title: Position-dependent Codon Usage Bias in the Human Transcriptome

abstract: All life depends on the reliable translation of RNA to protein according to complex interactions between translation machinery and RNA sequence features. While ribosomal occupancy and codon frequencies vary across coding regions, well-established metrics for computing coding potential of RNA do not capture such positional dependence. Here, we investigate positiondependent codon usage bias (PDCUB), which dynamically accounts for the position of proteincoding signals embedded within coding regions. We demonstrate the existence of PDCUB in the human transcriptome, and show that it can be used to predict translation-initiating codons with greater accuracy than other models. We further show that observed PDCUB is not accounted for by other common metrics, including position-dependent GC content, consensus sequences, and the presence of signal peptides in the translation product. More importantly, PDCUB defines a spectrum of translational efficiency supported by ribosomal occupancy and tRNA adaptation index (tAI). High PDCUB scores correspond to a tAI-defined translational ramp and low ribosomal occupancy, while low PDCUB scores exhibit a translational valley and the highest ribosomal occupancy. Finally, we examine the relationship between PDCUB intensity and functional enrichment. We find that transcripts with start codons showing the highest PDCUB are enriched for functions relating to neuropeptide signaling and nucleosome assembly, as well as development of musculoskeletal, cardiovascular, neurological, gastrointestinal, sensory, and other body systems. Furthermore, transcripts with high PDCUB are depleted for functions related to immune response and detection of chemical stimulus. These findings lay important groundwork for advances in our understanding of the regulation of translation, the calculation of coding potential, and the classification of RNA transcripts.


title: Invalidity of light sensor data in field studies and a proposal of an algorithmic approach for detection and filtering of non-wear time

abstract: Wearable light sensors are increasingly used in intervention and population-based studies investigating the consequences of environmental light exposure on human physiology. An important step in such analyses is the reliable detection of non-wear time. We observed in light data that days with less wear-time also have lower variability in the light signal, and we sought to test if the standard deviation of the change between subsequent samples can detect this condition. In this study, we propose and validate an easy-to-implement algorithm designed to discriminate between days with a non-wear time >4h ("invalid days") vs. [≤]4h ("valid days") and investigate to which extent values of commonly used physiologically meaningful light variables differ between invalid days, valid days, and algorithm-selected non-wear days. We used 83 days of light data from a field study with high participant compliance, complemented by 47 days of light data where free-living individuals were instructed not to wear the sensor for varying amounts of time. Light data were recorded every two minutes using the pendant-worn f.luxometer light sensor; validity was derived from daily logs where participants recorded all non-wear time. The algorithm-derived score discriminated well between valid and invalid days (area under the curve (AUC): 0.77, 95% CI: 0.67-0.87). The best cut-off value (i.e., highest Youden index) correctly recognized valid days with a probability of 87% ("sensitivity"), and invalid days with a probability of 63% ("specificity"). Values of various light variables derived from algorithm-selected days only (median: 264.3 (Q1: 153.6, Q3: 420.0) for 24h light intensity (in lux); 496.0 (404.0, 582.0) for time spent above 50-lux) gave values close to those derived from self-reported valid days only. However, these values did not significantly differ when derived across all days compared to self-reported valid days. Our results suggest that our proposed algorithm discriminates well between valid and invalid days. However, in high compliance cohorts, distortions in aggregated light measures of individual-level environmental light recordings across days appear to be small, making the application of our algorithm optional, but not necessary.

category: neuroscience


title: DEVELOPMENTAL AND EVOLUTIONARY COMPARATIVE ANALYSIS OF A HOXD REGULATORY LANDSCAPE IN MAMMALS AND BIRDS

abstract: Modifications in gene regulation during development are considered to be a driving force in the evolution of organisms. Part of these changes involve rapidly evolving cis-regulatory elements (CREs), which interact with their target genes through higher-order 3D chromatin structures. However, how such 3D architectures and variations in CREs contribute to transcriptional evolvability remains elusive. During vertebrate evolution, Hox genes were redeployed in different organs in a class-specific manner, while maintaining the same basic function in organizing the primary body axis. Since a large part of the relevant enhancers are located in a conserved regulatory landscape, this gene cluster represents an interesting paradigm to study the emergence of regulatory innovations. Here, we analysed Hoxd gene regulation in both murine vibrissae and chicken feather primordia, two mammalian- and avian-specific skin appendages which express different subsets of Hoxd genes, and compared their regulatory modalities with the regulations at work during the elongation of the posterior trunk, a mechanism highly conserved in amniotes. We show that in the former two structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, likely as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the regulations implemented in the mouse and chicken embryonic trunk partially rely on conserved CREs. Nevertheless, a high proportion of these non-coding sequences active in the trunk appear to have functionally diverged between the two species, suggesting that transcriptional robustness is maintained despite a considerable divergence in CREs sequence, an observation supported by a genome-wide comparative approach.

category: developmental biology


title: Robust and Functional Immunity up to 9 months after SARS-CoV-2 infection: a Southeast Asian longitudinal cohort

abstract:
Assessing the duration of humoral and cellular immunity remains key to overcome the current SARS-CoV-2 pandemic, especially in understudied populations in least developed countries. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for humoral immune response to the viral spike protein and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-spike (S) antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immunity was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased ADCC and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immunity. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection in the absence of re-infection.

One sentence summaryFunctional immune memory to SARS-CoV-2, consisting of polyfunctional antibodies, memory B cells and memory T cells are maintained up to nine months in a South-East Asian cohort in the absence of re-infection.

category: immunology


title: In vitro determination of inhibitory effects by humic substances complexing Zn and Se on SARS-CoV-2 virus replication

abstract: Humic substances are well known human nutritional supplement materials and play important performance-enhancing roles as animal feed additives, too. For decades, ingredients of humic substances have also been proven to carry potent antiviral effects against different viruses. Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern ("Alpha Variant") in a VeroE6 cell line. Results show that this combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. Even picomolar concentration ranges of humic substances, vitamin C and Zn/Se ions in the given composition were enough to achieve fifty percent viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.

category: microbiology


title: Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates

abstract:
Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 {micro}g of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.{beta} (heterologous), which encompasses the spike sequence of the B.1.351 (beta or {beta}) variant. Reciprocal ID50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the {beta} variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost {beta}-specific reciprocal ID50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the {beta} variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations.

One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.


title: Refining Bulk Segregant Analyses: Ontology-Mediated Discovery of Flowering Time Genes in Brassica oleracea

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSBulk segregant analysis (BSA) can help identify quantitative trait loci (QTLs), but this may result in substantial bycatch of functionally irrelevant genes. Here we develop a Gene Ontology-mediated approach to zoom in on specific markers implicated in flowering time from among QTLs identified by BSA of the giant woody Jersey kale phenotyped in four bulks of flowering onset.

ResultsOur BSA yielded tens of thousands of candidate genes. We reduced this by two orders of magnitude by focusing on genes annotated with terms contained within relevant subgraphs of the Gene Ontology. A further enrichment test led to the pathway for circadian rhythm in plants. The genes that enriched this pathway are attested from previous research as regulating flowering time. Some of these genes were also identified as having functionally significant variation compared to Arabidopsis

ConclusionsWe validated and confirmed our ontology-mediated results through a more targeted, homology-based approach. However, our ontology-mediated approach produced additional genes of putative importance, showing that the approach aids in exploration and discovery. We view our method as potentially applicable to the study of other complex traits and therefore make our workflows available as open-source code and a reusable Docker container.

category: bioinformatics


title: Wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish

abstract: Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors.

category: genetics


title: wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish


title: Maximizing GWAS discovery and genomic prediction accuracy in Biobank data

abstract: Genetically informed and deep-phenotyped biobanks are an important research resource. The cost of phenotyping far outstrips that of genotyping, and therefore it is imperative that the most powerful, versatile and efficient analysis approaches are used. Here, we apply our recently developed Bayesian grouped mixture of regressions model (GMRM) in the UK and Estonian Biobanks and obtain the highest genomic prediction accuracy reported to date across 21 heritable traits. On average, GMRM accuracies were 15% (SE 7%) greater than prediction models run in the LDAK software with SNP annotation marker groups, 18% (SE 3%) greater than a baseline BayesR model without SNP markers grouped into MAF-LD-annotation categories, and 106% (SE 9%) greater than polygenic risk scores calculated from mixed-linear model association (MLMA) estimates. For height, the prediction accuracy R2 was 47% in a UK Biobank hold-out sample, which was 76% of the estimated [Formula]. We then extend our GMRM prediction model to provide MLMA SNP marker estimates for GWAS discovery, which increased the independent loci detected to 7,910 in unrelated UK Biobank individuals, as compared to 5,521 from BoltLMM and 5,727 from Regenie, a 43% and 38% increase respectively. The average{chi} 2 value of the leading markers was 34% (SE 5.11) higher for GMRM as compared to Regenie, and increased by 17% for every 1% increase in prediction accuracy gained over a baseline BayesR model across the traits. Thus, we show that modelling genetic associations accounting for MAF and LD differences among SNP markers, and incorporating prior knowledge of genomic function, is important for both genomic prediction and for discovery in large-scale individual-level biobank-scale studies.

category: genomics


title: Improving GWAS discovery and genomic prediction accuracy in Biobank data


title: The Molecular Subtyping Resource (MouSR): a user-friendly tool for rapid biological discovery from human or mouse transcriptional data

abstract:
Generation of transcriptional data has dramatically increased in the last decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by "wet-lab" users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups.

