A novel sodium signaling complex regulates uterine activity Depolarization of the myometrial smooth muscle cell (MSMC) resting membrane potential is necessary for the transition of the uterus from a quiescent state to a contractile state. The molecular mechanisms involved in this transition are not completely understood. Here, we report a novel coupled system between the Na+-activated K+ channel (SLO2.1) and the non-selective Na+ leak channel (NALCN) which determines the MSMC membrane potential. We show that SLO2.1 currents are activated by an inward Na+ leak current carried by the NALCN channel leading to MSMC hyperpolarization. These results show an unanticipated role for the Na+ leak currents in activating a negative feedback system countering the excitable effects of Na+ currents. This is a novel role for the NALCN channel in which Na+ acts as an intracellular signaling molecule. In fact, we report here that the net effect of Na+ entry through NALCN channels is a hyperpolarization of the MSMCs plasma membrane because of the activation of SLO2.1 K channel. Importantly, we also report that a decrease in NALCN/SLO2.1 activity triggers both Ca2+ entries through VDCCs, promoting myometrial contraction. Consistently, with a functional coupling, our data show that NALCN and SLO2.1 are in proximity to one another in human MSMCs. We propose that the spatial arrangement of SLO2.1 and NALCN permits these channels to functionally interact in order to regulate human MSMC membrane potential and cell excitability to modulate uterine contractile activity.
Biomechanical property of limbal niche maintains stemness through YAP Stem cells (SCs) decision to self-renew or differentiate largely depends on the external control of their niche. However, the complex mechanisms that underlie this crosstalk are poorly understood. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit YAP localization and induce differentiation, a mechanism that is mediated by the TGFbeta-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.
Mechanical bistability of the mesoderm epithelium facilitates mesoderm invagination during Drosophila gastrulation Apical constriction driven by non-muscle myosin II ("myosin") provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of myosin, we find that during Drosophila mesoderm invagination, myosin contractility is critical to prevent tissue relaxation during the early, "priming" stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. The binary response suggests that the mesoderm is mechanically bistable during gastrulation. Combined modeling analysis and experimental measurements suggest that the observed mechanical bistability may arise from apicobasal shrinkage of the surrounding ectoderm, which promotes mesoderm invagination by facilitating a buckling transition. Our results suggest that Drosophila mesoderm invagination requires a joint action of local myosin contractility and mechanical bistability of the epithelium to trigger epithelial buckling. First detection of herpesvirus and frequency of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds. Environmental and genetic disease modifiers of haploinsufficiency of A20 Monogenic diseases can often manifest diverse clinical phenotypes and cause diagnostic dilemmas. While monoallelic loss-of-function variants in TNFAIP3 (Haploinsufficiency of A20; HA20) cause a highly penetrant autoinflammatory disease, the variable expressivity suggest a role for additional genetic and environmental disease modifiers. Here, we identify critically ill children who inherited a family-specific TNFAIP3 deletion from one of their otherwise healthy parents. Each of the probands also inherited in trans a subtle loss-of-function I207L TNFAIP3 variant that is common in Oceania, originally introgressed from Denisovans. Modelling this compound heterozgous state in mice under specific pathogen free conditions demonstrated a reduced threshold to break immune tolerance. Exaggerated immune responses were precipitated by inheriting the two genetic hits on the TNFAIP3 checkpoint coupled with increasing the microbial challenge to immune tolerance, either by co-housing with pet store mice carrying a wild microbial burden or by transient dietary exposure to a chemical that diminishes the intestinal mucin barrier separating gut microbes from immune sensing systems. These data illuminate second-hit genetic and environmental modifiers contributing to complex inflammatory and autoimmune disease. Increased mechanistic understanding of the presence and contribution of disease modifiers will aid diagnostic and prognostic patient stratification and potentially reveal novel therapeutic opportunities. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. Analysis of the leaf metabolome in Arabidopsis thaliana mutation accumulation lines reveals association of pleiotropy and fitness consequences Understanding the mechanisms by which mutations affect fitness and the distribution of mutational effects are central goals in evolutionary biology. Mutation accumulation (MA) lines have long been an important tool for understanding the effect of new mutations on fitness, phenotypic variation, and mutational parameters. However, there is a clear gap in predicting the effect of specific new mutations to their effects on fitness. Here, we complete gene ontology analysis and metabolomics experiments on Arabidopsis thaliana MA lines to determine how spontaneous mutations directly affect global metabolic output in lines that have measured fitness consequences. For these analyses, we compared three lines with relative fitness consistently higher than the unmutated progenitor and three lines with lower relative fitness as measured in four different field trials. In a gene ontology analysis, we find that the high fitness lines were significantly enriched in mutations in or near genes with transcription regulator activity. We also find that although they do not have an average difference in the number of mutations, low fitness lines have significantly more metabolic subpathways disrupted than high fitness lines. Taken together, these results suggest that the effect of a new mutation on fitness depends less on the specific metabolic pathways disrupted and more on the pleiotropic effects of those mutations, and that organisms can explore a considerable amount of physiological space with only a few mutations.

