Simpati: patient classifier identifies signature pathways based on similarity networks for the disease prediction BACKGROUNDPathway-based patient classification is a supervised learning task which implies a model learning pathways as features to predict the classes of patients. The counterpart of enrichment tools for the pathway analysis are fundamental methods for clinicians and biomedical scientists. They allow to find signature cellular functions which help to define and annotate a disease phenotype. They provide results which lead human experts to manually classify patients. It is a paradox that pathwaybased classifiers which natively resolve this objective are not strongly developed. They could simulate the human way of thinking, decipher hidden multivariate relationships between the deregulated pathways and the disease phenotype, and provide more information than a probability value. Instead, there are currently only two classifiers of such kind, they require a nontrivial hyperparameter tuning, are difficult to interpret and lack in providing new insights. There is the need of new classifiers which can provide novel perspectives about pathways, be easy to apply with different biological omics and produce new data enabling a further analysis of the patients.

RESULTSWe propose Simpati, an innovative and interpretable patient classifier based on pathway-specific patient similarity networks. The first classifier to adopt ad-hoc novel algorithms for such graph type. It standardizes the biological high-throughput dataset of patients profiles with a propagation algorithm that considers the interconnected nature of the cells molecules for inferring a new activity score. This allows Simpati to classify with dense, sparse, and non-homogenous omic data. Simpati organizes patients molecules in pathways represented by patient similarity networks for being interpretable, handling missing data and preserving the patient privacy. A network represents patients as nodes and a novel similarity measure determines how much every pair act co-ordinately in a pathway. Simpati detects signature biological processes based on how much the topological properties of the related networks separate the patient classes. In this step, it includes a new cohesive subgroup detection algorithm to handle patients not showing the same pathway activity as the other class members. An unknown patient is then classified by a unique recommender system which considers how much is similar to known patients and distant from being an outlier. Simpati outperforms previously published classifiers on five cancer datasets described with two biological omics, classifies well with sparse data, identifies more relevant pathways associated to the patients disease than the competitors and has the lowest computational requirements.

CONCLUSIONSimpati can serve as generic-purpose pathway-based classifier of patient classes. It provides signature pathways to unveil the altered biological mechanisms of a disease phenotype and to classify patients according to the learnt pathway-specific similarities. The signature condition and patient prediction can be deciphered considering the patient similarity networks which must reveal the members of a patient class more cohesive and similar than the non-members. Simpati divides the pathways in up and downinvolved. Upinvolved when the signaling cascades generated by the altered molecules of the disease patients impact stronger the pathway than the ones of the control class. We provide an R implementation, a graphical user interface and a visualization function for the patient similarity networks. The software is available at: https://github.com/LucaGiudice/Simpati
Exploratory analysis of multiple traits co-adaptations in the population history During the history of range expansion, the populations encounter with variety of environments. They respond to the local environments by modifying the mutually interacting traits. Therefore, to understand the whole life history of the populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the coadaptation of the multiple traits. Toward this end, we provide an exploratory analysis based on the features of populations: site frequency spectra of populations, population-specific FST, association between genes and environments, positive selections on traits mapped on the admixture graph, and GWAS results. Correspondence analysis of genes, environments, and traits provides a birds-eye view of the history of population differentiation and range expansion and various types of environmental selections at the times. Principal component analysis of the estimated trait-specific polygenic adaptations mapped on the admixture graph enables to understand the coadaptation of multiple traits. The potential usefulness was confirmed by analyzing a public dataset of wild poplar in northwestern America. In response to the northern cold temperature and longer daylength, the populations increased the photosynthetic activity and nutrient use efficiency at the expense of the risk of pathogen invasion, and in response to warm temperature, they increased the growth. At higher altitude, they shifted the maximum activity to earlier period in spring to reduce the activity in dry summer. The R codes for our representation method and simulations of population colonization used in this study are available as supplementary script.
