In addition to transcription factors from coding genes, non-coding RNAs may also play essential roles in shaping and directing the fate of germ cells. Here, we report the presence of many long non-coding RNAs (lncRNAs) which were specifically expressed in the germ cells during human gonadal development by single-cell profiling of the reported datasets, and the functional characterization of one divergent lncRNA, LNC1845. Comprehensive bioinformatic analysis of these lncRNAs indicates that divergent lncRNAs occupied the majority of female and male germ cells. Integrating lncRNA expression into the bioinformatic analysis also enhances the cell-type classification of female germ cells. Functional dissection using in vitro differentiation of human pluripotent stem cells to germ cells revealed the regulatory role of LNC1845 on a transcription factor essential for ovarian follicle development, LHX8, by modulating the levels of histone modifications, H3K4me3 and H3K27Ac. Hence, this study provides a comprehensive analysis of lncRNAs in developing germ cells and elucidates how a lncRNA function as a cis regulator during human germ cell development.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486705v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@31290forg.highwire.dtl.DTLVardef@17ec505org.highwire.dtl.DTLVardef@1b8cd0dorg.highwire.dtl.DTLVardef@19e4de3_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIlncRNAs are expressed at significantly higher levels in both male and female germ cells than in somatic cells during human gonadal development.
C_LIO_LIDivergent lncRNAs are enriched in the female meiotically and male mitotically arrested germ cells.
C_LIO_LILNC1845 regulates the transcriptional expression of LHX8 by modulating H3K4me3 and H3K27Ac levels.
C_LI

category: developmental biology


title: Asexual male production by ZW recombination in Artemia parthenogenetica

abstract: In some asexual species, parthenogenetic females occasionally produce males, which may strongly affect the evolution and maintenance of asexuality if they cross with related sexuals and transmit genes causing asexuality to their offspring ("contagious parthenogenesis"). How these males arise in the first place has remained enigmatic, especially in species with sex chromosomes. Here, we test the hypothesis that rare, asexually produced males of the crustacean Artemia parthenogenetica are produced by recombination between the Z and W sex chromosomes during non-clonal parthenogenesis, resulting in ZZ males through loss of heterozygosity at the sex determination locus. We used RAD-sequencing to compare asexual mothers with their male and female offspring. Markers on several sex-chromosome scaffolds indeed lost heterozygosity in all male but no female offspring, suggesting that they correspond to the sex-determining region. Other sex-chromosome scaffolds lost heterozygosity in only a part of the male offspring, consistent with recombination occurring at a variable location. Alternative hypotheses for the production of these males (such as partial or total hemizygosity of the Z) could be excluded. Rare males are thus produced because recombination is not entirely suppressed during parthenogenesis in A. parthenogenetica. This finding may contribute to explaining the maintenance of recombination in these asexuals.

category: evolutionary biology


title: Allele mining, evolutionary genetic analysis of TaHKT1;5 gene and evaluation of salinity stress in selected lines of wheat.

abstract: This study reports the novel allelic diversity in HKT 1;5 gene (High Affinity Potassium Transporter) in bread wheat and its phylogenetic relationship among the paralogs/orthologs of in Triticum aestivum and its wild relatives. HKT 1;5 gene is a known and pivotal gene associated with the salinity tolerance in plants upon the discrimination of K+ over Na+ in leaves without change in Na+ concentration in root. This gene was sequenced in a diverse collection of bread wheat, durum wheat, wild relatives, and ditelosomic lines. Sequence analysis in bread wheat led to the identification of four alleles, which could be distinguished by number of SNPs. Sequence comparison between monocot bread wheat and dicot Arabidopsis thaliana revealed that the HKT1;5 gene is conserved at level of exonic regions; however, the presence of transposable elements especially in intronic regions is further intriguing towards evolutionary relatedness. Two paralogous or major alleles observed in Triticum monoccum and Aegilops tauschii were further categorized as sub-alleles based on their SNPs comparison. This gene was absent in T. urartu in accordance with existing evidence, while it was found in A. speltoides (an allelic variant) with a few base pairs insertion in the exon1 region causing a frameshift mutation with an altered amino acids and genomic database mining unveiled additional alleles in this species. Ditelosomic lines with 4DL and 4DS chromosomes revealed a higher similarity with bread and durum wheat respectively. Phylogenetic studies of HKT1;5 orthologs from different Poaceae species revealed the occurrence of five different ortholog groups with taxonomic consistency. Phenotyping based salinity stress experiment distinguished the unknown lines for salinity tolerance and sensitiveness in comparison with known reference lines and possible allelic comparison was made. The salinity stress analysis further revealed that some known drought/heat tolerance lines showed slightly better salinity tolerance with mean values and variability of traits than known saline tolerant wheat line at controlled ambient.

category: genetics


title: The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi

abstract:
Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon, and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing in near-isogenic lines of cinerea was used to map a 60-kb genomic region for a vic locus. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

Author SummaryFungal colonies are characterised by radiating filaments, termed hyphae, which often fuse to form a highly interconnected individual. This is advantageous since it enables efficient water and nutrient utilisation across a colony network. However, hyphal fusion is not necessarily restricted to within an individual colony, with potential for hyphal fusion between individuals belonging to the same species. There are, however, drawbacks to this. For instance, viruses that detrimentally affect a colony may be transmitted, with their infection leading to a reduction in the virulence of a pathogenic species. Fungi have therefore developed complex systems to prevent fusion between genetically distinct individuals of the same species. This phenomenon is termed vegetative incompatibility and results in the death of fused cells and cessation of transfer of cellular contents from one individual to another. We have identified the first genes in the fungal plant pathogen Botrytis cinerea that control this phenomenon. They resemble genes that control vegetative incompatibility in other fungi, and genes involved in immunity in plants and animals. Uncovering further genes involved in vegetative incompatibility in B. cinerea may pave the way for the development of a  super donor strain capable of overriding vegetative incompatibility to transmit viruses, thus enabling their exploitation as potent control agents against this damaging plant pathogen.


title: Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain

abstract: Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of [~]1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including five that had not been previously associated with maize grain tocochromanols: four biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for co-localized eQTL and GWAS association signals. Our multi-omics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.


title: Loss-of-function mutations in novel triacylglycerol lipase genes are associated with low rancidity in pearl millet flour

abstract: Commercialization and utilization of pearl millet (Pennisetum glaucum L.) by consumers and processing industry is constrained due to rapid onset of rancidity in its milled flour. We studied the underlying biochemical and molecular mechanisms to flour rancidity in contrasting inbreds under 21-day accelerated storage. Rapid TAG decrease was accompanied by FFA increase in high rancidity genotype compared to the low rancidity line, that maintained lower FFA and high TAG levels, besides lower headspace aldehydes. DNA sequence polymorphisms observed in two lipase genes revealed loss-of-function mutations that were functionally confirmed in yeast system. We outline a direct mechanism for mutations in these key TAG lipases in pearl millet and the protection of TAG and fatty acids from hydrolytic and oxidative rancidity respectively. Natural variation in the PgTAGLip1 and PgTAGLip2 genes may be selected through marker assisted breeding or by precision genetics methods to develop hybrids with improved flour shelf life.