Significance StatementAs the source of all genetic variation, new mutations are crucial for understanding how organisms adapt to their environment. However, the ways in which new mutations affect the range of metabolic reactions that occur in the cell is unknown. With a combination of gene functional analyses and measurement of the small molecules that drive cellular function and physiology, we find that mutations associated with high fitness are disproportionately found in or near proteins-coding genes that regulate the specific timing and location of gene expression and are less disruptive of cellular physiology. Spontaneous Chiral Symmetry Breaking in a Random Driven Chemical System Living systems have evolved to efficiently consume available energy sources using an elaborate circuitry of chemical reactions which, puzzlingly, bear a strict restriction to asymmetric chiral configurations. While autocatalysis is known to promote such chiral symmetry breaking, whether a similar phenomenon may also be induced in a more general class of configurable chemical systems--via energy exploitation--is a sensible yet underappreciated possibility. This work examines this question within a model of randomly generated complex chemical networks. We show that chiral symmetry breaking may occur spontaneously and generically by harnessing energy sources from external environmental drives. Key to this transition are intrinsic fluctuations of achiral-to-chiral reactions and tight matching of system configurations to the environmental drives, which together amplify and sustain diverged enantiomer distributions. These asymmetric states emerge through steep energetic transitions from the corresponding symmetric states and sharply cluster as highly-dissipating states. The results thus demonstrate a generic mechanism in which energetic drives may give rise to homochirality in an otherwise totally symmetrical environment, and from an early-life perspective, might emerge as a competitive, energy-harvesting advantage. Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. Fitness-related traits are maximized in recently introduced, slow-growing populations of a global invasive clam Many species are shifting their ranges being forced to rapidly respond to novel stressful environmental conditions. Colonizing individuals experience strong selective forces that favor the expression of life history traits notably affecting dispersal and reproductive rates in newly invaded habitats. Limited information is currently available on trait variation within the invasive range despite being critical for understanding ecological and evolutionary factors that drive the process of range expansion of invasive species. Here we evaluated life history shifts of the widely introduced Asian clam Corbicula, within its invaded range. Through an exhaustive literature search, we obtained data for 17 invasive Corbicula populations from different ecosystems worldwide. We tested the relationship between population and individual parameters relevant to the process of range expansion. Our main results are that recently introduced Corbicula populations were characterized by (i) low density and low rate of population increase, (ii) clams reproduced earlier in slow-growing populations, and (iii) density had no effect on population increase. All Corbicula populations analyzed in this study, which are fixed for one genotype (lineage Form A/R), experienced different selective environments in the introduced range. These findings support the perspective that adaptive phenotypic plasticity favored the expression of traits that maximize fitness in recently established populations, which faced stronger r-selective forces relative to long-established ones. We discuss the role of plasticity in facilitating rapid adaptation and increasing the likelihood of populations to overcome difficulties associated with low densities and low population increase in newly invaded areas. LabGym: quantification of user-defined animal behaviors using learning-based holistic assessment Quantifying animal behavior is important for many branches of biological research. Current computational tools for quantifying behaviors typically rely on a few pre-defined, simplified features to identify a behavior. However, such an approach underuses the behavioral information and restricts the tools applicability to limited behavior types or animal species. Here we report a new tool, LabGym, for quantifying animal behaviors without such limitations. Combining a novel approach for effective evaluation of animal motion with customizable convolutional recurrent networks for capturing spatiotemporal details, LabGym provides holistic behavioral assessment and accurately identify user-defined animal behaviors without restrictions on behavior types or animal species. It then calculates various parameters to provide quantitative measurements of the intensity and dynamics of each behavior. LabGym requires neither any intermediate step that causes information loss nor programming knowledge from users for post-hoc analysis. It tracks multiple animals simultaneously in various experimental settings for high-throughput and versatile analysis, and provides users a way to generate visualizable behavioral datasets that are valuable resources for research in neuroscience, behavioral science, and computer science. We demonstrate its efficacy in capturing subtle behavioral changes in animals ranging from soft-bodied invertebrates to mammals. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Modulation Differentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors. Dug: A Semantic Search Engine Leveraging Peer-Reviewed Knowledge to Span Biomedical Data Repositories MotivationAs the number of public data resources continues to proliferate, identifying relevant datasets across heterogenous repositories is becoming critical to answering scientific questions. To help researchers navigate this data landscape, we developed Dug: a semantic search tool for biomedical datasets utilizing evidence-based relationships from curated knowledge graphs to find relevant datasets and explain why those results are returned.