Yersinia remodels epigenetic histone modifications in human macrophages Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61 % of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.

Author SummaryHuman pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages. In vitro synthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly Membrane proteins perform numerous important functions in cells and tissues. Approximately 20% of the human genome encodes for membrane proteins, which represent the majority of targets for clinically relevant small molecules. Consequently, understanding their structure and structure-function relationships is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional membrane proteins in supported lipid bilayers. Despite having shown the directional reconstitution of CFE-synthesized FL inner nuclear membrane SUN proteins (i.e. SUN1 and SUN2), which directly interact with outer nuclear membrane KASH proteins within the nuclear envelope lumen to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that mechanically couple the cytoskeleton and nucleus, the mechanism underlying regulated LINC complex assembly remains unclear. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment (e.g. calcium ions and redox state) on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and hetero-typic interactions that can occur between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies of membrane protein biochemistry and function, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic andtargetable tissue states Glioblastoma is an aggressive diffusely infiltrating neoplasm that spreads beyond surgical resection margins, where it intermingles with non-neoplastic brain cells. This complex microenvironment harbouring infiltrating glioma and non-neoplastic brain cells is the origin of tumor recurrence. Thus, understanding the cellular and molecular features of the glioma microenvironment is therapeutically and prognostically important. Here, we used single-nucleus RNA sequencing (snRNAseq) of primary and recurrent glioma to define three compositional  tissue-states rooted in cohabitation of cell-types and transcriptional states. These comprise a state featuring A) abundant normal brain microenvironment cells, B) reactive/inflamed brain microenvironment, and C) cellular/proliferative tumor. All these states exhibited variable degrees of infiltration by glioma cells. Spatial transcriptomics confirmed that the cell-types and transcriptomics states which compositionally cohabitate tissue states also colocalize in space. Tissue states are clinically significant because they correlate with radiographic, histopathologic, and prognostic features. We found that enrichment of tissue state B signature correlated with shorter survival. Importantly, we found that our compositionally defined tissue states are enriched in distinct metabolic pathways. One such pathway is fatty acid biosynthesis, which was enriched in tissue state B - a state enriched in recurrent glioblastoma and composed of astrocyte-like/mesenchymal glioma cells, reactive astrocytes resembling those seen in neurodegeneration, and monocyte-like myeloid cells. We showed that treating acute slices of GBM with a fatty acid synthesis inhibitor is sufficient to deplete the transcriptional signature of tissue state B. Our findings define a novel compositional approach to the glioma-infiltrated tissue which allows us to discover prognostic and targetable features, paving the way to new mechanistic and therapeutic discoveries. Orchestrated Excitatory and Inhibitory Learning Rules Lead to the Unsupervised Emergence of Self-sustained and Inhibition-stabilized Dynamics Self-sustaining neural activity maintained through local recurrent connections is of fundamental importance to cortical function. We show that Up-states--an example of self-sustained, inhibition-stabilized network dynamics--emerge in cortical circuits across three weeks of ex vivo development, establishing the presence of unsupervised learning rules capable of generating self-sustained dynamics. Previous computational models have established that four sets of weights (WE[&lt;-]E, WE[&lt;-]I, WI[&lt;-]E, WI[&lt;-]I) must interact in an orchestrated manner to produce Up-states, but have not addressed how a family of learning rules can operate in parallel at all four weight classes to generate self-sustained inhibition-stabilized dynamics. Using numerical and analytical methods we show that, in part due to the paradoxical effect, standard homeostatic rules are only stable in a narrow parameter regime. In contrast, we show that a family of biologically plausible learning rules based on "cross-homeostatic" plasticity robustly lead to the emergence of self-sustained, inhibition-stabilized dynamics. The role of Kabuki Syndrome genes KMT2D and KDM6A in development: Analysis in Human sequencing data and compared to mice and zebrafish KMT2D and KDM6A are epigenetic regulators that have been implicated in Kabuki Syndrome, a rare congenital birth defect with multiple tissue and organ abnormalities, including craniofacial and heart defects. Our previous study identified human