Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein coding-loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modelling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.

Contactpetr.pecinka@osu.cz; tlb20@cam.ac.uk
Identification and analysis of novel variants in SARS-COV-2 genomes isolated from the Kingdom of Bahrain The challenges imposed by the ongoing outbreak of severe acute respiratory syndrome coronavirus-2 affects every aspect of our modern world, ranging from our health to our socio-economic needs. Our existence highly depends on the vaccines availability, which demands in-depth research of the available strains and their mutations. In this work, we have analyzed all the available SERS-CoV2 genomes isolated from the Kingdom of Bahrain in terms of their variance and origin analysis. We have predicted various known and unique mutations in the SERS-CoV2 isolated from Bahrain. The complexity of the phylogenetic tree and dot plot representation of the strains mentioned above with other isolates of Asia indicates the versatility and multiple origins of Bahrains SERS-CoV2 isolates. We have also identified two high impact spike mutations from these strains which increase the virulence of SARS-CoV2. Our research could have a high impact on vaccine development and distinguishes the source of SERS-CoV2 in the Kingdom of Bahrain.
Programmed knockout mutation of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic opisthorchiasis Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to a nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We produced programmed gene knockout flukes ({Delta}Ov-grn-1) by delivery of a CRISPR/Cas9/gRNA system by electroporation. Genome sequencing confirmed Cas9-catalyzed mutations in the targeted genes, which was accompanied by rapid depletion of transcripts and the cognate proteins. Whereas Ov-grn-1 gene-edited parasites colonized the biliary tract and developed into adult flukes, less hepatobiliary tract disease manifested during chronic infection with{Delta} Ov-grn-1 worms in comparison to hamsters infected with control parasites. Specifically, immunohistochemical analysis of thin sections of livers revealed markedly less periductal fibrosis surrounding the flukes and less liver fibrosis globally during infection with{Delta} Ov-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and markedly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade cholangiocarcinoma when infected with the{Delta} Ov-grn-1 flukes compared to controls. The clinically-relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs. Tradeoff breaking as model of evolutionary transitions in individuality and the limits of the fitness decoupling metaphor Evolutionary transitions in individuality (ETIs) involve the formation of Darwinian collectives from Darwinian particles. The transition from cells to multicellular life is a prime example. During an ETI, collectives become units of selection in their own right. However, the underlying processes are poorly understood. One observation used to identify the completion of an ETI is an increase in collective-level performance accompanied by a decrease in particle-level performance, for example measured by growth rate. This seemingly counterintuitive dynamic has been referred to as "fitness decoupling" and has been used to interpret both models and experimental data. Using a mathematical approach, we show this concept to be problematic in that the fitness of particles and collectives can never decouple--calculations of particle and collective fitness performed over appropriate and equivalent time intervals are necessarily the same. By way of solution, we draw attention to the value of mechanistic approaches that emphasise traits, and tradeoffs among traits, as opposed to fitness. This trait-based approach is sufficient to capture dynamics that underpin evolutionary transitions. In addition, drawing upon both experimental and theoretical studies, we show that while early stages of transitions might often involve tradeoffs among particle traits, later--and critical--stages are likely to involve the rupture of such tradeoffs. Thus, tradeoff-breaking stands as a useful marker for ETIs. Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Similar to the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8 - all identified as multifunctional RBPs by our analysis - selectively influence target translation rates depending on 5 UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression. Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3'-to-5' exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Significance StatementThe ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2. Computationally scalable regression modeling for ultrahigh-dimensional omics data with ParProx Statistical analysis of ultrahigh-dimensional omics scale data has long depended on univariate hypothesis testing. With growing data features and samples, the obvious next step is to establish multivariable association analysis as a routine method for understanding genotype-phenotype associations. Here we present ParProx, a state-of-the-art implementation to optimize overlapping group lasso regression models for time-to-event and classification analysis, guided by biological priors through coordinated variable selection. ParProx not only enables model fitting for ultrahigh-dimensional data within the architecture for parallel or distributed computing, but also allows users to obtain interpretable regression models consistent with known biological relationships among the independent variables, a feature long neglected in statistical modeling of omics data. We demonstrate ParProx using three different omics data sets of moderate to large numbers of variables, where we use genomic regions and pathways to arrive at sparse regression models comprised of biologically related independent variables. ParProx is naturally applicable to a wide range of studies using ultrahigh-dimensional omics data, ranging from genome-wide association analysis to single cell sequencing studies where multivariable modeling is computationally intractable. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the kinetic mechanisms of complex eukaryotic gene regulation. Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses.

One Sentence SummaryIsolation of antibody germline precursors targeting the receptor binding domain of coronaviruses. Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning requires that ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate. How much does it take to be old? Modelling the time since the last harvesting to infer the distribution of overmature forests in France AimThe distribution of overmature forests in metropolitan France is poorly known, with only a few well-studied prominent sites, and has never been evaluated countrywide. Here, we modelled French forest reserves time since the last harvesting operation - a proxy for forest maturity - then inferred the current statistical distribution of overmature forests (i.e. forests over 50 years without harvesting) in France.