ResultsDeveloped through the National Heart, Lung, and Blood Institutes (NHLBI) BioData Catalyst ecosystem, Dug has indexed more than 15,911 study variables from public datasets. On a manually curated search dataset, Dugs total recall (total relevant results/total results) of 0.79 outperformed default Elasticsearchs total recall of 0.76. When using synonyms or related concepts as search queries, Dug (0.36) far outperformed Elasticsearch (0.14) in terms of total recall with no significant loss in the precision of its top results.

Availability and ImplementationDug is freely available at https://github.com/helxplatform/dug. An example Dug deployment is also available for use at https://search.biodatacatalyst.renci.org/.

Contactawaldrop@rti.org or scox@renci.org TAP: Targeting and analysis pipeline for optimization and verification of coil placement in transcranial magnetic stimulation ObjectiveTranscranial magnetic stimulation (TMS) can modulate brain function via an electric field (E-field) induced in a region of interest (ROI) in the brain. The ROI E-field can be computationally maximized and set to match a specific reference using individualized head models to find the optimal coil placement and stimulus intensity. However, the available software lacks many practical features for prospective planning of TMS interventions and retrospective evaluation of the experimental targeting accuracy.

ApproachThe TMS targeting and analysis pipeline (TAP) software uses an MRI/fMRI-derived brain target to optimize coil placement considering experimental parameters such as the subjects hair thickness and coil placement restrictions. The coil placement optimization is implemented in SimNIBS 3.2, for which an additional graphical user interface (TargetingNavigator) is provided to visualize and adjust procedural parameters. The coil optimization process also computes the E-field at the target, allowing the selection of the TMS device intensity setting to achieve a specific E-field strength. The optimized coil placement information is prepared for neuronavigation software, which supports targeting during the TMS procedure. The neuronavigation system can record the coil placement during the experiment, and these data can be processed in TAP to quantify the accuracy of the experimental TMS coil placement and induced E-field.

Main resultsTAP was demonstrated in a study consisting of three repetitive TMS sessions in five subjects. TMS was delivered by an experienced operator under neuronavigation with the computationally optimized coil placement. Analysis of the experimental accuracy from the recorded neuronavigation data indicated coil location and orientation deviations up to about 2 mm and 2{degrees}, respectively, resulting in an 8% median decrease in the target E-field magnitude compared to the optimal placement.

SignificanceTAP supports navigated TMS with a variety of features for rigorous and reproducible stimulation delivery, including planning and evaluation of coil placement and intensity selection for E-field-based dosing. RapidoPGS: A rapid polygenic score calculator for summary GWAS data without a test dataset MotivationPolygenic scores (PGS) aim to genetically predict complex traits at an individual level. PGS are typically trained on genome-wide association summary statistics and require an independent test dataset to tune parameters. More recent methods allow parameters to be tuned on the training data, removing the need for independent test data, but approaches are computationally intensive. Based on fine-mapping principles, we present RapidoPGS, a flexible and fast method to compute PGS requiring summary-level GWAS datasets only, with little computational requirements and no test data required for parameter tuning.