To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called "MouSR". This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive "point and click" interface, which includes a suite of molecular characterisation options including QC, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA-Seq. Users are provided with adjustable options for analysis parameters to generate results that can be saved as publication-quality images. To highlight its ability to perform high quality data analysis, we utilise the MouSR tool to interrogate our recently published tumour dataset, derived from genetically engineered mouse models and matched organoids, where we rapidly reproduced the key transcriptional findings.

The MouSR online tool provides a unique freely-available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery.


title: No evidence for three functionally specialized subregions in the subthalamic nucleus: A model-based 7 T fMRI study

abstract: The subthalamic nucleus (STN) is a small, subcortical brain structure. It is a target for deep brain stimulation, an invasive treatment that reduces motor symptoms of Parkinsons disease. Side effects of DBS are commonly explained using the tripartite model of STN organization, which proposes three functionally distinct subregions in the STN specialized in cognitive, limbic, and motor processing. However, evidence for the tripartite model exclusively comes from anatomical studies and functional studies using clinical patients. Here, we provide the first experimental tests of the tripartite model in healthy volunteers using ultra-high field 7 Tesla (T) functional magnetic resonance imaging (fMRI). 34 participants performed a random-dot motion decision-making task with a difficulty manipulation and a choice payoff manipulation aimed to differentially affect cognitive and limbic networks. Moreover, participants responded with their left and right index finger, differentially affecting motor networks. We analysed BOLD signal in three subregions of equal volume of the STN along the dorsolateral-ventromedial axis, identified using manually delineated high resolution anatomical images. Our results indicate that all segments responded equally to the experimental manipulations, and did not support the tripartite model.


title: Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern

abstract: To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naive and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naive people. Of these, 944 from naive and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naive people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naive people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.


title: Autophagy cargo receptors are secreted via extracellular vesicles and particles in response to endolysosomal inhibition or impaired autophagosome maturation

abstract: The endosome-lysosome (endolysosome) system plays central roles in both autophagic degradation and secretory pathways, including the exocytic release of extracellular vesicles and particles (EVPs). Although previous work has revealed important interconnections between autophagy and EVP-mediated secretion, our molecular understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition that mediates the EVP-associated extracellular release of autophagic cargo receptors, including p62/SQSTM1. This extracellular secretion is highly regulated and critically dependent on multiple ATGs required for the progressive steps of early autophagosome formation as well as Rab27a-dependent exocytosis. Furthermore, the disruption of autophagosome maturation, either due to genetic inhibition of the autophagosome-to-autolyosome fusion machinery or blockade via the SARS-CoV2 viral protein ORF3a, is sufficient to induce robust EVP-associated secretion of autophagy cargo receptors. Finally, we demonstrate that this ATG-dependent, EVP-mediated secretion pathway buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Based on these results, we propose that secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis in response to endolysosomal dysfunction or impaired autophagosome maturation.

category: cell biology


title: Grandparent inference from genetic data: The potential for parentage-based tagging programs to identify offspring of hatchery strays

abstract: Fisheries managers routinely use hatcheries to increase angling opportunity. Many hatcheries operate as segregated programs where hatchery-origin fish are not intended to spawn with natural-origin conspecifics in order to prevent potential negative effects on the natural-origin population. Currently available techniques to monitor the frequency with which hatchery-origin strays successfully spawn in the wild rely on either genetic differentiation between the hatchery- and natural-origin fish or extensive sampling of fish on the spawning grounds. We present a method to infer grandparent-grandchild trios using only genotypes from two putative grandparents and one putative grandchild. We developed estimators of false positive and false negative error rates and showed that genetic panels containing 500 - 700 single nucleotide polymorphisms or 200 - 300 microhaplotypes are expected to allow application of this technique for monitoring segregated hatchery programs. We discuss the ease with which this technique can be implemented by pre-existing parentage-based tagging programs and provide an R package that applies the method.


title: Proteomic analysis reveals different sets of proteins expressed during high temperature stress in two thermotolerant isolates of Trichoderma

abstract: Several species of the soil borne fungus of the genus Trichoderma are known to be versatile, opportunistic plant symbionts, and are the most successful biocontrol agents used in todays agriculture. To be successful in the field conditions, the fungus must endure varying climatic conditions. Studies have indicated that high atmospheric temperature coupled with low humidity is a major limitation for the inconsistent performance of Trichoderma under field conditions. Understanding the molecular modulation associated with such Trichoderma that persist and deliver under abiotic stress condition will aid in exploiting the worth of these organisms for such use. In this study, comparative proteomic analysis using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/time-of-flight (MALDI-TOF-TOF) mass spectrometry was used to identify proteins associated with thermotolerance in two thermotolerant isolates of Trichoderma--T. longibrachiatum 673, TaDOR673 and T. asperellum 7316, TaDOR7316--and 32 differentially expressed proteins were identified. Sequence homology and conserved domains were used to identify these proteins and to assign probable function to them. Thermotolerant isolate, TaDOR673, seemed to employ the stress signaling MAPK pathways and heat shock response pathways to combat the stress condition whereas the moderately tolerant isolate, TaDOR7316, seemed to adapt to high temperature conditions by reducing the accumulation of mis-folded proteins through unfolded protein response pathway and autophagy. Also, there were unique as well as common proteins that were differentially expressed in the two isolates studied.

category: molecular biology


title: Overexpression of the SETD2 WW domain inhibits the phosphor-IWS1/SETD2 interaction and the oncogenic AKT/IWS1 RNA splicing program.

abstract:
Our earlier studies had shown that AKT phosphorylates IWS1, and that following phosphorylation, IWS1 recruits the histone methyltransferase SETD2 to an SPT6/IWS1/ALY complex, which assembles on the Ser2-phosphorylated CTD of RNA Pol II. Recruited SETD2 methylates histone H3 at K36, during transcriptional elongation of target genes, and this regulates multiple steps in RNA metabolism. By regulating the RNA splicing of U2AF2, it controls cell proliferation. Importantly, pathway activity correlates with histological grade, clinical stage, and metastatic potential of lung adenocarcinomas, especially those with EGFR mutations. By regulating nucleocytoplasmic mRNA transport of intronless genes, including those encoding type I IFNs, it regulates sensitivity to viral infection. Here, we show that SETD2 interacts with IWS1 via its WW domain, that the interaction is IWS1 phosphorylation-dependent, and that WW domain overexpression blocks the interaction and inhibits the pathway and its biological outcomes. We conclude that blocking the phosphor-IWS1/SETD2 interaction is feasible and has significant therapeutic potential in human cancer.