LocationMetropolitan France

MethodsWe used inventories from forest reserves and managed forests to calibrate a generalised linear mixed model explaining the time since the last harvesting with selected structural attributes and environmental variables. We then projected this model on the independent National Forest Inventory dataset. We thus obtained an updated estimation of the proportion and a rough distribution of overmature forest stands in metropolitan France.

ResultsWe found that high basal area of very large trees, high volumes of standing and downed deadwood, high diversity of tree-related microhabitats and more marginally diversity of decay stages best characterized the time since the last harvesting. Volumes of stumps and high density of coppices translating legacy of past forest management also distinguished more overmature plots. Our projection yielded an estimated 3% of French forests over 50 years without harvesting mostly located in more inaccessible areas (i.e. mountainous areas) and a promising proportion of future overmature forests if left unharvested.

Main conclusionsOur study showed that the time since the last harvesting is a good proxy for a combination of stand structure attributes key in characterising overmature temperate forests. It gives the first robust statistical estimate of the proportion of overmature forests and may serve to report on their status in metropolitan France. Our method could be implemented at a larger spatial scale, notably in countries with accessible National Forest Inventory and calibration data, to produce indicators at international level. Material-mediated histogenesis using mechano-chemically microstructured cell niches Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models. A small molecule antagonizes jasmonic acid perception and auxin responses in vascular and nonvascular plants The phytohormone JA-Ile regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein COI1 (Coronatine Insensitive 1) and a JAZ (Jasmonate ZIM-domain) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signalling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1- JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signalling, J4 works as an antagonist of the closely-related auxin signalling pathway, preventing TIR1/Aux-IAA interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways.

One sentence summaryA chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and non-vascular plants. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Graph Ricci Curvatures Reveal Atypical Functional Connectivity in Autism Spectrum Disorder While standard graph-theoretic measures have been widely used to characterize atypical resting-state functional connectivity in autism spectrum disorder (ASD), geometry-inspired network measures have not been applied. In this study, we apply Forman-Ricci and Ollivier-Ricci curvatures to compare networks of ASD and typically developing individuals (N = 1112) from the Autism Brain Imaging Data Exchange I (ABIDE-I) dataset. We find brain-wide and region-specific ASD-related differences for both Forman-Ricci and Ollivier-Ricci curvatures, with region-specific differences concentrated in Default Mode, Somatomotor and Ventral Attention networks for Forman-Ricci curvature. We use meta-analysis decoding to demonstrate that brain regions with curvature differences are associated to those cognitive domains known to be impaired in ASD. Further, we show that brain regions with curvature differences overlap with those brain regions whose non-invasive stimulation improves ASD-related symptoms. These results suggest the utility of graph Ricci curvatures in characterizing atypical connectivity of clinically relevant regions in ASD and other neurodevelopmental disorders. Large Bi-ethnic Study of Plasma Proteome Leads to Comprehensive Mapping of cis-pQTL and Models for Proteome-wide Association Studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Modulation of P2X4 pore closure by magnesium,potassium, and ATP The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+. Genome editing excisase origins illuminated by somatic genome of Blepharisma Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout a copy of the ciliate germline genome are deleted during development of the streamlined somatic genome. Blepharisma represents one of the two earliest diverging ciliate classes, and, unusually, has dual pathways of somatic nuclear development, making it ideal for investigating the functioning and evolution of these processes. Here, we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes. This genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We propose PiggyBac homologs were ancestral excisases that enabled evolution of extensive, natural genome editing. The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testins mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state. Effect of tag attachment on the flight performance of five raptor species Bio-logging devices play a fundamental and indispensable role in movement ecology studies, particularly in the wild. However, researchers are becoming increasingly aware of the influence that attaching devices can have on animals, particularly on their behaviour, energy expenditure and survival. The way a device is attached to an animals body has also potential consequences for the collected data, and quantifying the type and magnitude of such potential effects is fundamental to enable researchers to combine and compare data from different studies, as much as it is to improve animal welfare.

For over two decades, large terrestrial birds have been in the focus of long-term movement ecology research, employing bio-logging devices attached with different types of harnesses. However, comparative studies investigating the effects of different harness types used on these species are scarce.

In this study, we tested for potential differences in data collected by two commonly used harness types, backpack and leg-loop, on the flight performance of 10 individuals from five raptor species, equipped with high resolution bio-logging devices, in the same area and time. We explored the effect of harness type on vertical speed, horizontal speed, glide ratio, height above sea level, distance travelled, proportion of soaring and flapping behaviour, and VeDBA (a proxy for energy expenditure) between and within individuals, all used as fine-scale measures of flight performance. Birds equipped with leg-loops climbed up to 0.65 ms-1 faster, reached 19% greater heights while soaring, and spent less time in active flight compared to birds equipped with backpacks, suggesting that backpack harnesses, compared to leg-loops, might cause additional drag affecting the birds flight performance. A lower rate of sinking while gliding, a slightly higher glide ratio, higher horizontal speed while soaring, and lower VeDBA, were also indicative of less drag using leg-loops. Our results add to the existing literature highlighting the design-related advantages of leg-loops, and support the use of leg-loops as a better alternative to backpack harnesses for large soaring birds, when possible. Our study also highlights how apparently small changes in device attachment can lead to notable improvements in tagging practice, with implications for animal welfare, data interpretation and comparability. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land.