ResultsWe show that RapidoPGS performs slightly less well than two out of three other widely-used PGS methods (LDpred2, PRScs, and SBayesR) for case-control datasets, with median r2 difference: -0.0092, -0.0042, and 0.0064, respectively, but up to 17,000-fold faster with reduced computational requirements. RapidoPGS is implemented in R and can work with user-supplied summary statistics or download them from the GWAS catalog.

Availability and implementationOur method is available with a GPL license as an R package from GitHub. Large protein complex interfaces have evolved to promote cotranslational assembly Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromers in bacterial, yeast, and human complexes. We detect a clear preference in N-terminal interfaces, which will be translated first and thus more likely to form cotranslationally, to be larger than C-terminal interfaces, suggesting that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding. 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. S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signalling. Plant receptor kinases are key transducers of physical extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signalling molecules. Receptor kinases are regulated by numerous post-translational modifications. Here, using the bacterial flagellin perceiving receptor kinase FLS2, we show that S-acylation at an evolutionarily conserved cysteine is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biophysical properties and behaviour within the membrane environment. We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes of treatment with flg22 ligand, and in a BAK1 co-receptor dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signalling, including anti-bacterial immunity. Similarly, mutating the corresponding conserved cysteine residue in the immune receptor kinase EFR supressed elf18 mediated signalling. Biochemical analysis of unstimulated and activated FLS2 containing complexes using detergents and native membrane DIBMA nanodiscs indicates that S-acylation assists the stabilization of activated receptor kinase complexes withing the membrane environment to increase signalling efficiency. Modular Dynamic Biomolecular Modelling with Bond Graphs: The Unification of Stoichiometry, Thermodynamics, Kinetics and Data. Renewed interest in dynamic simulation models of biomolecular systems has arisen from advances in genome-wide measurement and applications of such models in biotechnology and synthetic biology. In particular, genome-scale models of cellular metabolism beyond the steady state are required in order to represent transient and dynamic regulatory properties of the system. Development of such whole-cell models requires new modelling approaches. Here we propose the energy-based bond graph methodology, which integrates stoichiometric models with thermo-dynamic principles and kinetic modelling. We demonstrate how the bond graph approach intrinsically enforces thermodynamic constraints, provides a modular approach to modelling, and gives a basis for estimation of model parameters leading to dynamic models of biomolecular systems. The approach is illustrated using a well-established stoichiometric model of Escherichia coli (E. coli) and published experimental data. Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present  universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an  end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and  end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems.

Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of  saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this  universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer  end on-binding to sialoside sugars was revealed contrasting with  extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems. Induction of exaggerated cytokine production in human peripheral blood mononuclear cells by a recombinant SARS-CoV-2 spike glycoprotein S1 and its inhibition by dexamethasone An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNF, IL-6, IL-1{beta} and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-{kappa}B p65 and I{kappa}B, and I{kappa}B degradation. DNA binding of NF-{kappa}B p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 {micro}M) resulted in inhibition of spike glycoprotein S1-induced NF-{kappa}B activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNF, IL-6, IL-1{beta} and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1{beta} production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-{kappa}B, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 is possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm. Effect of a low concentration in chloride, sodium and potassium on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout. Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species.

Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation.

Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout
C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration
C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes
C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging
C_LI Natural tolerance to transposition is associated with double-strand break repair and germ-cell differentiation Transposable elements (TE) are mobile genetic parasites whose unregulated activity in the germline causes DNA damage and sterility. While the regulation of TE mobilization by hosts is studied extensively, little is known about mechanisms that could allow germline cells to persist in the face of genotoxic stress imposed by active transposition. Such tolerance mechanisms are predicted to be beneficial when new TEs invade and host repression has not yet evolved. Here we use hybrid dysgenesis--a sterility syndrome of Drosophila caused by transposition of invading DNA transposons--to uncover genetic variants that confer tolerance to transposition. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, we identified two linked quantitative trait loci (QTL), that determine tolerance in young and old females, respectively. Through transcriptomic and phenotypic comparisons, we provide evidence that young tolerant females exhibit enhanced repair of double-stranded breaks, explaining their ability to withstand high germline transposition rates. We furthermore identify the germline differentiation factor brat as an independent tolerance factor, whose activity may promote germline maintenance in aging dysgenic females. Together, our work reveals the diversity of potential tolerance mechanisms across development, as well as tolerant variants that may be beneficial in the context of P-element transposition.