SignificanceIWS1 phosphorylation by AKT, primarily AKT3, promotes tumor cell proliferation and correlates positively with the histological grade, clinical stage, and metastatic potential of lung adenocarcinomas, especially those with EGFR mutations. Therefore, targeting the phosphor-IWS1 pathway may benefit patients with these tumors. Inhibiting AKT3 should block this pathway. However, we do not currently have clinical grade AKT3-specific inhibitors, and even if we did, inhibition of AKT3 would have off target effects. Here, we show that SETD2 binds phosphor-IWS1 via its 33 aa-long WW domain and that overexpression of this domain blocks the phosphor-IWS1/SETD2 interaction, inhibiting the pathway and tumor cell proliferation in culture and in animals. Therefore, blocking the phospho-IWS1/SETD2 interaction is feasible and has significant therapeutic potential in human cancer.

category: cancer biology


title: Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii

abstract: Genetic compensation through transcriptional adaptation has been proposed to explain phenotypic differences between gene knockouts and knockdowns. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, e.g., CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a {Delta}vtc1 mutant, in which PDAT was up-regulated under stress. Knockout of PDAT in the {Delta}vtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in {Delta}vtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress with a mechanism distinct from that found in plants. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis, and provides guidance for future genetic engineering and mutant characterization efforts.

category: plant biology


title: Reconstitution of monoterpene indole alkaloid biosynthesis in Nicotiana benthamiana

abstract: Monoterpene indole alkaloids (MIAs) are a diverse and important class of plant natural products that include a number of medicinally significant compounds, often present at low concentrations within their native plant species. The complex biosynthesis of MIAs requires the assembly of tryptamine with a secoiridoid to produce the central intermediate, strictosidine, from which all known MIAs derive. Structural complexity makes chemical synthesis challenging, but recent efforts to identify the biosynthetic enzymes provide options for pathway reconstruction in a heterologous host. Previous attempts have had limited success, with yield in microorganisms limited by the poor expression of some enzymes. Here, we reconstitute the pathway for strictosidine biosynthesis from central metabolism without the need for supplementation of any metabolite precursors or intermediates in Nicotiana benthamiana. The best yields were obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the secoiridoid pathway. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

category: synthetic biology


title: Simultaneous infection with porcine reproductive and respiratory syndrome and influenza viruses abrogates clinical protection induced by live attenuated porcine reproductive and respiratory syndrome vaccination

abstract: The porcine respiratory disease complex (PRDC) is responsible for significant economic losses in the pig industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus are major viral contributors to PRDC. Vaccines are cost-effective measures for controlling PRRS, however, their efficacy in the context of co-infections has been poorly investigated. In this study, we aimed to determine the effect of PRRSV-2 and swine influenza H3N2 virus co-infection on the efficacy of PRRSV modified live virus (MLV) vaccination, which is widely used in the field. Following simultaneous challenge with contemporary PRRSV-2 and H3N2 field isolates, we found that the protective effect of PRRS MLV vaccination on clinical disease and pathology was abrogated, although viral load was unaffected and antibody responses were enhanced. In contrast, co-infection in non-immunized animals reduced PRRSV-2 viremia and H3N2 virus load in the upper respiratory tract and potentiated T cell responses against both PRRSV-2 and H3N2 in the lung. Further analysis suggested that an upregulation of inhibitory cytokines gene expression in the lungs of vaccinated pigs may have influenced responses to H3N2 and PRRSV-2. These findings provide important insights into the effect of viral co-infections on PRRS vaccine efficacy that may help identity more effective vaccination strategies against PRDC in the field.


title: Paramecium Polycomb Repressive Complex 2 physically interacts with the small RNA binding PIWI protein to repress transposable elements

abstract: Polycomb Repressive Complex 2 (PRC2) maintains transcriptionally silent genes in a repressed state via deposition of histone H3 K27 trimethyl (me3) marks. PRC2 has also been implicated in silencing transposable elements (TEs) yet how PRC2 is targeted to TEs remains unclear. To address this question, we performed tandem affinity purification combined with mass spectrometry and identified proteins that physically interact with the Paramecium Enhancer-of-zeste Ezl1 enzyme, which catalyzes H3K9me3 and H3K27me3 deposition at TEs. We show that the Paramecium PRC2 core complex comprises four subunits, each required in vivo for catalytic activity. We also identify PRC2 cofactors, including the RNA interference (RNAi) effector Ptiwi09, which are necessary to target H3K9me3 and H3K27me3 to TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3-9 enzymes.


title: Sphingosine 1-Phosphate Mediates Adiponectin Receptor Signaling Essential For Lipid Homeostasis And Embryogenesis

abstract:
Cells and organisms require proper membrane composition to function and develop. Phospholipids are the major component of membranes and are primarily acquired through the diet. Given great variability in diet composition, cells must be able to deploy mechanisms that correct deviations from optimal membrane composition and properties. Here, using unbiased lipidomics and proteomics, we found that the embryonic lethality in mice lacking the fluidity regulators Adiponectin Receptors 1 and 2 (AdipoR1/2) is associated with aberrant high saturation of the membrane phospholipids. Using mouse embryonic fibroblasts (MEFs) derived from AdipoR1/2-KO embryos, human cell lines and the model organism C. elegans we found that, mechanistically, AdipoR1/2-derived sphingosine 1-phosphate (S1P) signals in parallel through S1PR3-SREBP1 and PPAR{gamma} to sustain the expression of the fatty acid desaturase SCD and maintain membrane properties. Thus, our work identifies an evolutionary conserved pathway by which cells and organism maintain membrane homeostasis and adapt to a variable environment.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/456080v1_ufig1.gif" ALT="Figure 1">
View larger version (52K):
org.highwire.dtl.DTLVardef@b6730aorg.highwire.dtl.DTLVardef@3a95e3org.highwire.dtl.DTLVardef@469027org.highwire.dtl.DTLVardef@a21790_HPS_FORMAT_FIGEXP  M_FIG C_FIG In BriefCells and organisms require proper membrane composition to function and develop. Ruiz et al. identified AdipoR1/2-derived S1P as essential for membrane homeostasis in mammals and invertebrates. S1P signals through SREBP and PPAR{gamma} to sustain fatty acid desaturation. Targeting AdipoR1/2 pathway may be a therapeutic approach for disorders with dysfunctional membranes.

Highlights- Excess membrane phospholipid saturation explains AdipoR1/2 DKO embryonic lethality
- AdipoR1/2 DKO membrane defects are rescued by S1P
- Parallel activation of S1PR3 and PPAR{gamma} by S1P mediates AdipoR2 membrane protection
- Fatty acid desaturation is the final outputs of the AdipoR2-S1P signaling pathway


title: Sclerostin Small Molecule Inhibitors Promote Osteogenesis by Activating Canonical Wnt and BMP Pathways

abstract:
BackgroundThe healing environment within the posterolateral lumbar spine is one of the most clinically challenging bone healing environments in all of orthopaedics due to a lack of a contained space and the need to form de novo bone in a non-bony environment. Our group has previously published data that suggests that sclerostin in expressed locally at high levels throughout the process of a spinal fusion mass maturing.

MethodsWe computationally identified multiple FDA-approved drugs, as well as a novel drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of {beta}-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion.

ResultsWe show that by controlling GSK3b phosphorylation, these SMIs simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft.

ConclusionsFew if any osteogenic small molecules have been described that possess the osteoinductive potency of BMP itself - that is, the ability to form de novo ectopic bone as a standalone agent, particularly in stringent in vivo environments. Herein, we describe two such SMIs that have this unique ability and thus may have potential application as novel cost-effective biologic bone graft substitutes for achieving consistent spinal fusion or even or critical-sized fracture defects.

FundingThis work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).


title: Microtubule deacetylation reduces cell stiffness to allow the onset of collective cell migration in vivo

abstract: Embryogenesis, tissue repair and cancer metastasis rely on collective cell migration (CCM). In vitro studies propose that migrating cells are stiffer when exposed to stiff substrates, known to allow CCM, but softer when plated in compliant non-permissive surfaces. Here, by combining in vivo atomic force microscopy (iAFM) and modelling we reveal that to collectively migrate in vivo, cells require to dynamically decrease their stiffness in response to the temporal stiffening of their native substrate. Moreover, molecular and mechanical perturbations of embryonic tissues uncover that this unexpected cell mechanical response is achieved by a new mechanosensitive pathway involving Piezo1-mediated microtubule deacetylation. Finally, lowering microtubule acetylation and consequently cell stiffness was sufficient to allow CCM in soft non-permissive substrates, suggesting that a fixed value of substrate stiffness is not as essential for CCM as it is reaching an optimal cell-to-substrate stiffness value. These in vivo insights on cell-to-substrate mechanical interplay have major implications to our re-interpretation of physiological and pathological contexts.


title: Neuronal mTORC1 inhibition promotes longevity without suppressing anabolic growth and reproduction in C. elegans

abstract: One of the most robust and reproducible methods to prolong lifespan in a variety of organisms is inhibition of the mTORC1 (mechanistic target of rapamycin complex 1) pathway. mTORC1 is a metabolic sensor that promotes anabolic growth when nutrients are abundant. Inhibition of mTORC1 extends lifespan, but also frequently has other effects such as stunted growth, slowed development, reduced fertility, and disrupted metabolism. It has long been assumed that suppression of anabolism and resulting phenotypes such as impaired growth and reproduction may be causal to mTORC1 longevity, but this hypothesis has not been directly tested. RAGA-1 is an upstream activator of TORC1. Previous work from our lab using a C. elegans model of mTORC1 longevity, the long-lived raga-1 null mutant, found that the presence of raga-1 only in the neurons suppresses longevity of the null mutant. Here, we use the auxin-inducible degradation (AID) system to test whether neuronal mTORC1 inhibition is sufficient for longevity, and whether any changes in lifespan are also linked to stunted growth or fertility. We find that life-long AID of RAGA-1 either in all somatic tissue or only in the neurons of C. elegans is sufficient to extend lifespan. We also find that AID of RAGA-1 or LET-363/mTOR beginning at day 1 of adulthood extends lifespan to a similar extent. Unlike somatic degradation of RAGA-1, neuronal degradation of RAGA-1 doesnt impair growth, slow development, or decrease the reproductive capacity of the worms. Lastly, while AID of LET-363/mTOR in all somatic cells shortens lifespan, neuronal AID of LET-363/mTOR slows aging. This work demonstrates that targeting mTORC1 specifically in the neurons uncouples longevity from growth and reproductive impairments, challenging previously held ideas about the mechanisms of mTORC1 longevity and elucidating the promise of tissue-specific aging therapeutics.


title: SUMOylation of Jun fine-tunes the Drosophila gut immune response

abstract: Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins. The transcriptional regulator Jun, a member of the AP-1, complex is one such SUMO target. We find that Jra, the Drosophila Jun ortholog, is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, that include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. SUMOylation attenuates Jra activity, with the master immune regulator Relish being an important transcriptional target. Our study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation modulating the kinetics of the gut transcriptional immune response.


title: Long reads and Hi-C sequencing illuminate the two-compartment genome of the model arbuscular mycorrhizal symbiont Rhizophagus irregularis

abstract: Chromosome folding links genome structure with gene function by generating distinct nuclear compartments and topologically associating domains (TADs). In mammals, these undergo preferential interactions and regulate gene expression. However, their role in fungal genome biology is unclear. Here, we combine Nanopore (ONT) sequencing with chromatin conformation capture sequencing (Hi-C) to reveal chromosome and epigenetic diversity in a group of obligate plant symbionts; the arbuscular mycorrhizal fungi (AMF). We find that five phylogenetically distinct strains of the model AMF Rhizophagus irregularis carry 33 chromosomes with substantial within species variability in size, as well as in gene and repeat content. Strain-specific Hi-C contact maps all reveal a  checkerboard pattern that underline two dominant euchromatin (A) and heterochromatin (B) compartments. Each compartment differs in the level of gene transcription, regulation of candidate effectors and methylation frequencies. The A-compartment is more gene-dense and contains most core genes, while the B-compartment is more repeat-rich and has higher rates of chromosomal rearrangement. While the B-compartment is transcriptionally repressed, it has significantly more secreted proteins and in planta up-regulated candidate effectors, suggesting a possible host-induced change in chromosome conformation. Overall, this study provides a fine-scale view into the genome biology and evolution of prominent plant symbionts, and opens avenues to study the epigenetic mechanisms that modify chromosome folding during host-microbe interactions.


title: Heterochromatin rewiring and domain disruption-mediated chromatin compaction during erythropoiesis

abstract: Development of mammalian red blood cells involves progressive chromatin compaction and subsequent enucleation in terminal stages of differentiation, but the molecular mechanisms underlying the three-dimensional chromatin reorganization and compaction remains obscure. Here, we systematically analyze the distinct features of higher-order chromatin in purified populations of primary human erythroblasts. Our results reveal that while heterochromatin regions undergo substantial compression, select transcription competent regions with active transcription signature are preferentially maintained to achieve a highly-compacted yet functional chromatin state in terminal erythropoiesis, which is about 20-30% of the nuclear volume compared to that of erythroid progenitors. While the partition of euchromatic and heterochromatic regions (compartment A and B) remain mostly unchanged, H3K9me3 marks relocalize to the nuclear periphery and a significant number of H3K9me3 long-range interactions are formed in the three-dimensional rewiring during terminal erythroid chromatin condensation. Moreover, [~]63% of the topologically associating domain (TAD) boundaries are disrupted, while certain TADs with active chromatin modification are selectively maintained during terminal erythropoiesis. The most well-maintained TADs are enriched for chromatin structural factors CTCF and SMC3, as well as factors and marks of the active transcription state. Finally, we demonstrate that the erythroid master regulator GATA1 involves in safeguarding select essential chromatin domains during terminal erythropoiesis. Our study therefore delineate the molecular characteristics of a development-driven chromatin compaction process, which reveals transcription competence as a key determinant of the select domain maintenance to ensure appropriate gene expression during immense chromatin compaction.


title: A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

abstract:
The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development.

Key PointsO_LIMeta-analysis of 9 mouse bone marrow scRNAseq identifies markers for cell types and hematopoietic development
C_LIO_LICharacterize a replicable functional landscape of cell types by exploiting co-expression
C_LI


title: The longitudinal dynamics and natural history of clonal haematopoiesis

abstract: Human cells acquire somatic mutations throughout life, some of which can drive clonal expansion. Such expansions are frequent in the haematopoietic system of healthy individuals and have been termed clonal haematopoiesis (CH). While CH predisposes to myeloid neoplasia and other diseases, we have limited understanding of how and when CH develops, what factors govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression. Here, we track 697 CH clones from 385 individuals aged 55 or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A, TP53) to over 50%/yr (SRSF2-P95H). Growth rates of clones with the same mutation differed by approximately +/-5%/yr, proportionately impacting "slow" drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole genome-sequenced haematopoietic colonies from 7 older individuals, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations only drove expansion later in life, while growth of TET2-mutant clones showed minimal age-dependency. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterise the lifelong natural history of CH and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.


title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

abstract: The induction of apoptosis upon USP7 (HAUSP) inhibition is established in cancers that contain a wild-type p53 (p53Wt) through the  USP7-Mdm2-p53 axis, but no clear explanation has yet been reported for the same to occur in cancers containing mutant 53 (p53Mut) or even p53 null (p53Null) systems. Instead of this  USP7-Mdm2-p53 axis USP7 also works through an alternative new pathway identified in this study. Here in this study, we observed that the magnitude of apoptosis induction in response to USP7 inhibition was remarkably similar between cancer cells showing p53Null or p53Mut and those with p53Wt. Through a proteomics-based approach, we were able to identify XIAP as a novel interacting partner for USP7. XIAP is a potent and well-characterized member of the inhibitor of apoptosis proteins (IAPs), which function through caspase inhibition. We successfully identified USP7 as a positive regulator of XIAP at post-translational but not at its transcriptional level. Using molecular modelling coupled with domain deletion studies, we show that the first three Ubl domains in association with the catalytic domain of USP7 interact with the BIR2 and the linker region between BIR2 and BIR3 domains of XIAP. Modulation of expression and catalytic activity of USP7 in multiple type of cancer cell lines showed that USP7 stabilizes XIAP through its deubiquitinase activity. We have also observed that USP7 sensitizes cells against chemotherapeutic drugs through stabilization of XIAP. Thus, USP7 promotes tumorigenesis in multiple cancers, via stabilization of XIAP that results in apoptosis inhibition in caspase dependent pathway. Moreover, we observed that combinatorial inhibition of USP7 and XIAP can induce cellular apoptosis in a higher magnitude than their individual inhibition. Additionally, our results indicates that nanoformulated P5091 and P22077 showed higher potency for killing C6 cells in comparison to normal drugs. To the best of our knowledge, this is the first report on identification and validation of XIAP, a crucial E3 ubiquitin ligase, as a novel substrate of the deubiquitinase USP7 and they together involve in empowerment of the tumorigenic potential of cancer cells.


title: Phylogenetic, ecological and intraindividual variability patterns in grass phytolith shape

abstract:
O_LIGrass silica short cell (GSSC) phytoliths appear to be the most reliable source of fossil evidence for tracking the evolutionary history and paleoecology of grasses. In recent years, modern techniques have been used to quantitatively assess phytolith shape variation. This progress has widened opportunities with respect to the classification of grass fossil phytoliths. However, phylogenetic, ecological and intraindividual variability patterns in phytolith shape remain largely unexplored.
C_LIO_LIThe full range of intraindividual phytolith shape variation (3650 2D outlines) from 73 extant grass species, 48 genera, 18 tribes, and 8 subfamilies (with special attention paid to Pooideae) was analysed using the geometric morphometric analysis based on the semilandmarks spanning phytolith outlines.
C_LIO_LIAlthough we showed that 2D phytolith shape is mainly driven by deep-time diversification of grass subfamilies, a closer look uncovered distinct phytolith shape variation in early-diverging lineages of Pooideae.
C_LIO_LIThe phylogenetic pattern in phytolith shape was successfully revealed by applying geometric morphometrics to 2D phytolith shape outlines. This finding strengthens the potential of phytoliths to track the evolutionary history and paleoecology of grasses. Moreover, geometric morphometrics of 2D phytolith shape proved to be an excellent tool for analysis requiring large sums of phytolith outlines, making it useful for quantitative palaeoecological reconstruction.
C_LI


title: Peripheral sTREM2-related inflammatory activity alterations in early stage Alzheimer's disease

abstract: Alzheimers disease (AD) has been linked to multiple immune system genetic variants, implicating potential broad alterations in inflammatory profiles in the disease. Triggering receptor expressed on myeloid cells 2 (TREM2) genetic variants are risk factors for AD and other neurodegenerative diseases. A soluble TREM2 isoform (sTREM2) is elevated in cerebrospinal fluid in the early stages of AD suggesting it may be a biomarker of progressive alterations in immune response to AD-related pathology. Multiple studies have reported an altered peripheral immune response in AD. However, less is known about the relationship between plasma sTREM2 and the altered peripheral immune response in AD. The objective of this exploratory study was to examine the relationship between sTREM2 and inflammatory activity in human participants defined by clinically characterized cognitive symptoms and groups defined by the cerebrospinal fluid biomarkers amyloid beta, phosphorylated tau, and neurodegeneration (NIA-AA Research Framework: "ATN continuum".) The hypothesis of this exploratory study was that sTREM2 related inflammatory activity differs by AD stage. We observed different patterns of inflammatory activity across disease groups and ATN categories that implicates peripheral sTREM2 related inflammatory activity as altered in the early stages of AD. Notably, fractalkine showed a significant relationship with sTREM2 across different analyses in the control groups that was lost as disease progressed, and fractalkine, IL-5 and IL-17A were decreased in AD. These preliminary data provide important support to the hypothesis that sTREM2-related inflammatory activity is a stage-specific biomarker of AD progression, providing the groundwork for future studies and therapeutic strategies.


title: Genome sequencing of the vermicompost strain Stenotrophomonas maltophilia UENF-4GII and population structure analysis of the S. maltophilia Sm3 genogroup

abstract: The Stenotrophomonas maltophilia complex (Smc) is a cosmopolitan bacterial group that has been proposed an emergent multidrug-resistant pathogen. Taxonomic studies support the genomic heterogeneity of Smc, which comprises genogroups exhibiting a range of phenotypically distinct strains from different sources. Here, we report the genome sequencing and in-depth analysis of S. maltophilia UENF-4GII, isolated from vermicompost. This genome harbors a unique region encoding a penicillin-binding protein (pbpX) that was carried by a transposon, as well as horizontally-transferred genomic islands involved in anti-phage defense via DNA modification, and pili glycosylation. We also analyzed all available Smc genomes to investigate genes associated with resistance and virulence, niche occupation, and population structure. S. maltophilia UENF-4GII belongs to genogroup 3 (Sm3), which comprises three phylogenetic clusters (PC). Pan-GWAS analysis uncovered 471 environment-associated and 791 PC-associated genes, including antimicrobial resistance (e.g. blaL1 and blaR1) and virulence determinants (e.g. treS and katG) that provide insights on the resistance and virulence potential of Sm3 strains. Together, the results presented here provide the grounds for more detailed clinical and ecological investigations of S. maltophilia.


title: A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

abstract:
In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

SignificancePhenotypic heterogeneity benefits the proliferation of microbial populations in changing environments. Such heterogeneity can be created by recruitment of enzymatic activity to specific cellular compartments, e.g., the cell pole. Here we show how a GGDEF domain of a multidomain phosphodiesterase has adopted the function as a spatial on-switch that is specifically activated upon direct interaction with a polar landmark protein.


title: Matrix stiffness controls ciliogenesis and centriole position

abstract:
Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including diseases such as osteoarthritis. Substrate stiffness is one of the well-known mechanical properties of the matrix that enabled establishing the central dogma of an integrin-mediated mechanotransduction using stem cells. However, how specific cells  feel or sense substrate stiffness requires further study. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesised that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignalling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of substrate stiffness on cilia frequency, length and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates demonstrating tissue-specific mechanosensing which is aligned with the tissue stiffness the cells originate from. We further show that stiffness alone determines centriole positioning to either the basal or apical membrane during attachment and spreading, with centriole positioned towards the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibres in a time-dependent manner. Based on these findings, we propose that substrate stiffness plays a central role in cilia positioning, regulating cellular response to external forces, and may be a key driver of mechanosignalling-associated diseases.

Significance StatementThe primary cilium has been thrust into the limelight owing to its role as a cellular sensor in embryonic development and adult tissue maintenance. How the primary cilium interacts with the mechanical environment still remains unclear. We show that substrate stiffness dynamically regulates primary cilium length and position through integrin-mediated traction forces, the cilia are a key determinant of cell shape on certain stiffnesses. Our data support the promising potential of primary cilia as a novel target in mechanotherapy for improved clinical outcomes in cartilage pathobiology.


title: BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

abstract: During mitosis, unattached kinetochores in a dividing cell activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset by generating the anaphase-inhibitory Mitotic Checkpoint Complex (MCC). These kinetochores generate the MCC by recruiting MCC constituent proteins, including BubR1. In principle, BubR1 recruitment to signaling kinetochores should increase its local concentration and promote MCC formation. However, in human cells BubR1 is mainly thought to sensitize the SAC to silencing; whether BubR1 localization to signaling kinetochores per se enhances SAC signaling activity remains unknown. Therefore, we used ectopic SAC activation systems (eSAC) to isolate two molecules that recruit BubR1 to the kinetochore: the checkpoint protein Bub1 and the KI and MELT motifs in the kinetochore protein KNL1, and observed their contribution to eSAC signaling. Our quantitative analyses and mathematical modeling show that the Bub1-mediated BubR1 recruitment to the human kinetochore promotes SAC signaling and highlight BubR1s dual role of directly strengthening the SAC and indirectly silencing it.


title: Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

abstract:
NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing reactive oxygen species. Since coenzyme Q (CoQ) mimics affect NOX4 activity, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, modulates inflammatory activation.

Mice lacking endothelial CYB5R3 (R3 KO), under lipopolysaccharides (LPS) challenge, showed exacerbated hypotension, decreased acetylcholine-induced vasodilation, and elevated vascular adhesion molecule 1 (Vcam-1) mRNA in aorta. In vitro, silencing Cyb5r3 enhanced LPS-induced VCAM-1 protein in a NOX4 dependent manner. APEX2- based electron microscopy and proximity biotinylation demonstrated CYB5R3s localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 silenced HAECs had less total H2O2 but more mitochondrial O2*-. Using inactive or non-membrane bound active CYB5R3, we found CYB5R3 activity and membrane translocation were needed for optimal generation of H2O2 by NOX4. Lastly, CoQ deficient cells showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity.

In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

NOVELTY AND SIGNIFICANCEO_ST_ABSWhat Is Known?C_ST_ABSNADPH oxidase 4 (NOX4) reportedly produces primarily hydrogen peroxide (H2O2) and, to a lesser extent, superoxide (O2*-) and has been shown to have both beneficial and deleterious effects in the cardiovascular system.

NOX4 activity can be affected by NAD(P)H quinone oxidoreductase 1 (NQO1), a CoQ reductase, and synthetic quinone compounds used to mimic CoQ.

Cytochrome b5 reductase 3 (CYB5R3) is known to reduce CoQ and is highly expressed in endothelial cells.

What New Information Does This Article Contribute?In vivo, the lack of endothelial CYB5R3 causes exacerbated lipopolysaccharides (LPS)-induced inflammatory signaling, endothelial dysfunction, and hypotension.

Endothelial CYB5R3 mitigates inflammatory signaling by LPS and tumor necrosis factor  (TNF-) in a NOX4 dependent manner.

In endothelial cells, CYB5R3 and NOX4 reside in close proximity on the mitochondrial outer membrane.

NOX4s ability to generate H2O2 depends on the membrane translocation and activity of CYB5R3 and the presence of endogenous CoQ.

NONSTANDARD Abbreviations and Acronyms
[Table 1]

Protein names are abbreviated as capital letters (e.g., CYB5R3), while the corresponding gene names are annotated as in italic lower cases (e.g., Cyb5r3).


title: Loss of the extracellular matrix protein DIG-1 causes glial fragmentation, dendrite breakage, and dendrite extension defects

abstract: The extracellular matrix (ECM) guides and constrains the shape of the nervous system. In C. elegans, DIG-1 is a giant ECM component that is required for fasciculation of sensory dendrites during development and for maintenance of axon positions throughout life. We identified four novel alleles of dig-1 in three independent screens for mutants affecting disparate aspects of neuronal and glial morphogenesis. First, we find that disruption of DIG-1 causes fragmentation of the amphid sheath glial cell in larvae and young adults. Second, it causes severing of the BAG sensory dendrite from its terminus at the nose tip, apparently due to breakage of the dendrite as animals reach adulthood. Third, it causes embryonic defects in dendrite fasciculation in inner labial (IL2) sensory neurons, as previously reported, as well as rare defects in IL2 dendrite extension that are enhanced by loss of the apical ECM component DYF-7, suggesting that apical and basolateral ECM contribute separately to dendrite extension. Our results highlight novel roles for DIG-1 in maintaining the cellular integrity of neurons and glia, possibly by creating a barrier between structures in the nervous system.


title: Large-scale integration of DNA methylation and gene expression array platforms

abstract: AO_SCPLOWBSTRACTC_SCPLOWEpigenome-wide association studies (EWAS) aim to provide evidence that marks of DNA methylation (DNAm) have downstream consequences that can result in the development of human diseases. Although these methods have been successful in identifying DNAm patterns associated with disease states, any further characterization of etiologic mechanisms underlying disease remains elusive. This knowledge gap does not originate from a lack of DNAm-trait associations, but rather stems from study design issues that affect the interpretability of EWAS results. Despite known limitations in predicting the function of a particular CpG site, most EWAS maintain the broad assumption that altered DNAm results in a concomitant change of transcription at the most proximal gene. This study integrated DNAm and gene expression (GE) measurements in two cohorts, the Adolescent and Young Adult Twin Study (AYATS) and the Pregnancy, Race, Environment, Genes (PREG) study, to improve the understanding of epigenomic regulatory mechanisms. CpG sites associated with GE in cis were enriched in areas of transcription factor binding and areas of intermediate-to-low CpG density. CpG sites associated with trans GE were also enriched in areas of known regulatory significance, including enhancer regions. These results highlight issues with restricting DNAm-transcript annotations to small genomic intervals and question the validity of assuming a canonical cis DNAm-GE pathway. Based on these findings, the interpretation of EWAS results is limited in studies without multi-omic support and further research should identify genomic regions in which GE-associated DNAm is overrepresented.


title: De novo chromosome level assembly of the mule deer (Odocoileus hemionus) genome

abstract: The mule deer (Odocoileus hemionus) is an ungulate species that ranges from western Canada to central Mexico. Mule deer are an essential source of food for many predators, are relatively abundant, and commonly make broad migration movements. A clearer understanding of the mule deer genome can help facilitate knowledge of its population genetics, movements, and demographic history, aiding in conservation efforts. While mule deer are excellent candidates for population genomic studies because of their large population size, continuous distribution, and diversity of habitat, few genomic resources are currently available for this species. Here, we sequence and assemble the mule deer genome into a highly contiguous chromosome-length assembly for use in future research using long-read sequencing and Hi-C. We also provide a genome annotation and compare demographic histories of the mule deer and white-tail deer using PSMC. We expect this assembly to be a valuable resource in the continued study and conservation of mule deer.


title: De novo chromosome-length assembly of the mule deer (Odocoileus hemionus) genome


title: Probing 3'UTRs as modular regulators of gene expression

abstract: Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.

category: systems biology


title: Limitations of composability of cis-regulatory elements in messenger RNA


title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

abstract:
Neural crest cells (NCCs) are a migratory population that gives rise to a diverse cell lineage, including the craniofacial complex, the peripheral nervous system, and a part of the heart. Hyaluronan (HA) is a major component of the extracellular matrix, and its tissue levels are dynamically regulated in during development. Although the synthesis of HA has been shown to exert substantial influence on embryonic morphogenesis, the functional importance of the catabolic side of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in NCC development and the morphogenesis of their derivatives. Wnt1-Cre-mediated Tmem2 knockout (Tmem2CKO) mice exhibit severe craniofacial and cardiovascular abnormalities. Analysis of Tmem2 expression using Tmem2 knock-in reporter mice reveals that Tmem2 is expressed at the site of NCC delamination in the neural tube and in Sox9-positive emigrating NCCs, suggesting that Tmem2 is critical for NCC development. Consistent with this possibility, linage tracing analysis reveals that the contribution of Wnt1-Cre-labeled cells to NCC derivatives is significantly reduced in a Tmem2-deficient background. Moreover, the emigration of NCCs from the neural tube is greatly reduced in Tmem2CKO mice. In vitro assays demonstrate that Tmem2 expression is essential for the ability of mouse O9-1 NCCs to form focal adhesion on and migrate into HA-containing substrates. Tmem2CKO mice also exhibit increased apoptotic cell death in NCC-derived tissues. Collectively, our data demonstrate that Tmem2 is essential for normal development of NCC-derivatives, including the craniofacial complex, and that TMEM2-mediated HA degradation allows NCCs to generate a tissue environment suitable for efficient focal adhesion assembly and migration. This study reveals the hitherto unrecognized functional importance of the catabolic side of HA metabolism in embryonic development and highlights the pivotal role of Tmem2 in the process.

Author SummaryThe functional significance of hyaluronan (HA) in embryonic developmental processes has been demonstrated by studies using genetic manipulation of HA synthesis. However, the expression of HA is regulated not only by its synthesis, but also by its degradation. This issue is of particular importance due to the extremely rapid metabolic turnover of HA. Curiously, mice with mutations/ablations of known hyaluronidase molecules, such as the lysosomal hyaluronidases HYAL1 and HYAL2, exhibit little embryonic phenotypes. This suggests the existence of yet another hyaluronidase molecule that plays a key role in regulating extracellular HA balance in developing tissues. In this context, transmembrane protein 2 (TMEM2) is a novel hyaluronidase that functions on the cell surface. Here, we demonstrate that TMEM2 is expressed at the site of neural crest development and in neural crest cell (NSC)-derived craniofacial tissue, and that NCC-targeted Tmem2 conditional knockout mice develop severe craniofacial defects, which attests to a requirement for TMEM2-mediated extracellular HA degradation in neural crest development. Our in vitro and in vivo analyses on the underlying mechanisms of the phenotype demonstrate that TMEM2 is essential for generating a tissue environment suitable for efficient focal adhesion formation by NCCs. This paper reveals for the first time that the catabolic machinery for HA exerts a specific regulatory role in embryonic morphogenesis, and that its dysregulation of HA degradation leads to severe developmental defects.


title: Effects of land-use and landscape drivers in the species richness and distribution of carnivores in Faragosa-Fura Landscape of Southern Rift Valley, Ethiopia

abstract: Understanding the species richness and distribution of carnivores across anthropogenic land-use types in an area is an essential first step for biodiversity conservation and human-carnivore coexistence. However, quantitative data on carnivore species coexisting with humans in different land-use types remain largely missing. Thus, this paper investigated the effect of anthropogenic land-use and landscape drivers on carnivore species richness and distribution in the Faragosa-Fura Landscape, Gamo Zone, southern Ethiopia. To collect data, we employed the line transect method using three complementary field surveys techniques: sign survey, camera-trapping, and opportunistic sighting survey during wet and dry seasons in 2020 and 2021. We stratified the study landscape into five land-use types-forest, wetland, grassland, agricultural land, and settlement. The result proved the occurrence of 12 carnivore species belonging to six families, including vulnerable Felidae species - Panthera pardus. Family Felidae and Herpestidae were composed of a greater number of species, while Hyaenidae and Mustelidae were each represented by single species. Out of identified species, only two species (Panthera pardus and Crocuta crocuta) were large-sized, while the rest were medium and small-sized carnivores. Overall, the mean richness of the study area was 5.73{+/-}0.284(SE). The species richness was highest in the wetland (n = 12, mean = 7.67{+/-}0.494(SE)) and lowest in the settlement (n = 5, mean = 4.25{+/-}0.479(SE)). The regression analysis showed that most of the carnivores displayed a strong negative relationship with agriculture, roads, and settlement while displayed a strong positive relationship with wetland and forest. In general, out of 32 species recorded in Ethiopia, this study quantified 12 carnivore species that signify the area is an important area for wildlife conservation in Ethiopia. Further, the study concluded that the wetland is the most important habitat, particularly for larger-sized and habitat specialists while anthropogenic land-uses types adversely affecting species richness. Thus, a generic paradigm to reconcile land management and biodiversity conservation is highly important.

category: ecology


title: New land use change scenarios for Brazil: refining global SSPs with a regional spatially-explicit allocation model

abstract: The future of land use and cover change in Brazil, in particular due to deforestation and forest restoration processes, is critical for the future of global climate and biodiversity, given the richness of its five biomes. These changes in Brazil depend on the interlink between global factors, due to its role as one of the main exporters of commodities in the world, and the national to local institutional, socioeconomic and biophysical contexts. Aiming to develop scenarios that consider the balance between global and local factors, a new set of land use change scenarios for Brazil were developed, aligned with the global structure Shared Socio-Economic Pathways (SSPs) and Representative Concentration Pathway (RCPs) developed by the global change research community. The narratives of the new scenarios align with SSP1/RCP 1.9, SSP2/RCP 4.5, and SSP3/RCP 7.0. The scenarios were developed combining the LuccME spatially explicit land change allocation modeling framework and the INLAND surface model to incorporate the climatic variables in water deficit. Based on detailed biophysical, socio-economic and institutional factors for each biome in Brazil, we have created spatially-explicit scenarios until 2050, considering the following classes: forest vegetation, grassland vegetation, planted pasture, agriculture, mosaic of small land uses, and forestry. The results aim at regionally detailing global models and could be used both regionally to support decision-making, but also to enrich global analysis.


title: Virulence determinant, PTP7, controls vesicle budding from the Maurers clefts, adhesin protein trafficking and host cell remodeling in Plasmodium falciparum

abstract:
Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins P. falciparum malaria pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurers cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurers clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, found only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.

Author SummaryWe describe a malaria parasite protein involved in virulence factor trafficking (PTP7) that moves between different compartments in the host red blood cell cytoplasm in a stage-dependent manner. Upon disruption of the PTP7 locus, the Maurers cleft trafficking compartments become decorated with vesicles; the knobby protrusions on the host red blood cell surface are depleted and distorted; and trafficking of the virulence protein, EMP1, to the host red blood cell surface is ablated. We provide evidence that a region of PTP7 with low sequence complexity plays an important role in driving fission of vesicles from the Maurers clefts.

category: biochemistry


title: Distinguishing and employing sister species of fish in assessment of stream quality

abstract: Biological indicators (bioindicators) can be individual species or species groups used to assess habitat quality. Unfortunately, conservationists often lack information on species distribution, how to differentiate between similar species, and environmental conditions associated with the presence of a species. We addressed these problems using two "sister" species of fish, the Mottled Sculpin (Cottus bairdii) and the Slimy Sculpin (Cottus cognatus), as stream quality indicators in the Manistee River watershed in the Huron-Manistee National Forests in Michigan, USA. We determined the abundance and distribution of these species and related their presence to concurrent in-stream measurements of temperature, dissolved oxygen, pH, conductivity, turbidity, and stream quality score based on macroinvertebrate diversity. To be certain of identification, we sequenced the Cytochrome c Oxidase Subunit I (CO1) molecular marker for specimens and used it as a DNA barcode to determine a specimens species. Cladistic analyses of CO1 unambiguously supported recognition of Mottled Sculpin and Slimy Sculpin as distinct species, confirming initial 87.5% correct identification using morphological characteristics, with uncertainty limited to juvenile fish. Field determinations increased to 100% correct identification as investigators gained more experience. Both species were most abundant in headwater regions, decreased downstream, and were sympatric at several locations. Mottled Sculpin were more likely to be found at stream locations with lower conductivity, pH, and stream quality scores, whereas Slimy Sculpin presence was more strongly associated higher levels of DO and lower levels of turbidity. Such findings are important because Mottled Sculpin are a designated management indicator species of the US Forest Service in the Huron-Manistee National Forests, but may be ineffective as a habitat quality indicator when used alone. Concurrent use of Mottled Sculpin and Slimy Sculpin as a management indicator sister-species complex could allow sufficient landscape coverage to permit habitat assessment if species-specific differences in environmental tolerances are precisely determined.


title: High molecular weight hyaluronan - a potential adjuvant to fluid resuscitation in porcine abdominal sepsis

abstract:
While fluid resuscitation is fundamental in the treatment of sepsis-induced tissue hypo-perfusion, a sustained positive fluid balance is associated with excess mortality. Crystalloids are the mainstay of fluid resuscitation and use of either synthetic colloids or albumin is controversial. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water, has not been tested as adjuvant in fluid resuscitation.

We sought to evaluate the effects of hyaluronan as an adjuvant to fluid resuscitation in peritonitis induced sepsis. In a prospective, parallel-grouped, blinded model of porcine peritonitis-sepsis, we randomized animals to intervention with adjuvant hyaluronan (add-on to standard therapy) (n=8) or 0.9% saline (n=8). After the onset of hemodynamic instability the animals received an initial bolus of 0.1 % hyaluronan 1 mg/kg/10 min or placebo (saline) followed by a continuous infusion of 0.1% hyaluronan (1 mg/kg/h) or saline during the experiment. We hypothesized that the administration of hyaluronan would reduce the volume of fluid administered (aiming at stroke volume variation <13%) and/or attenuate the inflammatory reaction.

Total volumes of intravenous fluids infused were 17.5 {+/-} 11 ml/kg/h vs. 19.0 {+/-} 7 ml/kg/h in intervention and control groups, respectively (p = 0.442). Plasma IL-6 increased to 2450 (1420 - 6890) pg/ml and 3700 (1410 - 11960) pg/ml (18 hours of resuscitation) in the intervention and control groups (NS). In a post-hoc analysis, modified shock index remained lower in intervention group (p = 0.011 - 0.037).

In conclusion adjuvant hyaluronan did not reduce the volume needed for fluid administration or decrease the inflammatory reaction. Adjuvant hyaluronan was, however, associated with lower modified shock index. Bearing in mind that the experiment has a limited group-size we suggest that further studies on hyaluronan in sepsis are warranted.

category: pharmacology and toxicology


title: Mechanisms of Coupling between Angiotensin Converting Enzyme 2 and Nicotinic Acetylcholine Receptors

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus encapsulated by a spike (S) glycoprotein envelope, binds with high affinity to angiotensin converting enzyme 2 (ACE2) during cell entry of a susceptible host. Recent studies suggest nicotinic acetylcholine receptors (nAChRs) play a role in functional ACE2 regulation and nicotine may contribute to the progression of coronavirus disease 2019 (COVID-19). Here, we present evidence for coupling between ACE2 and nAChR through bioinformatic analysis and cell culture experiments. Following molecular and structural protein comparison of over 250 ACE2 vertebrate orthologues, a region of human ACE2 at positions C542-L554 was identified to have sequence similarity to nAChR-binding neurotoxin and rabies virus glycoproteins (RBVG). Furthermore, experiments conducted in PC12 cells indicate a potential for physical interaction between ACE2 and 7 nAChR proteins. Our findings support a model of nAChR involvement in in COVID-19.


title: Integrin signaling via actin cytoskeleton activates MRTF/SRF to entrain circadian clock

abstract:
The circadian clock is entrained to daily environmental cues. Integrin-linked intracellular signaling via actin cytoskeleton dynamics transduces cellular niche signals to induce Myocardin-related Transcription Factor (MRTF)/Serum Response Factor (SRF)-mediated transcription. So far, how the integrin-associated signaling cascade may transmit cellular physical cues to entrain circadian clock remains to be defined. Using combined pharmacological and genetic approaches, here we show that the transcription factors mediating integrin to actin cytoskeleton signaling, MRTF-A and SRF, exert direct transcriptional control of core clock components, and that this signaling cascade modulates key properties of clock circadian activity. Pharmacological inhibition of MRTF/SRF activity by disrupting actin polymerization significantly augmented clock amplitude with period shortening, whereas an actin polymerizing compound attenuated oscillation amplitude with period lengthening. Genetic loss-of-function of Srf or Mrtf mimics that of actin-depolymerizing agents, validating the role of actin dynamics in driving clock function. Furthermore, integrin-mediated focal adhesion with extracellular matrix and its downstream signaling modulates the circadian clock, as blockade of integrin, focal adhesion kinase or Rho-associated kinase (ROCK) increased clock amplitude and shortened period length. Mechanistically, we identify specific core clock transcription regulators, Per1, Per2 and Nr1d1, as direct target genes of MRTF-A/SRF. Collectively, our findings uncovered an integrin-actin cytoskeleton-MRTF/SRF signaling cascade in linking clock entrainment to its extracellular microenvironment, which may mediate cellular adaptation to its physical niche.

Author SummaryThe circadian clock anticipates and adapts to environmental changes. Interestingly, serum, as a universal clock synchronizing signal, drives intracellular actin cytoskeleton reorganization through modulation of MRTF/SRF activity. However, mechanisms that may transduce extracellular niche signals to circadian clock remains to be defined. We hypothesize that integrin-mediated intracellular signaling to actin cytoskeleton links extracellular microenvironment with MRTF/SRF transcriptional regulation to control clock function. Using small molecules and genetic approaches targeting distinct steps of integrin-actin cytoskeleton-MRTF/SRF signaling cascade, we uncover the effects of this pathway in controlling circadian clock oscillation. We also identify specific core clock regulators as direct gene target genes of MRTF and SRF-mediated transcriptional control. Our study revealed how integrin-mediated cellular interaction with its physical environment influences its intrinsic clock properties through signaling transduction via actin cytoskeleton remodeling, and that this mechanism may facilitate circadian clock adaptation to cellular physical niche.


title: Predicting the distributions of Pouteria adolfi-friederici and Prunus africana iconic tree species under current and future climate change scenarios in Ethiopia

abstract: The distributions of the potential adaptive ranges of iconic plant species are not yet fully known especially in regions such as Ethiopia where high climatic variability and vegetation types are found. This study was undertaken to predict the distributions of the potential suitable habitats of Pouteria adolfi-friederici and Prunus africana tree species under the current and two future climate scenarios (i.e., RCP 4.5 and RCP 8.5 in 2050 and 2070) using MaxEnt software (version: 3.4.4.). Eleven less correlated environmental variables (r<0.7) were identified and used to make the prediction models. Elevational shifts of the highly suitable habitats, effects of elevation, solar radiation and topographic position in relation to the current and future climatic scenarios on the habitats were statistically analyzed using independent t-test and linear model. Under all climate scenarios, we found a decrease in the proportion of areas of highly suitable habitats for both study species. High potentials of suitable habitats for Pouteria adolfi-friederici are predicted to be confined to southwest, west central and south parts of Ethiopia in fragmented moist afromontane forest patches, while it is in the southwest and west central parts of Ethiopia for Prunus africana. On basis of vegetation types of the country, potential suitable habitats for Pouteria adolfi-friederici are predicted to occur in moist evergreen forest, dry evergreen forest and grassland complex and Combretum-Terminalia woodland vegetation types. Whereas, moist evergreen forest, dry evergreen forest and grassland complex and riverine vegetation types are predicted to comprise potential suitable habitats for Prunus africana, showing considerable spatial dynamics. Overall, our results suggest that the strategies deem to design biodiversity conservation should take into account the dynamics of the suitable niches of different species under different future climate scenarios.


title: An efficient method to identify, date and describe admixture events using haplotype information

abstract: We present fastGLOBETROTTER, an efficient new haplotype-based technique to identify, date and describe admixture events using genome-wide autosomal data. With simulations, we demonstrate how fastGLOBETROTTER reduces computation time by 4-20 fold relative to the haplotype-based technique GLOBETROTTER without suffering loss of accuracy. We apply fastGLOBETROTTER to a cohort of >6000 Europeans from ten countries, revealing previously unreported admixture signals. In particular we infer multiple periods of admixture related to East Asian or Siberian-like sources, starting >2000 years ago, in people living in countries north of the Baltic Sea. In contrast, we infer admixture related to West Asian, North African and/or Southern European sources in populations south of the Baltic Sea, including admixture dated to {approx}300-700CE, overlapping the fall of the Roman Empire, in people from Belgium, France and parts of Germany. Our new approach scales to analysing hundreds to thousands of individuals from a putatively admixed populations and hence is applicable to emerging large-scale cohorts of genetically homogeneous populations.


title: Mixed selectivity in the cerebellar Purkinje-cell response during visuomotor association learning

abstract: Although the cerebellum has been traditionally considered to be exclusively involved in motor control, recent anatomical and clinical studies show that it also has a role in reward-processing. However, the way in which the movement related and the reward related neural activity interact at the level of the cerebellar cortex and contribute towards learning is still unclear. Here, we studied the simple spike activity of Purkinje cells in the mid-lateral cerebellum when monkeys learned to associate a right or left-hand movement with one of two visual symbolic cues. These cells had distinctly different discharge patterns between an overtrained symbol-hand association and a novel symbol-hand association, responding in association with the movement of both hands, although the kinematics of the movement did not change between the two conditions. The activity change was not related to the pattern of the visual symbols, the movement kinematics, the monkeys reaction times or the novelty of the visual symbols. The simple spike activity changed with throughout the learning process, but the concurrent complex spikes did not instruct that change. Although these neurons also have reward-related activity, the reward-related and movement-related signals were independent. We suggest that this mixed-selectivity may facilitate the flexible learning of difficult reinforcement learning problems.


title: Binding specificity of ASHH2 CW-domain towards H3K4me1 ligand is coupled to its structural stability through its α1-helix

abstract: The CW-domain binds to histone-tail modifications found in different protein families involved in epigenetic regulation and chromatin remodelling. CW-domains recognize the methylation state of the fourth lysine on histone 3, and could therefore be viewed as a reader of epigentic information. The specificity towards different methylation states such as me1, me2 or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2-methyltransferase binds preferentially to H3K4me1, MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings are not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity towards H3K4me1 and the stabilization of the domain and loops implicated in binding. Key elements of the binding site - the two tryptophans and the 1-helix form and maintain the binding pocket were perturbed by mutagenesis and investigated. Results show that 1-helix maintains the overall stability of the fold via the I915 and L919 residues, and that correct binding consolidates the loops designated {eta}1, {eta}3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability upon binding. Moreover, loop-mutations not directly involved in the binding site nonetheless affect the equillibrium positions of key residues.

category: biophysics


title: Phylogenetic reconciliation reveals extensive ancestral recombination in Sarbecoviruses and the SARS-CoV-2 lineage

abstract: An accurate understanding of the evolutionary history of rapidly-evolving viruses like SARS-CoV-2, responsible for the COVID-19 pandemic, is crucial to tracking and preventing the spread of emerging pathogens. However, viruses undergo frequent recombination, which makes it difficult to trace their evolutionary history using traditional phylogenetic methods. Here, we present a phylogenetic workflow, virDTL, for analyzing viral evolution in the presence of recombination. Our approach leverages reconciliation methods developed for inferring horizontal gene transfer in prokaryotes, and, compared to existing tools, is uniquely able to identify ancestral recombinations while accounting for several sources of inference uncertainty, including in the construction of a strain tree, estimation and rooting of gene family trees, and reconciliation itself. We apply this workflow to the Sarbecovirus subgenus and demonstrate how a principled analysis of predicted recombination gives insight into the evolution of SARS-CoV-2. In addition to providing confirming evidence for the horseshoe bat as its zoonotic origin, we identify several ancestral recombination events that merit further study.

category: evolutionary biology


title: MorphoGraphX 2.0: Providing context for biological image analysis with positional information

abstract: Positional information is a central concept in developmental biology. In developing organs, positional information can be idealized as a local coordinate system that arises from morphogen gradients controlled by organizers at key locations. This offers a plausible mechanism for the integration of the molecular networks operating in individual cells into the spatially-coordinated multicellular responses necessary for the organization of emergent forms. Understanding how positional cues guide morphogenesis requires the quantification of gene expression and growth dynamics in the context of their underlying coordinate systems. Here we present recent advances in the MorphoGraphX software (Barbier de Reuille et al. eLife 2015;4:e05864) that implement a generalized framework to annotate developing organs with local coordinate systems. These coordinate systems introduce an organ-centric spatial context to microscopy data, allowing gene expression and growth to be quantified and compared in the context of the positional information thought to control them.

category: developmental biology