families with mutations in the epigenetic modifiers KMT2D and KDM6A, which is implicated in 32% and 10% of Kabuki Syndrome patients respectively. To understand the connection to Kabuki syndrome patients, and the transcriptional targets of KMT2D and KDM6A in humans, we performed RNA sequencing (seq) of lymphoblastoid cells from Kabuki Syndrome patients carrying mutations in KMT2D and KDM6A. We identified 1995 significant changes in transcriptional targets for KMT2D and 1917 for KDM6A, as compared to control. When compared with RNA-seq datasets obtained from other mouse and zebrafish studies, our analysis revealed KMT2D mutations affect the expression of 76 orthologous genes across all three datasets. Similarliy, KDM6A afftects the expprssion of 7 orthologous genes across three datasets. Despite the differences in cell types, stages, and species in the comparison between the transcriptomic datasets, there are common gene expression changes associated with KMT2D and KDM6A mutations. qPCR on novel zebrafish mutants confirmed the differentially expression in KMT2D or KDM6A mutant backgrounds. Taken together, our results show that KMT2D and KDM6A regulate common and unique genes across humans, mice, and zebrafish for early craniofacial and cardiac development and this information contributes to the understanding of epigenetic dysregulation during development of Kabuki syndrome. Joint analysis of microsatellites and flanking sequences enlightens complex demographic history of interspecific gene flow and vicariance in rear-edge oak populations. Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in seven Quecus canariensis and four Quercus faginea rear-edge populations across Algeria, to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we measure variable importance from random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas the analysis of microsatellite variation alone indicates recent interspecific gene flow, additional information gained from combining with nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters. 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. The genome of the cereal pest Sitophilus oryzae: a transposable element haven BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis.

ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes. E156G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.617.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type.



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org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP  M_FIG C_FIG "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. 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 CIITA induces expression of MHC-I and MHC-II in transmissible cancers MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The two monoclonal transmissible devil facial tumours (DFT1, DFT2) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. We have previously shown that constitutive expression of NLRC5 can induce stable upregulation of MHC-I on DFT1 and DFT2 cells, but unlike IFNG-treated cells, NLRC5 does not upregulate PDL1. MHC-II expression is crucial for CD4+ T cell activation and is primarily confined to haematopoietic antigen presenting cells. Transcriptomic analysis of DFT1 and DFT2 cell lines showed that several genes of the MHC-I and MHC-II pathways were upregulated in response to constitutive overexpression of the class II transactivator (CIITA) gene. This was further supported by upregulation of MHC-I protein on DFT1 and DFT2 cells, but interestingly MHC-II protein was upregulated only on DFT1 cells. The functional significance of the MHC upregulation on DFT cells was shown using serum from devils with natural or immunotherapy-induced DFT1 regressions; binding of serum IgG was stronger in CIITA-transfected cells than wild type cells, but was less than binding to NLRC5 transfected cells. This new insight into regulation of MHC-I and MHC-II in cells that naturally overcome allogeneic barriers can inform vaccine, immunotherapy, and tissue transplant strategies for human and veterinary medicine. Protective host-dependent antagonism among 	Pseudomonas in the Arabidopsis phyllosphere The plant microbiome is a rich biotic environment, comprising numerous taxa. The community structure of these colonizers is constrained by multiple factors, including host-microbe and microbe-microbe interactions, as well as the interplay between the two. While much can be learned from pairwise relationships between individual hosts and microbes, or individual microbes with themselves, the ensemble of interrelations between the host and microbial consortia may lead to different outcomes that are not easily predicted from the individual interactions. Their study can thus provide new insights into the complex relationship between plants and microbes. Of particular importance is how strain-specific such plant-microbe-microbe interactions are, and how they eventually affect plant health. Here, we test strain-level interactions in the phyllosphere between groups of co-existing commensal and pathogenic Pseudomonas among each other and with A. thaliana, by employing synthetic