category: biochemistry


title: Protein-Peptide Turnover Profiling reveals wiring of phosphorylation during protein maturation

abstract: Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods that rely on pulsed metabolic labeling are very popular to quantify turnover rates on a proteome-wide scale. Such data have often been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a proteins lifetime--a fundamentally different metric. We use this framework to identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results open up an entirely new aspect in the study of PTMs, by tying them into the context of a proteins lifetime.


title: Evolutionary adaptation of the folding pathway for secretability

abstract: Secretory preproteins of the Sec pathway bear signal peptides and are targeted post-translationally to cross the plasma membrane or ER through translocases. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or after further trafficking. During cytoplasmic transit, mature domains must remain non-folded for translocase recognition and translocation. Here, we sought the structural basis for the delayed folding mechanism of mature domains and how this is regulated by signal peptides. To address this, we compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural twin cytoplasmic PpiB. Using global and local hydrogen deuterium exchange mass spectrometry we showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, that displayed different order and rates. Folding is delayed in PpiA by less hydrophobic/bulky native contacts, frustrated residues and a critical {beta} -turn in the early folding region and by signal peptide-driven disorder, which disrupts foldon hierarchy. When selected PpiA residues and its signal peptide were grafted onto PpiB they converted it into a slow folder with enhanced in vivo secretion. These data reveal the structural basis of non-folding in a secretory protein, that allows its trafficking.


title: Near-neutral pH increased n-caprylate production in a microbiome with product inhibition of methanogenesis

abstract: The pH is a critical parameter in chain-elongating bioreactors, affecting: (1) the concentration of inhibitory undissociated carboxylic acids, which in turn affects the efficiency of product extraction; (2) the thermodynamics; and (3) the kinetics. Here, we examined the effect of five different pH levels (5.5 to 7.0) on n-caprylate (C8) production using an anaerobic sequencing batch reactor (ASBR) with continuous membrane-based liquid-liquid extraction (pertraction). We found that the product spectrum was directed by pH: mildly acidic pH (5-6) led to n-caproate (C6) production, while near-neutral and neutral pH (6.75-7) favored n-caprylate production. In particular, the pH of 6.75 led to the maximum values of volumetric n-caprylate production rate (75.6 {+/-} 0.6 mmol C L-1 d-1; 0.06 g L-1 d-1) and n-caprylate concentration in the fermentation broth (420 mM C; 7.57 g L-1). Given that methane production remained low at near-neutral and neutral pH, we theorized that the high concentration of undissociated n-caprylic acid (5.71 mM C) inhibited methanogenesis. We then demonstrated such an inhibitory effect at neutral pH in: (1) microcosm experiments; and (2) the continuous bioreactor by adding methanogenic sludge. Furthermore, 16S rRNA gene sequencing analysis revealed that near-neutral and neutral pH led to more diverse microbial communities than at mildly-acidic pH. For the first time, we report predominant n-caprylate production in a microbiome at near-neutral and neutral pH conditions where methanogenesis was controlled by the inhibitory effects of undissociated n-caprylic acid. At the same time, extraction of this species occurred even at near-neutral and neutral pH.

category: bioengineering


title: Prediction of designer-recombinases for DNA editing with generative deep learning

abstract:
Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gathered the sequence information of over two million Cre-like recombinase sequences evolved for 89 different target sites with which we trained Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrated that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486669v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@c08326org.highwire.dtl.DTLVardef@e26984org.highwire.dtl.DTLVardef@7d7542org.highwire.dtl.DTLVardef@9d6642_HPS_FORMAT_FIGEXP  M_FIG C_FIG Teaser Figure: Recombinase prediction generates active recombinases for a desired target site, while existing libraries with a similar target site need to go through directed evolution to achieve activity on the new site. While evolution takes weeks, prediction and synthesis of recombinases can be done in days.

category: bioinformatics


title: Viability of AMURA biomarkers from single-shell diffusion MRI in Migraine Clinical Studies

abstract: Diffusion Tensor Imaging (DTI) is the most employed method to assess white matter properties using quantitative parameters derived from diffusion MRI, but it presents known limitations that restricts the evaluation of complex structures. The objective of this study was the assessment of complementary diffusion measures extracted with a novel approach, Apparent Measures Using Reduced Acquisitions (AMURA), in comparison with DTI in clinical studies. Fifty healthy controls, 51 episodic migraine and 56 chronic migraine patients underwent single-shell diffusion MRI. Three DTI-based and eight AMURA-based parameters were compared between groups with tract-based spatial statistics. On the other hand, following a region-based analysis, the measures were assessed for multiple subsamples with diverse reduced sample sizes and their stability was evaluated with the coefficient of quartile variation. Compared to DTI, statistically significant differences in additional regions and group comparisons were found with AMURA-based measures. Additionally, some AMURA-based measures showed a remarkable discriminative power for quite reduced sample size and, simultaneously, lower stability (higher coefficient of quartile variation) than DTI-based measures. These findings suggest that AMURA presents favorable characteristics to identify differences of specific microstructural properties between clinical groups in regions with complex fiber architecture and lower dependency on the sample size or assessing technique than DTI.


title: Unravelling specific diet and gut microbial contributions to inflammatory bowel disease

abstract: Inflammatory bowel disease (IBD) is a chronic condition characterized by periods of spontaneous intestinal inflammation and is increasing in industrialized populations. Combined with host genetic predisposition, both diet and gut bacteria are thought to be prominent environmental features contributing to IBD, but little is known about the precise mechanisms. We show that low dietary fiber promotes bacterial erosion of the protective colonic mucus layer, leading to lethal colitis in interleukin-10-deficient mice. This diet-induced inflammation is specifically driven by mucin-degrading bacteria-mediated Th1 immune responses and is preceded by expansion of natural killer T cells and reduced immunoglobulin A coating of specific bacterial species. Low fiber, exclusive enteral nutrition reduced disease in part by increasing bacterial production of the metabolite isobutyrate, which was dependent on the presence of soy protein in the diet. Our results highlight a mechanistic framework to unravel the complex web of potentially opposing diet, host and microbial factors that combine to influence IBD development.


title: Saving the Devils is in the details: Tasmanian Devil facial tumor disease can be eliminated with interventions

abstract:
AO_SCPLOWBSTRACTC_SCPLOWTasmanian Devils facial tumor disease is severely impacting the population of this wild animal. We developed a computational model of the population of Tasmanian Devils, and the change induced by DFTD. We use this model to test possible intervention strategies Tasmanian conservationists could do. We investigate bait drop vaccination programs, diseased animal removals programs, and evolution of natural immunity. We conclude that a combination of intervention strategies gives the most favorable outcome.

An additional goal of this paper is for reproducibility of our results. Our StochSS software platform features the ability to share and reproduce the computational notebooks that created all of the results in the paper. We endeavor that all readers should be able to reproduce our results with minimum effort.

category: ecology


title: Population estimation of a cryptic moss frog using acoustic spatially explicit capture recapture

abstract: Cryptic amphibians pose a problem for conservation managers as they are difficult to find to assess initial populations, and monitor changes during potentially threatening processes. The rough moss frog, Arthroleptella rugosa, is small and occurs in seepages on a single unprotected mountain in South Africas fire prone, biodiverse fynbos biome. The area is heavily impacted by invasive plants, which dry seepages and increase the frequency and intensity of fires, leading to the assessment of this species as Critically Endangered. We aimed to test the efficacy of acoustic spatially explicit capture recapture (aSCR) to estimate the entire population of calling adult A. rugosa, and assess the impacts by invasive plants. Using aSCR, our estimates suggest that the population of A. rugosa is more than five times that previously estimated using aural calling surveys on the mountain, at [~]2000 individuals. This despite an intense fire over the entire area three years earlier that reduced the calling population to a few tens of individuals. Our vegetation surveys suggest that the ongoing removal of invasive plants from the mountain is successful in areas occupied by A. rugosa, but that adjacent areas invaded by pines and hakea have a negative impact on calling density. The private public conservancy partnership on Klein Swartberg Mountain, is conserving this frog but will require ongoing management and monitoring to ensure conservation in the future.


title: Fungal States of Minds

abstract: Fungal organisms can perceive the outer world in a way similar to what animals sense. Does that mean that they have full awareness of their environment and themselves? Is a fungus a conscious entity? In laboratory experiments we found that fungi produce patterns of electrical activity, similar to neurons. There are low and high frequency oscillations and convoys of spike trains. The neural-like electrical activity is yet another manifestation of the fungal intelligence. In this paper we discuss fungal cognitive capabilities and intelligence in evolutionary perspective, and question whether fungi are conscious and what does fungal consciousness mean, considering their exhibiting of complex behaviours, a wide spectrum of sensory abilities, learning, memory and decision making. We overview experimental evidences of consciousness found in fungi. Our conclusions allow us to give a positive answer to the important research questions of fungal cognition, intelligence and forms of consciousness.

category: animal behavior and cognition


title: Rapid loss of fine motor skills after low dose space radiation exposure

abstract: Sensorimotor function, motivation, and attentional processes are fundamental aspects of behavioral organization during skilled tasks. NASAs planned expedition to Mars will expose astronauts to space radiation (SR) that has the potential to impair performance in mission critical tasks. Impairments in task accuracy and movement kinematics have been previously reported during string-pulling behavior ~7 months after SR exposure. If similar SR-induced sensorimotor deficits emerge at earlier times, then astronauts may have compromised in-flight performance disruptions while performing skilled tasks in critical situations, such as when manipulating controls or performing seat egress. Due to the possibility that such performance losses may compromise mission success, it is critical to determine if sensorimotor, motivation, or attentional deficits occur acutely after SR exposure at a time point that corresponds to in-flight performance. Male Wistar rats were thus exposed to either 10 cGy simplified galactic cosmic radiation (GCRsim), 10 cGy 4Helium (4He), or no radiation at all (Sham), and string-pulling behavior was assessed approximately 72 hours later. Following exposure to SR, rats (4He) took more time to approach the string to initiate string-pulling behavior and to pull in the string to reach the Cheerio (4He and GCRsim) relative to Sham rats. 4He-exposed rats also exhibited a greater number of misses and less contacts relative to both Sham and GCRsim-exposed rats. Further, rats exposed to 4He demonstrated less concentrated reach endpoints with both the left and right hands compared to GCR-exposed rats. This work suggests that sensorimotor function and motivation and/or attentional processes were impaired 72 hours after 4He-radiation exposure.


title: Nonlethal effects of predation: Presence of insectivorous birds affects the behaviour and level of stress in insects

abstract: Insect exposure to their predators can affect individuals and community processes, through direct consumption or nonlethal (i.e., nonconsumptive) effects. However, the links between behavioural and physiological responses and stimuli needed for development of the fear are not clear. We therefore subjected the desert locusts (Schistocerca gregaria) to three nonlethal treatments, using the great tits (Parus major) as a potential predator. The treatments involved: (1) bird - presence of a live great tit and its calls, (2) call - great tit calls only, (3) control - without any treatment. In the first behavioural laboratory experiment, hungry locusts were kept in an experimental cage with a shelter and food on opposite sides of the cage. The duration of hiding and feeding were considered as an indicator of fear responses. In the second laboratory experiment with the same three treatments, levels of the adipokinetic hormone (AKH) were evaluated in the central nervous system (CNS) and haemolymph. In the third experiment in an outdoor aviary, birds were free to fly in larger distances from locusts, before hormone levels were measured as response to bird and control treatments. In the first behavioural experiment, the presence of tits and their call resulted in significantly longer hiding time and significantly shorter feeding time than in the call/control treatments. The proximity of birds and locusts in the laboratory experiment elicited a significant increase in the AKH levels in the CNS and haemolymph as compared to the call/control treatments. In the outdoor experiment, the AKH levels were significantly higher in the CNS of locusts exposed to the bird than to control; no difference was recorded in their haemolymph. We showed that predator exposure quickly affected behavioural responses and physiological processes of locusts. Playback of the avian calls was not an appropriate stimulus to induce stress responses in desert locusts.


title: A Miniaturized 3D-Printed Pressure Regulator (μPR) for Microfluidic Cell Culture Applications

abstract: Controlled fluid flows are the hallmark feature of microfluidic culture systems and provide precise definition over the biophysical and biochemical microenvironment. Flow control is commonly achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-based techniques. These methods offer complex flow capabilities but can be challenging to integrate into incubators or other confined environments due to their large form factors and accompanying peripheral equipment. Since many microfluidic cell culture studies use a single controlled flow rate to maintain or stimulate cells, a portable flow control platform that fits easily into an incubator will benefit the microfluidic community. Here, we demonstrate that a tunable, 3D printed micro pressure regulator (PR), combined with a battery-powered miniature air pump, can operate as a stand-alone pneumatic flow control platform for microfluidic applications. We detail the design and fabrication of the PR and demonstrate: i) a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), ii) highlight dynamic control in a microfluidic network, and iii) maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment membrane-based culture device under continuous flow conditions. We anticipate that our 3D-printed fabrication approach and open access designs will allow other laboratories to rapidly customize PRs to support a broad range of applications.


title: Thermal Effects on Neurons During Stimulation of the Brain

abstract: All artificial stimulation of the brain deposits thermal energy in the brain. This occurs through either Joule heating of the conductors carrying current through electrodes and magnetic coils, or through dissipation of energy in the conductive brain. Similarly, temperature affects all biological processes and chemical reactions. Although electrical interaction with brain tissue is inseparable from thermal effects when electrodes are used, magnetic induction enables us to separate Joule heating from induction effects by contrasting AC and DC driving of magnetic coils using the same energy deposition within the conductors. Since mammalian cortical neurons have no known sensitivity to static magnetic fields, and if there is no evidence of effect on spike timing to oscillating magnetic fields, we can presume that the induced electrical currents within the brain are below the molecular shot noise where any interaction with tissue is purely thermal. In this study, we examined a range of frequencies produced from micromagnetic coils operating below the molecular shot noise threshold for electrical interaction with single neurons. We found that small temperature increases and decreases of 1{degrees}C caused consistent transient suppression and excitation of neurons during temperature change. Numerical modeling of the biophysics demonstrated that the Na-K pump, and to a lesser extent the Nernst potential, could account for these transient effects. Such effects are dependent upon compartmental ion fluxes, and the rate of temperature change. A new bifurcation is described in the model dynamics that accounts for the transient suppression and excitation; in addition, we note the remarkable similarity of this bifurcations rate dependency with other thermal rate-dependent tipping points in planetary warming dynamics. Furthermore, bifurcations in the steady state dynamics leading to stable firing suppression are described for slightly higher temperatures. These experimental and theoretical findings demonstrate that stimulation of the brain must take into account small thermal effects that are ubiquitously present in electrical and magnetic stimulation. More sophisticated models of electrical current interaction with neurons combined with thermal effects will be required in order to more accurately enable model-based control of neuronal circuitry.