communities of genome-barcoded isolates. We found that commensal Pseudomonas prompted a host response leading to a selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of plant protection, however, was dependent on plant genotype, indicating that these effects are host-mediated. There were similar genotype-specific effects on the microbe side, as we could pinpoint an individual Pseudomonas isolate as the predominant cause for this differential interaction. Collectively, our work highlights how within-species genetic differences on both the host and microbe side can have profound effects on host-microbe-microbe dynamics. The paradigm that we have established provides a platform for the study of host-dependent microbe-microbe competition and cooperation in the A. thaliana-Pseudomonas system. Ribosome actuator via the SpoT/ppGpp pathway to mitigate gene overexpression burden Gene overexpression causes a burden on ribosomes, which decreases the expression of other genes and growth rate. Here, we engineer a genetic ribosome actuator that increases gene expression rate and growth rate by over 150% and 80%, respectively. The actuator expresses the hydrolysis domain of the SpoT enzyme (SpoTH) in a bacterial strain with high basal levels of ppGpp, thus increasing ribosome availability. We use this actuator in a feedforward control arrangement, wherein SpoTH is co-expressed with a gene of interest (GOI). Without SpoTH co-expression, activation of the GOI decreases the expression of a constitutive gene and growth rate by over 80% and 40%, respectively. By contrast, with SpoTH co-expression, the ribosome binding site (RBS) of SpoTH can be tuned to achieve inappreciable change of either the expression of the constitutive gene or growth rate when the GOI is activated to the same level. We finally employ the actuator to keep growth rate constant while expressing dCas9 to a level that would otherwise decrease growth rate by more than 40% due to toxicity. This actuator will be relevant in applications where high cellular protein concentrations are desired without growth defects or global reduction of gene expression. 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. Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands. Visualization of loop extrusion by DNA nanoscale tracing in single human cells The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensation and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random chromatin structures such as topologically associating domains2, 3. However, whether the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we developed LoopTrace, a fluorescence imaging workflow for high-resolution reconstruction of 3D genome architecture that conserves chromatin structure at the nanoscale and can resolve the 3D-fold of chromosomal DNA with better than 5-kb precision in single human cells. Our results show that the chromatin fibre behaves as a random coil up to the megabase scale and is further structured by contacts between sites that anchor loops. Our single cell folds reveal that such looping interactions are sparse and lead to a large heterogeneity of folds with one or two dynamically positioned loop bases as the main reproducible feature of megabase scale chromosomal regions. Clustering folds by their 3D conformations revealed a series of structures consistent with progressive loop extrusion between major anchor sites. Consistently, the looping interactions and their non-random positioning depend on the presence of the loop extrusion enzyme cohesin and its anchor protein CTCF, respectively. Our approach is scalable and will be instrumental to image the functional 3D architecture of the genome directly at the nanoscale. Variation in female songbird state determines signal strength needed to evoke copulation It is the female response to male signals that determines courtship success. In most songbirds, females control reproduction via the copulation solicitation display (CSD), an innate, stereotyped posture produced in direct response to male displays. Because CSD can be elicited in the absence of males by the presentation of recorded song, CSD production enables investigations into the effects of underlying signal features and behavioral state on female mating preferences. Using computer vision to quantify CSD trajectory in female brown-headed cowbirds (Molothrus ater), we show that both song quality and a females internal state predict CSD production, as well as the onset latency and duration of the display. We also show that CSD can be produced in a graded fashion based on both signal strength and internal state. These results emphasize the importance of underlying receiver state in determining behavioral responses and suggest that female responsiveness acts in conjunction with male signal strength to determine the efficacy of male courtship. Reproduction in deep-sea vent shrimps is shaped by diet and phylogeny, with rhythms unlinked to surface production Variations in offspring production according to feeding strategies or food supply have been recognized in many animals from various ecosystems. Despite an unusual trophic structure based on