category: biophysics


title: Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging

abstract: Over the last decade, single molecule localization microscopy (SMLM) has developed into a set of powerful techniques that has improved spatial resolution over diffraction-limited microscopy and has demonstrated the ability to resolve biological features at molecular scale. We introduce a single molecule based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Using a standard widefield illumination, the system employs a scanning based detection 4f-sub-system suited for volume interrogation. The 4f system comprises of a combination of electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of electrically-tunable lens (ETL) in a 4f detection system, the selectivity of axial (Z) plane can be achieved in the object plane, for which the corresponding image forms in the image/detector plane. So, in-principle one can scan the object volume by just changing the aperture of ETL. To carry out volume imaging, a cyclic scanning scheme is developed and compared with conventional scanning routinely used in SMLM. The scanning scheme serves the purpose of distributing photobleaching evenly by ensuring uniform dwell time on each frame for collecting data (single molecule events) throughout the specimen volume. With minimal change in the system hardware (requiring an addition of ETL lens and related hardware for step-voltage generation and time synchronization) in the existing SMLM system, volume scanning can be achieved. To demonstrate, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to understand clustering of HA single molecules in a transfected cell (Influenza A disease model). The system for the first time enables visualization of HA distribution in a 3D cells that reveal its clustering across the cell volume. Critical biophysical parameters related to HA clusters (density, #HA/cluster and cluster fraction) are also determined for a single NIH3T3 cell transfected with photoactivable Dendra2-HA plasmid DNA.


title: Simultaneous adjustments of major mitochondrial pathways through redox regulation of dihydrolipoamide dehydrogenase (mtLPD1)

abstract:
Thioredoxins (TRX) are pivotal for the redox regulation of enzyme activities to adjust metabolic fluxes towards environmental changes. Previous reports demonstrated TRX o1 and h2 impact on mitochondrial metabolism including photorespiration and the tricarboxylic acid (TCA) cycle. Here, we aimed to unravel potential specificities between regulation modes of both TRXs, especially under conditions with short-term changes in photorespiration. Therefore, short-term metabolite responses of single TRX mutants were analyzed after exposure to altered CO2/O2 ratios during darkness and illumination. This approach was complemented by comprehensive characterization of multiple Arabidopsis mutants lacking either one or both TRX in the wild-type Arabidopsis or the glycine decarboxylase (GDC) T-protein knock down line (gldt1). The results provided evidence for additive effects of combined TRX o1 and h2 deficiency to suppress growth, photosynthesis and mitochondrial metabolism. Quantification of pyrimidine nucleotides in conjunction with metabolite and 13C-labelling approaches revealed a rather uniform impact on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) dependent pathways. Biochemical analysis of recombinant mtLPD1 demonstrated its inhibition by NADH, pointing at an additional measure to fine-tune its in vivo activity. Collectively, we propose that TRX o1 and h2 contribute to the communication of altered subcellular redox-states through direct and indirect regulation of mtLPD1. This regulation module might represent a common intercept for simultaneous adjustments in the operation of photorespiration, the TCA-cycle and the degradation of branched chain amino acids.

One-sentence summaryRedox regulation of mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) simultaneously modulates photorespiration, the tricarboxylic acid (TCA)-cycle and branched chain amino acid (BCAA) degradation in response to rapid environmental changes.

category: plant biology


title: Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling

abstract: The spatiotemporal organisation of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting and endocytosis. Earlier studies have shown that multiple types of membrane proteins including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered GPI-anchored proteins (GPI-APs) form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drive the creation of active emulsions, mesoscale liquid ordered (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid-phase segregation. To test these ideas we use spatial imaging of homo-FRET combined with local membrane order and demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions, and exhibit significant lipid order, consistent with predictions of the active composite model.

category: cell biology


title: Gain-of-function genetic screens in human cells identify SLC transporters overcoming environmental nutrient restrictions

abstract: Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of members of this family in response to environmental stresses remain poorly elucidated. In order to infer functional relationships between SLCs and metabolites, we developed a strategy to identify human SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from the growth-limiting metabolic bottleneck. We identified the cationic amino acid transporter SLC7A3 as a gene that, when upregulated, overcame low availability of arginine and lysine by increasing their uptake. SLC7A5 (LAT1), on the other hand, was able to sustain cellular fitness upon deprivation of several neutral amino acids. A genome-wide screen identified SLC7A3 as the single main gene product able to rescue cell survival in the limiting arginine conditions tested, demonstrating the potentially decisive role of transporters in overcoming nutrient limitations. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells led to the definition of functional transporter-nutrient relationships and revealed that upregulation of transport activity may be sufficient to overcome environmental metabolic restrictions.


title: A comparison of feature selection methodologies and learning algorithms in the development of a DNA methylation-based telomere length estimator

abstract: The field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine-guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, we test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We found that principal component analysis in advance of elastic net regression led to the overall best performing estimator when evaluated using a nested cross-validation analysis and two independent test cohorts. In contrast, the baseline model of elastic net regression with no prior feature reduction stage performed worst - suggesting a prior feature-selection stage may have important utility. The variance in performance across tested approaches shows that estimators are sensitive to data set heterogeneity and the development of an optimal DNA methylation-based estimator should benefit from the robust methodological approach used in this study. Additionally, we observed that different DNA methylation-based TL estimators, which have few common CpGs, are associated with many of the same biological entities. Moreover, our methodology which utilises a range of feature-selection approaches and ML algorithms could be applied to other biological markers and disease phenotypes, to examine their relationship with DNA methylation and predictive value.


title: Vimentin Intermediate Filaments Can Enhance or Abate Active Cellular Forces in a Microenvironmental Stiffness-Dependent Manner

abstract:
The mechanical properties of cells are largely determined by the cytoskeleton, which is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. While disruption of the actin filament and microtubule networks is known to decrease and increase cell-generated forces, respectively, the effect of intermediate filaments on cellular forces is not well understood. Using a combination of theoretical modeling and experiments, we show that disruption of vimentin intermediate filaments can either increase or decrease cell-generated forces, depending on microenvironment stiffness, reconciling seemingly opposite results in the literature. On the one hand, vimentin is involved in the transmission of actomyosin-based tensile forces to the matrix and therefore enhances traction forces. On the other hand, vimentin reinforces microtubules and their stability under compression, thus promoting the role of microtubules in suppressing cellular traction forces. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. For low matrix stiffness, the force-transmitting role of vimentin dominates over their microtubule-reinforcing role and therefore vimentin increases traction forces. At high matrix stiffness, vimentin decreases traction forces as the microtubule-reinforcing role of vimentin becomes more important with increasing matrix stiffness. Our theory reconciles seemingly disparate experimental observations on the role of vimentin in active cellular forces and provides a unified description of stiffness-dependent chemo-mechanical regulation of cell contractility by vimentin.

SignificanceVimentin is a marker of the epithelial to mesenchymal transition which takes place during important biological processes including embryogenesis, metastasis, tumorigenesis, fibrosis, and wound healing. While the roles of the actin and microtubule networks in the transmission of cellular forces to the extracellular matrix are known, it is not clear how vimentin intermediate filaments impact cellular forces. Here, we show that vimentin impacts cellular forces in a matrix stiffness-dependent manner. Disruption of vimentin in cells on soft matrices reduces cellular forces, while it increases cellular forces in cells on stiff matrices. Given that cellular forces are central to both physiological and pathological processes, our study has broad implications for understanding the effect of vimentin on cellular forces in different microenvironments.


title: Perturbed actin cap and nuclear morphology in primary fibroblasts of Huntington's disease patients as a new phenotypic marker for personalized drug evaluation

abstract: Human primary skin fibroblast cells from patients skin biopsies were used previously as a model to study different neurodegenerative diseases, including Huntingtons Disease (HD). These cells are directly isolated from the patients tissue without any alteration in the genome, retaining in culture conditions their endogenous cellular characteristics and biochemical properties, as well as their cellular proliferation capacity for several passages. The aim of this study was to identify a distinctive cellular phenotype in primary skin fibroblasts from various HD patients, using image-based high content analysis, which could be used in the future for personalized drug screening treatment evaluation. We show that HD fibroblasts have a distinctive nuclear morphology associated with a nuclear actin cap deficiency, which in turn affects cell motility in a similar manner to primary skin fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients used as known actin cap deficient cells. Moreover, treatment of the HD cells with either Latrunculin B, used to disrupt actin cap formation, or the antioxidant agent Mitoquinone, used to improve mitochondrial activity, show opposite effects on actin cap associated morphological features and cell motility. The former exacerbates the HD phenotype while the latter improves it. Deep data analysis of the HD nuclear and actin cap features using custom developed image analysis algorithms allow strong cluster classification distinct from HGPS and healthy matching controls, supporting the finding of a novel HD cellular phenotypic marker that could be modulated by pharmacological agents in this patient-based disease model.


title: Integrative analysis of metabolite GWAS illuminates the molecular basis of pleiotropy and genetic correlation

abstract: Pleiotropy and genetic correlation are widespread features in GWAS, but they are often difficult to interpret at the molecular level. Here, we perform GWAS of 16 metabolites clustered at the intersection of amino acid catabolism, glycolysis, and ketone body metabolism in a subset of UK Biobank. We utilize the well-documented biochemistry jointly impacting these metabolites to analyze pleiotropic effects in the context of their pathways. Among the 213 lead GWAS hits, we find a strong enrichment for genes encoding pathway-relevant enzymes and transporters. We demonstrate that the effect directions of variants acting on biology between metabolite pairs often contrast with those of upstream or downstream variants as well as the polygenic background. Thus, we find that these outlier variants often reflect biology local to the traits. Finally, we explore the implications for interpreting disease GWAS, underscoring the potential of unifying biochemistry with dense metabolomics data to understand the molecular basis of pleiotropy in complex traits and diseases.

category: genomics


title: A brain-inspired object-based attention network for multi-object recognition and visual reasoning

abstract: The visual system uses sequences of selective glimpses to objects to support goal-directed behavior, but how is this attention control learned? Here we present an encoder-decoder model inspired by the interacting bottom-up and top-down visual pathways making up the recognitionattention system in the brain. At every iteration, a new glimpse is taken from the image and is processed through the "what" encoder, a hierarchy of feedforward, recurrent, and capsule layers, to obtain an object-centric (object-file) representation. This representation feeds to the "where" decoder, where the evolving recurrent representation provides top-down attentional modulation to plan subsequent glimpses and impact routing in the encoder. We demonstrate how the attention mechanism significantly improves the accuracy of classifying highly overlapping digits. In a visual reasoning task requiring comparison of two objects, our model achieves near-perfect accuracy and significantly outperforms larger models in generalizing to unseen stimuli. Our work demonstrates the benefits of object-based attention mechanisms taking sequential glimpses of objects.


title: Growth rate controls the sensitivity of gene regulatory circuits

abstract: Unicellular organisms adapt to their changing environments by gene regulatory switches that sense chemical cues and induce specific target genes when the inducing signal is over a critical threshold. Using mathematical modeling we here show that, because growth rate sets the dilution rate of intra-cellular molecules, the sensitivity of gene regulatory switches generally decreases with growth rate, independent of their precise architecture. We confirm the modeling predictions by experimentally demonstrating that the concentration of inducer required for activating the lac operon in E. coli decreases quadratically with growth rate at the population level, and that growth-arrested cells become hyper-sensitive to inducer at the single-cell level. Moreover, we establish that this growth-coupled sensitivity allows bacteria to implement concentration-dependent sugar preferences, in which a new carbon source is used only if its concentration is high enough to improve upon the current growth rate of the cells. Using microfluidics in combination with time-lapse microscopy, we validate experimentally that this strategy governs how mixtures of glucose and lactose are used in E. coli and that the central regulator CRP plays a key role in implementing this strategy. Overall growth-coupled sensitivity provides a general mechanism through which cells can  mute external signals in beneficial conditions when growth is fast, and become highly sensitive to alternative nutrients or stresses when growth is slow or arrested.

category: systems biology


title: Dominance vs. epistasis: the biophysical origins and plasticity of genetic interactions within and between alleles

abstract: A central challenge in genetics, evolutionary biology and biotechnology is to understand and predict how mutations combine to alter phenotypes, including molecular activities, fitness and disease. In diploid organisms, two mutations in the same gene can either combine on the same chromosome or on different chromosomes, with interactions between the mutations quantified as epistasis and dominance, respectively. However, a direct comparison of the extent, sign and stability of interactions within and between alleles is lacking. Here we show that, even in the simplest biophysical systems, interactions between mutations are frequent, context-dependent and different when variants are combined within and between alleles. Whereas protein folding alone generates epistasis, the addition of a single molecular interaction is sufficient to cause dominance. Epistasis and dominance interactions change quantitatively, qualitatively and differently as a system becomes more complicated or the conditions change. Altering the concentration of a ligand can, for example, switch an allele from dominant to recessive. Our results show that epistasis and dominance should be widely expected in even the simplest biological systems but also reinforce the view that they are plastic system properties and so a formidable challenge to predict. Accurate prediction of epistasis and dominance will require either detailed mechanistic understanding and experimental parameterization or brute-force measurement and learning.


title: Antibodies to Coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared Fibrinogen Binding repeats

abstract:
Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of multiple staphylococcal virulence proteins. Amongst its secreted virulence factors, Coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminus of Coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naive antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminus of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

ImportanceThe death toll related to methicillin-resistant S. aureus piled to almost 1 million people in only one year (2019), ascribing S. aureus to the second leading cause of deaths associated with antimicrobial resistance. Therefore, new therapeutic strategies must be investigated. Blocking the adhesion step with the use of monoclonal antibodies is one promising alternative and Fg is a central plasma protein involved in staphylococcal infection. We present here a panel of monoclonal antibodies raised against Coa, cross-reacting to Efb and targeting the shared Fg binding repeats of Coa. In addition, we describe new epitope determinants in the repeated region of Coa, highlighted by differential binding of the newly selected antibodies.


title: Tegaserod maleate suppresses the growth of gastric cancer in vivo and in vitro by targeting MEK1/2

abstract: Gastric cancer (GC), ranking fifth in global incidence and fourth in mortality. The current treatments for GC include surgery, chemotherapy, and radiotherapy. Although management and treatment strategies for GC have been improved over the last decade, the overall five-year survival rate remains less than 30%. Therefore, there is an urgent need to find novel therapeutic or preventive strategies that can increase GC patient survival rates. In the current study, we found that tegaserod maleate, a FDA-approved drug, could inhibit the proliferation of gastric cancer cells. Tegaserod maleate binds to MEK1 /2 and inhibits MEK1 /2 kinase activity. Moreover, the construction of CRISPER/Cas9 cell line further verified that tegaserod maleate depended on MEK1 /2 to inhibit the progress of gastric cancer. Notably, we found that tegaserod maleate suppressed tumor growth in patient-derived gastric xenograft (PDX) mouse model. We also compared tegaserod maleate with trametinib, a clinical MEK1/2 inhibitor, and comfirmed that tegaserod maleate have the same effect in inhibiting tumor volume and tumor weight. Our findings suggest that tegaserod maleate can inhibit GC proliferation by targeting MEK1/2.

category: pathology


title: Toxicity of extracellular alpha-synuclein is independent of intracellular alpha-synuclein

abstract: Parkinson's disease (PD) pathology progresses throughout the nervous system affecting numerous neuronal structures. It has been postulated that the progression of the pathology is based on a prion-like disease mechanism partly due to the seeding effect of endocytosed alpha-synuclein (ASYN) on the endogenous ASYN. The appearance of the pathology in dopaminergic neurons leads to neuronal cell death and motor symptoms. However, the effect on other neuronal structures is more inconsistent, leading to a higher variability in the prevalence of non-motor symptoms. Thus, the sensitivity to the pathology seems to vary among neuronal subtypes. Here, we analyzed the role of endogenous ASYN in the progression of PD-like pathology and the effect of monomeric and oligomeric ASYN as well as paraquat and rotenone on primary enteric, dopaminergic and cortical neurons from wild-type mice. Our results showed that pathology progression did not occur in the absence of endogenous ASYN and that dopaminergic neurons were more sensitive to ASYN and rotenone when compared to all other neuronal subtypes. Remarkably, the toxic effect of ASYN was independent of the presence of endogenous ASYN and directly related to the disturbance of the mitochondrial membrane potential. Thus, we suggest that the interaction between ASYN and mitochondria plays an important role in the toxicity of trans-synaptically transported ASYN and in the progression of PD pathology. These results question the prion-disease hypothesis and propose that endocytosed ASYN impairs the host's mitochondrial function thereby also contributing to PD-pathology progression.

category: neuroscience


title: Accumulated 3-phosphoglycerate in darkness is a primary starting substrate for stepwise photosynthetic induction process in cyanobacteria

abstract:
In photosynthetic organisms, a smooth metabolic transition from dark to light conditions is essential for efficient CO2 fixation and growth. Dark-to-light transition leads to activation of the enzymes of the Calvin-Benson-Bassham (CBB) cycle and initiates CO2 fixation metabolism using energy and redox equivalents provided by the photosynthetic electron transfer reaction. While many enzymatic mechanisms of the light activation of the CBB cycle have been clarified to date, the metabolic distribution and dynamics underlying the smooth start of photosynthetic metabolism remain unclear. Here, we show rapid (on a time scale of seconds) dynamics of changes in absolute metabolite concentration and 13C tracer incorporation during photosynthetic induction (PI) in the cyanobacterium Synechocystis sp. PCC 6803. By combining these data, we were able to estimate time-resolved nonstationary dynamics of metabolic fluxes during PI. This novel metabolic analysis indicated that the initial step of PI consists of the reduction of 3-phosphoglycerate (3PG), a metabolite that accumulates in dark conditions; This step occurs before the subsequent generation of ribulose-1,5-bisphosphate (RuBP) and CO2 fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In vivo oxygen evolution measurement in the presence of a CBB cycle inhibitor further confirmed that the accumulated 3PG is a major initial substrate for RuBP generation and that 3PG functions as a key electron sink before the initiation of CO2 fixation. Our results thus illustrate a fundamental mechanism supporting the seamless activation of photosynthetic metabolism after dark-to-light transitions.

Significance StatementPlants, algae and cyanobacteria activate photosynthetic CO2 fixation after transition from dark to light conditions. Although this metabolic regulation is the fundamental system which enables photosynthetic organisms to grow in fluctuating light environment, metabolite distribution and dynamics essential for smooth initiation of photosynthetic metabolism remain unclear. Here we determined nonstationary metabolic flux dynamics during photosynthetic induction by quantifying changes in absolute metabolite concentrations at second-scale time intervals after the dark-to-light transition in cyanobacteria. This novel metabolic analytical technique revealed that 3-phosphoglycerate that has accumulated in the dark period is a primary starting substrate for fast ribulose 1,5-bisphosphate generation and the subsequent initiation of CO2 fixation. This metabolic principle can be used for constructing efficient chemical production systems using CO2 fixation.


title: Emergence and clonal expansion in Europe of Vibrio aestuarianus lineages pathogenic for oysters

abstract: Crassostrea gigas oysters, with 4.7 million tons harvested per year, represent a considerable food supply worldwide. Since 2001 Vibrio aestuarianus has emerged as a pathogenic bacterium causing adult oyster mortality events in France and Ireland, and its impact on oyster aquaculture has increased in Europe since its reemergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey with national reference laboratories (NRLs) at the European scale for mollusk diseases. We analyzed genomes of V. aestuarianus isolated from multiple countries, since 2001, with or without bivalve mortalities, and from multiple environmental compartments. Using a combination of comparative genomics and population genetics approaches, we show that two oyster pathogen lineages have almost clonally expanded in Europe, after a probable recent selective bottleneck. Low mutation and recombination rates, as well as purifying selective pressure, have preserved virulent selected genotypes. Furthermore, we identified a specific cus-cop-containing island conferring copper resistance to V. aestuarianus that could have favored the emergence of V. aestuarianus pathogenic lineages adapted to oysters.


title: Anatomy and Symbiosis of the digestive system of the vent shrimps Rimicaris exoculata and Rimicaris chacei revealed through imaging approaches.

abstract: The shrimps Rimicaris exoculata and Rimicaris chacei are visually dominant fauna co-occurring at deep-sea hydrothermal sites of the Mid-Atlantic Ridge (MAR). Their co-existence was related to contrasted life-history traits, among which differences in their diet and reliance on chemoautotrophic symbionts at adult stage. Both shrimps are colonized by diversified chemosynthetic symbiotic microbial communities in their cephalothoracic cavity. Symbiotic association with bacteria was also evidenced in their digestive system, and the major lineages were identified through sequencing (Mycoplasmatales lineages mainly in the foregut and Deferribacteres lineages mainly in the midgut) but their clear distribution within each host species was not assessed. For the first time, we used Fluorescence in situ Hybridization (FISH) to visualize these lineages. Then, we described their association with digestive structures of both Rimicaris species. The aim was to identify possible differences between host species that could be related to their different life-history traits. For this purpose, we first developed specific FISH probes targeting Deferribacteres and Mycoplasmatales lineages identified in the digestive system of these shrimps. After signal specificity validation for each the new probe, we showed a partitioning of the bacterial lineages according to the digestive organ. Despite morphological differences between the foregut of R. exoculata and R. chacei that could be related to the adult diet, our FISH results showed overall similar distribution of digestive symbionts for the two host species. However, a more comprehensive study is needed with specimens at different life or molt stages to bring potentially host specific patterns out. Such comparative approach using FISH is now warranted thanks to our newly designed probes. These will be valuable tools to track symbiont lineages in the environment, allowing a better understanding of their relationship with their host along its life cycle, including acquisition mechanisms.


title: Evolutionary relations between mycorrhizal symbiosis and plant-plant communication in trees

abstract: Ecological factors that drive the evolution of plant-plant communication via volatile organic compounds (VOCs) have not been elucidated. Here, we examined the relationship between type of mycorrhizal symbiosis (arbuscular mycorrhiza, AM; ectomycorrhizal mycorrhiza, ECM) and plant-plant communication within tree species. We hypothesized that ECM promotes plant-plant communication among conspecific individuals in trees, because it promotes their cooccurrence through positive plant-soil feedback. We tested communication using saplings of nine tree species with either AM or ECM, either exposed for 10 days to volatiles from an injured conspecific or not exposed. We evaluated the number of insect-damaged leaves and the area of leaf damage after 1 and 2 months in the field. Most exposed ECM-associated trees had less leaf damage than controls. However, AM-associated trees did not differ in leaf damage between treatments. We combined our results with those of previous studies and analysed the evolutionary relation between mycorrhizal type and the presence or absence of plant-plant communication within tree species. ECM symbiosis is associated with the evolution of plant-plant communication within species. These results suggest that the evolution of types of mycorrhizal symbiosis associates with the evolution of plant-plant communications within tree species.


title: Genomic insights into present local adaptation and future climate change vulnerability of a keystone forest tree species in East Asian

abstract: Rapid global climate change is posing a huge threat to biodiversity. Assessments of the adaptive capacity for most taxa is usually performed on the species as a whole, but fails to incorporate intraspecific adaptive variation that may play a fundamental role in buffering future shifting climates. Here we generate a chromosome-scale genome assembly for Populus koreana, a pioneer and keystone tree species in East Asia temperate forests. We also obtain whole-genome sequences of 230 individuals collected from 24 natural populations. An integration of population genomics and environmental variables was performed to reveal the genomic basis of local adaptation to diverse climate variable. We identify a set of climate-associated single nucleotide polymorphisms (SNPs), insertions-deletions (Indels) and structural variations (SVs), in particular numerous adaptive non-coding variants distributed across the genome of P. koreana. We incorporate these variants into an environmental modelling scheme to predict spatiotemporal responses of P. koreana to future climate change. Our results highlight the insights that the integration of genomic and climate data can shed on the future evolutionary adaptive capacities of a species to changing environmental conditions.


title: Genomic regions associate with major axes of variation in gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.)

abstract:
Stomata and leaf veins play an essential role in transpiration and the movement of water throughout leaves. These traits are thus thought to play a key role in the adaptation of plants to drought and a better understanding of the genetic basis of their variation and coordination could inform efforts to improve drought tolerance. Here, we explore patterns of variation and covariation in leaf anatomical traits and analyze their genetic architecture via genome-wide association (GWA) analyses in cultivated sunflower (Helianthus annuus L.). Traits related to stomatal density and morphology as well as lower order veins were manually measured from digital images while the density of minor veins was estimated using a novel deep learning approach. Leaf, stomatal, and vein traits exhibited numerous significant correlations that generally followed expectations based on functional relationships. Correlated suites of traits could further be separated along three major principal component (PC) axes that were heavily influenced by variation in traits related to gas exchange, leaf hydraulics, and leaf construction. While there was limited evidence of colocalization when individual traits were subjected to GWA analyses, major multivariate PC axes that were most strongly influenced by several traits related to gas exchange or leaf construction did exhibit significant genomic associations. These results provide insight into the genetic basis of leaf trait covariation and showcase potential targets for future efforts aimed at modifying leaf anatomical traits in sunflower.

Significance StatementUsing traditional and automated/high-throughput (using a novel deep learning approach) phenotyping methods we studied leaf anatomical variation in sunflower. Genome-wide association (GWA) analyses identified numerous genomic regions underlying individual trait variation and regions underlying major multivariate axes of phenotypic variation. These results illustrate the value of employing a multivariate approach to GWA analyses and shed light on the extent to which leaf trait (co-)variation can be genetically decoupled to explore novel phenotypic space.


title: Genomic regions associate with major axes of variation driven by gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.)


title: Combined drought resistance strategies and the hydraulic limit in co-existing Mediterranean woody species

abstract:
O_LIWoody species employ various strategies to cope with drought stress. We investigated similarities and differences in response to chronic drought to understand resistance strategies in co-occurring Mediterranean species.
C_LIO_LIWe studied five predominant Mediterranean species; Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia over two summers at three sites with different aridities. We measured key hydraulic and osmotic traits related to drought resistance, including resistance to embolism ({Psi}50), carbon isotope signature ({delta}13C), pre-dawn ({Psi}PD) and mid-day ({Psi}MD) water potentials, and native ({Psi}s) and full turgor ([Pcy]0) osmotic potentials.
C_LIO_LISignificant differences among species appeared in resistance to embolism. The species also showed differences in the water potential plastic response over the dry season. This interspecific variation increased at the end of the dry season and resulted in very narrow hydraulic safety margins (HSM). Consequently, predicted loss of hydraulic conductivity revealed species with significant native embolism. Two of the species also had seasonal changes in osmotic adjustment.
C_LIO_LIOur detailed analysis indicates that co-existing Mediterranean woody species combine various drought resistance strategies to minimize mortality risk. However, all of them risk mortality as they approach their hydraulic limit near the dry margin of their distribution.
C_LI


title: Cellular dynamics of endosperm development in Arabidopsis thaliana

abstract: After double fertilization, the endosperm in the seeds of many flowering plants undergoes repeated mitotic nuclear divisions without cytokinesis, resulting in a large coenocytic endosperm that then cellularizes. Growth during the coenocytic phase is strongly associated with the final seed size; however, a detailed description of the cellular dynamics controlling the unique coenocytic development in flowering plants has remained elusive. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm of Arabidopsis thaliana including nuclear divisions, their timing intervals, nuclear movement, and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive F-actin organization. Microtubules promote nuclear movement after division while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and mature seed. Characterization the of cytoskeleton dynamics in real-time throughout the entire coenocyte endosperm period provides foundational knowledge of plant coenocytic development, insights into the coordination of F-actin and microtubules in nuclear dynamics, and new opportunities to increase seed size and our food security.


title: A scalable phenotyping approach for female floral organ development and senescence in the absence of pollination in wheat

abstract: In the absence of pollination, female reproductive organs senesce leading to an irrevocable loss in the reproductive potential of the flower and directly affecting seed set. In self-pollinating crops like wheat (Triticum aestivum), the post-anthesis viability of the unpollinated carpel has been overlooked, despite its importance for hybrid seed production systems. To advance our knowledge of carpel development in the absence of pollination, we created a relatively high-throughput phenotyping approach to quantify stigma and ovary morphology. We demonstrate the suitability of the approach, which is based on light microscopy imaging and machine learning, for the detailed study of floral organ traits in field grown plants using both fresh and fixed samples. We show that the unpollinated carpel undergoes a well-defined initial growth phase, followed by a peak phase (in which stigma area reaches its maximum and the radial expansion of the ovary slows), and a final deterioration phase. These developmental dynamics were largely consistent across years and could be used to classify male sterile cultivars, however the absolute duration of each phase varied across years. This phenotyping approach provides a new tool for examining carpel morphology and development which we hope will help advance research into this field and increase our mechanistic understanding of female fertility in wheat.


title: Toxoplasma gondii Coq1 is a heptaprenyl diphosphate synthase and its inhibition by a lipophilic bisphosphonate protects mice against T. gondii infection

abstract:
Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in e.g. sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species, heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion, and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low nM concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis.

ImportanceMillions of people are infected with Toxoplasma gondii and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection and treatment can trigger serious side-effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present several evidences that demonstrate that inhibition of the enzyme is linked to parasite death. In addition, the drug is able to protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.


title: Intracellular acidification is a hallmark of thymineless death in E. coli

abstract: Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD. Here, we used a diverse set of unbiased approaches to systematically determine the genetic and regulatory underpinnings of TLD in Escherichia coli. In addition to discovering novel genes in previously implicated pathways, our studies revealed a critical and previously unknown role for intracellular acidification in TLD. We observed that a decrease in cytoplasmic pH is a robust early event in TLD across diverse genetic backgrounds. Furthermore, we show that acidification is a causal event in the death process, as chemical and genetic perturbations that increase intracellular pH substantially reduce killing. We also observe a decrease in intracellular pH in response to exposure to the antibiotic gentamicin, suggesting that intracellular acidification may be a common mechanistic step in the bactericidal effects of other antibiotics.


title: Unique genomic sequences in novel Mycobacterium avium subsp. hominissuis lineage enabled fine scale tracing the transmission route during pig movement

abstract:
Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial (NTM) disease in human and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig and human MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. We conducted a variable number of tandem repeat (VNTR), genome phylogenetic tree analysis, historical recombination analysis and genome content analysis. Consistent with the results of previous studies, VNTR revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human. Recombination analysis indicated that most pig isolates could be classified into SC2/4 and SC3. To our surprise, four isolates belonged to a new lineage in the global MAH population. Members of this group had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. As the members of this new lineage were rare, we were able to trace the putative transmission route via their host pigs. Together, these results reveal the diversity in the pig MAH population and clarify the difference of source of infection and the vector of transmission between humans and pigs.

ImportanceMycobacterium avium subsp. hominissuis (MAH) is a dominant cause of non-tuberculous mycobacterial (NTM) disease in human and animals. In this study, in order to clarify the epidemiological relatedness between MAH isolates from human and pigs, and to determine the MAH transmission route among pigs, we conducted molecular and genomic-based epidemiological research. The pig isolates were genetically distant from those of humans in Japan, however, they were closely related to human isolates from North America, Europe and Russia, suggesting a proportion of pig MAH isolates are imported from abroad. Importantly, we identified a novel lineage of MAH that was distributed in a specific area in Japan. Epidemiological survey and its unique genomic features allowed us to trace putative transmission routes via pig movement. Our study provides critical information for understanding the diversity of MAH and will be useful for countermeasures of pig and human NTM disease.


title: Differential pathogenesis of SARS-CoV-2 variants of concern in human ACE2-expressing mice

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in millions of deaths worldwide. Increasingly contagious variants of concern (VoC) have fueled recurring global infection waves. A major question is the relative severity of disease caused by the previous and currently circulating variants of SARS-CoV-2. In this study, we evaluated the pathogenesis of SARS-CoV-2 variants in human ACE-2-expressing (K18-hACE2) mice. Eight-week-old K18-hACE2 mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta) or B.1.1.529 (omicron) lineages. We also infected a group of mice with the mouse-adapted SARS-CoV-2 (MA10). Our results demonstrate that B.1.1.7, B.1.351 and B.1.617.2 viruses are significantly more lethal than B.1 strain in K18-hACE2 mice. Infection with B.1.1.7, B.1.351 and B.1.617.2 variants resulted in significantly higher virus titers in the lungs and brain of mice compared to the B.1 virus. Interestingly, mice infected with the B.1.1.529 variant exhibited less severe clinical signs and high survival rate. We found that B.1.1.529 replication was significantly lower in the lungs and brain of infected mice in comparison to other VoC. Transcription levels of cytokines and chemokines in the lungs of the B.1.1.529-infected mice were significantly less when compared to those challenged with the B.1.1.7 virus. Together, our data provide insights into the pathogenesis of the previous and circulating SARS-CoV-2 VoC in mice.


title: Neuroendocrine control of the proteostatic network by HPK-1 delays aging

abstract:
The nervous system systemically coordinates proteostasis to maintain organismal health. In this work, we identify the transcriptional cofactor HPK-1 (homeodomain-interacting protein kinase) as a key neuronal component of the proteostatic network: its overexpression produces a paracrine signal to hyper-induce molecular chaperones and a neuroendocrine signal to induce autophagy in peripheral tissues. Neuronal HPK-1 signaling improves proteostasis in distal tissues through neurotransmitters. These pro-longevity modalities are independently regulated within serotonergic and GABAergic neurons, respectively, through distinct adaptive responses, either of which improve proteostasis in a cell non-autonomous manner. The former protects the proteome from acute stress while the latter fortifies the proteome to delay aging. Thus, HPK-1 acts as a central regulatory node upstream of the greater PN, exerting complementary roles in distinct neuronal cell-types.

One-Sentence SummaryNeuronal control of proteostasis and aging


title: Transmembrane helices 5 and 12 control transport dynamics, substrate affinity and specificity in the elevator-type UapA transporter

abstract: An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a novel genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead to increased rate of transport, but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding, and thus broadened specificity and UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function or how their transport characteristics might be altered genetically or have been modified in the course of evolution.


title: Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan

abstract: The adhesion receptor dystroglycan provides a critical mechanical link between the extracellular matrix (ECM) and the actin cytoskeleton to help muscle cells withstand contraction and neural cells maintain the blood brain barrier. Disrupting the link is associated cancer and muscular dystrophy. Proteolysis of dystroglycan by matrix metalloproteinases (MMPs) provides a mechanism to break the mechanical anchor and is amplified in several pathogenic states, yet how MMP cleavage is regulated is largely unexplored. We solved the crystal structure of the extracellular membrane-adjacent domains of dystroglycan containing the MMP site and find the tandem Ig-like and SEA-like domains structurally interact in an L-shape to facilitate a permissive conformation for /{beta} auto-catalytic processing. The MMP cleavage site is located in a structural motif C-terminal to the SEA-like domain that folds back and nestles into a pocket within the SEA-like domain to protect the cleavage site from MMPs. We then show while the intact proteolytic switch domain is resistant to MMP cleavage, structurally disruptive muscular dystrophy-related mutations sensitize dystroglycan to proteolysis in secreted constructs and the full-length receptor. Intriguingly, previously uncharacterized cancer-associated mutations that map to the proteolytic switch domain similarly lead to increases in proteolysis. Taken together, this study demonstrates dystroglycan proteolysis is conformationally regulated and mutations in the domain lead to enhanced proteolysis.