non-photosynthetic primary production, these relationships remain largely under-studied in chemosynthetic ecosystems. Here, we use Rimicaris shrimps from deep-sea hydrothermal vents as a study case to explore relations between reproduction, diets and food supply in these environments. For that, we compared reproductive outputs of three congeneric shrimps differing by their diets. They inhabit vents located under oligotrophic waters of tropical gyres with opposed latitudes, allowing us to also examine the prevalence of phylogenetic vs environmental drivers in their reproductive rhythms. For this we used both our original data and a compilation of published observations on the presence of ovigerous females covering various seasons over the past 35 years. We report distinct egg production trends between Rimicaris species relying solely on chemosymbiosis - R. exoculata and R. kairei - and those relying on mixotrophy - R. chacei - where R. chacei produces broods with higher numbers of smaller eggs. Besides, our data and historical records suggest a reproductive period with substantial proportions of brooding females mostly between January and early April for all examined species whatever the region. Intriguingly, this periodicity does not correspond to seasonal variations in surface production with presence of brooding females during either boreal winter or austral summer. These observations contrast with the long-standing paradigm in deep-sea species for which periodic reproductive patterns have always been attributed to seasonal variations of photosynthetic production sinking from surface. Our results suggest the presence of intrinsic basis for biological rhythms in the deep sea, and bring to light the importance of having year-round observations in order to understand life history of vent animals. N-VEGF, the forgotten isoform of VEGF-A, activate cellular stress survival circuits Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long isoform called L-VEGF is concomitantly expressed with VEGF-A. Once translated, L-VEGF it is proteolytically cleaved to generate N-VEGF and VEGF-A. Interestingly, while VEGF-A is secreted and affects the surrounding cells, N-VEGF is mobilized to the nucleus. This suggests that N-VEGF participates in transcriptional response to hypoxia. In this study, we performed a series of complementary experiments to examine the functional role of N-VEGF. Strikingly, we found that the mere expression of N-VEGF followed by its hypoxia-independent mobilization to the nucleus was sufficient to induce key genes associated with angiogenesis, such as Hif1, VEGF-A isoforms, as well as genes associated with cell survival under hypoxia. Complementarily, when N-VEGF was genetically depleted, key hypoxia-induced genes were downregulated and cells were significantly susceptible to hypoxia-mediated apoptosis. This is the first reports of N-VEGF serving as an autoregulatory arm of VEGF-A. Further experiments will be needed to determine the role of N-VEGF in cancer and embryogenesis. Microsecond simulations and CD spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) in isolation All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions. Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region. This curiosity has led us to investigate the C terminal cytosolic region of the spike glycoprotein of SARS-CoV-2 in isolation. The cytosolic regions is supposed to be from 1235-1273 residues or 1242-1273 residues depending on what prediction tool we use. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown its conformational dynamics. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm which is also adding the analysis as its intrinsically disordered nature. We believe that our findings will surely help us understand the structure-function relationship of the spike proteins cytosolic region.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs Human in vitro models of neural tissue with controllable cellular identity, tunable microenvironment, and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing in jammed microgel supports (i.e., granular gels) holds great promise as it allows precise and programmable patterning of extremely soft and compliant tissue constructs. However, in contrast to the vast material landscape available for bulk hydrogels, granular printing support formulations are restricted to a handful of materials without the ability for facile adjustment of biofunctional properties of the cellular microenvironment. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise freeform patterning of human stem cells and consequent generation and long-term maintenance of mature subtype-specific neurons that extend projections within the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential. Deep learning: step forward to high-resolution in vivo shortwave infrared imaging Shortwave infrared window (SWIR: 1000-1700 nm) represents a major improvement compared to the NIR-I region (700-900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIRIIb region (1500-1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images. A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors.