RNA splicing analysis using heterogeneous and large RNA-seq datasets The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we demonstrate that the approaches in MAJIQ v2 outperform existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer new insights into brain subregion-specific splicing regulation.
A novel, computationally tractable algorithm flags in big matrices every column associated in any way with others or a dependent variable, with much higher power when columns are linked like mutations in chromosomes When a data matrix DM has many independent variables IVs, it is not computationally tractable to assess the association of every distinct IV subset with the dependent variable DV of the DM, because the number of subsets explodes combinatorially as IVs increase. But model selection and correcting for multiple tests is complex even with few IVs.

DMs in genomics will soon summarize millions of mutation markers and genomes. Searching exhaustively in such DMs for markers that alone or synergistically with others are associated with a trait is therefore computationally tractable only for 1- and 2-marker effects. Also population geneticists study mainly 2-marker combinations.

I present a computationally tractable, fully parallelizable Participation in Association Score (PAS) that in a DM with markers detects one by one every column that is strongly associated in any way with others. PAS does not examine column subsets and its computational cost grows linearly with the number of columns, remaining reasonable even when DMs have millions of columns.

PAS exploits how associations of markers in the rows of a DM cause associations of matches in the rows pairwise comparisons. For every such comparison with a match at a tested column, PAS computes the matches at other columns by modifying the comparisons total matches (scored once per DM), yielding a distribution of conditional matches that reacts diagnostically to the associations of the tested column. Equally computationally tractable is dvPAS that flags DV-associated IVs by also probing the matches at the DV.

P values for the scores are readily obtained by permutation and accurately Sidak-corrected for multiple tests, bypassing model selection. The P values of a columns PASs for different orders of association are i.i.d. and readily turned into a single P value.

Simulations show that i) PAS and dvPAS generate uniform-(0,1)-distributed type I error in null DMs and ii) detect randomly encountered binary and trinary models of significant n-column association and n-IV association with a binary DV, respectively, with power in the order of magnitude of exhaustive evaluations and false positives that are uniform-(0,1)-distributed or straightforwardly tuned to be so. Power to detect 2-way associations that extend over 100+ columns is non-parametrically ultimate but that to detect pure n-column associations and pure n-IV DV associations sinks exponentially as n increases.

Important for geneticists, dvPAS power increases about twofold in trinary vs. binary DMs and by orders of magnitude with markers linked like mutations in chromosomes, specially in trinary DMs where furthermore dvPAS fine-maps with highest resolution.
Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Sources of information waste in neuroimaging:mishandling structures, thinking dichotomously, and over-reducing data Neuroimaging relies on separate statistical inferences at tens of thousands of spatial locations. Such massively univariate analysis typically requires an adjustment for multiple testing in an attempt to maintain the family-wise error rate at a nominal level of 5%. First, we examine three sources of substantial information loss that are associated with the common practice under the massively univariate framework: (a) the hierarchical data structures (spatial units and trials) are not well maintained in the modeling process; (b) the adjustment for multiple testing leads to an artificial step of strict thresholding; (c) information is excessively reduced during both modeling and result reporting. These sources of information loss have far-reaching impacts on result interpretability as well as reproducibility in neuroimaging. Second, to improve inference efficiency, predictive accuracy, and generalizability, we propose a Bayesian multilevel modeling framework that closely characterizes the data hierarchies across spatial units and experimental trials. Rather than analyzing the data in a way that first creates multiplicity and then resorts to a post hoc solution to address them, we suggest directly incorporating the cross-space information into one single model under the Bayesian framework (so there is no multiplicity issue). Third, regardless of the modeling framework one adopts, we make four actionable suggestions to alleviate information waste and to improve reproducibility: 1) abandon strict dichotomization, 2) report full results, 3) quantify effects, and 4) model data hierarchies. We provide examples for all of these points using both demo and real studies, including the recent NARPS investigation. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. Cis-Regulatory Hubs: a new 3D Model of Complex Disease Genetics with an Application to Schizophrenia The 3-dimensional (3D) conformation of the chromatin creates complex networks of noncoding regulatory regions (distal elements) and promoters impacting gene regulation. Despite the importance of the role of noncoding regions in complex diseases, little is known about their interplay within regulatory hubs and implication in multigenic diseases like schizophrenia. Here we show that cis-regulatory hubs (CRHs) in neurons highlight functional interactions between distal elements and promoters, providing a model to explain epigenetic mechanisms involved in complex diseases. CRHs represent a new 3D model, where distal elements interact to create a complex network of active genes. In a disease context, CRHs highlighted strong enrichments in schizophrenia-associated genes, schizophrenia-associated SNPs and schizophrenia heritability compared to equivalent structures. Finally, CRHs exhibit larger proportions of genes differentially expressed in schizophrenia compared to promoter-distal element pairs or TADs. CRHs thus capture causal regulatory processes improving the understanding of complex disease etiology such as schizophrenia. These multiple lines of genetic and statistical evidence support CRHs as 3D models to study dysregulation of gene expression in complex diseases more generally.

Summary BlurbGenes and their regulatory elements are organized within 3D networks in neurons which model functional structures and explain schizophrenia genetic etiology. Learned deconvolution using physics priors for light-sheet microscopy with propagation-invariant beams Deconvolution is a challenging inverse problem, particularly in techniques that employ complex engineered point-spread functions, such as microscopy with propagation-invariant beams. Here, we present a deep learning method that uses known physics priors, in lieu of any ground truths, to rapidly and robustly deconvolve and super-resolve these microscopy images. Specifically, we constrain a generative adversarial network with images generated with the known point-spread function of the system, and combine this with unpaired experimental data that preserves perceptual content. Our network overcomes a major present barrier to deep learning adoption, namely, the need for 1,000-10,000s of paired training images. It is experimentally unsupervised and can be trained solely on a few hundred regions of interest. We demonstrate its performance on light-sheet microscopy with propagation-invariant Airy beams, including in calibration beads, oocytes, preimplantation embryos, and excised brain tissue, as well as illustrate its utility for Bessel-beam LSM. This method aims to democratise learned methods for deconvolution, as it does not require data acquisition outwith the conventional imaging protocol. Erythropoietin supplementation ameliorates the immunological and hematological deficiencies of lysinuric protein intolerance in mice SLC7A7 encodes for y+LAT1, a cationic amino acid transporter expressed on the basolateral membrane of epithelial cells and macrophages. Mutations in human SLC7A7 cause lysinuric protein intolerance (LPI), a rare autosomal disorder with a variety of clinical symptoms. Citrulline and a low protein diet are widely used to treat the main symptoms of LPI; however, the hematological and immunological abnormalities in LPI remain poorly understood and difficult to manage by established treatments. Here, we show that erythropoietin levels are significantly diminished in mice lacking Slc7a7, which reduces the number of erythroblast precursors, resulting in mature red blood cells with a smaller mean corpuscular volume. Ex vivo analysis demonstrated that erythrocytes from Slc7a7-deficient mice were phagocytosed more by bone marrow-derived macrophages. Gene expression analysis of sorted red pulp macrophages (RPMs) from Slc7a7-deficient mice indicated that this cell population had higher erythrophagocytic activity, but preserved heme catabolic activity. The increase in erythrophagocytosis resulted in iron accumulation in macrophages and tissues, and ultimately led to RPM apoptosis. Surprisingly, ablation of Slc7a7 in myeloid cells failed to trigger tissue iron accumulation or changes in the RPM population, indicating that the expression of this gene in macrophages is not key for the development of iron accumulation in tissue. Finally, restoration of serum erythropoietin levels in Slc7a7-deficient mice re-established normal erythropoiesis and erythrophagocytosis and prevented the accumulation of iron in tissues. Thus, erythropoietin has a fundamental role in the immune-hematological alterations of LPI in mice, revealing a new crucial role for this hormone in this disease. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. E-Cadherin mediated AMIS localisation Individual cells within de novo polarising tubes and cavities must integrate their forming apical domains into a centralised apical membrane initiation site (AMIS). This is necessary to enable organised lumen formation within multi-cellular tissue. Despite the well documented importance of cell division in localising the AMIS, we have found a division-independent mechanism of AMIS localisation that relies instead on Cadherin-mediated cell-cell adhesion. Our study of de novo polarising mouse embryonic stem cells (mESCs) cultured in 3D suggest that cell-cell adhesion directs the localisation of apical proteins such as PAR-6 to a centralised AMIS. Unexpectedly, we also found that mESC cell clusters lacking functional E-Cadherin were still able to form a lumen-like cavity in the absence of AMIS localisation and did so at a later stage of development via a  closure mechanism, instead of via hollowing. This work suggests that there are two, interrelated mechanisms of apical polarity localisation: cell adhesion and cell division. Alignment of these mechanisms in space allows for redundancy in the system and ensures the development of a coherent epithelial structure within a growing organ. Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK (Alpha), South Africa (Beta), and India (Delta) We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC50: as low as 13 nM), SARS-CoV-2 (IC50: as low as 23 nM), and MERS-CoV (IC50: as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC100) at 1.25 {micro}M against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates. Extreme parallel evolution of flagellar motility facilitated by silent mutations Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes. The Shigella type III effector protein OspB is a cysteine protease The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes.

ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. Estimating the fundamental niche: accounting for the uneven availability of existing environmental conditions in the calibration area Studies that question important conceptual and methodological aspects of the field of ecological niche modeling (and species distribution modeling) have cast doubts on whether it is possible to estimate the fundamental niche of a species using presence-only data. The main limitation in niche estimation is that presence data come from the realized niche, which is only a subset of the fundamental niche. Most of the existing methods lack the ability to overcome this limitation and therefore fit niches that resemble the realized niche. To obtain a more accurate estimate of the fundamental niche, we propose using the geographic region that is accessible to a species (based on its dispersal ability) to determine a sampling distribution, in environmental space, from which we can quantify the likelihood of observing a particular environmental combination in a sample of presence points. We incorporate this sampling distribution into a multivariate normal model (i.e., a Mahalanobis distance model) by creating a weight function that modifies the probabilities of observing different environmental combinations in a sample of presences. This modification accounts for the uneven availability of environmental conditions. We show that the parameters of this weighted-normal model can be approximated with a maximum likelihood estimation approach; and then used to draw ellipsoids (confidence regions) that represent the fundamental niche of the species. We illustrate the application of our model with two worked examples. First, we use presence data for an invasive species and an accessible area that includes only its native range to evaluate whether the fitted model predicts confirmed establishments of the species outside its native range. Second, we use presence data for closely related species with known accessible areas to demonstrate how the different dispersal abilities of the species constrain a classic Mahalanobis distance model. Overall, we show that accounting for the distribution of environmental conditions that are accessible to a species indeed affects the estimation of the ellipsoids used to model its fundamental niche. Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. 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. A unicellular walker controlled by a microtubule-based finite state machine Cells are complex biochemical systems whose behavior emerges from interactions among myriad molecular components. Computation is often invoked as a general framework for navigating this cellular complexity. However, the manner in which cells might embody computational processes such that theories of computation, including finite state machine models, could be productively applied, remains to be seen. Here we demonstrate finite state machine-like processing embodied in cells using the walking behavior of Euplotes eurystomus, a ciliate that walks across surfaces using fourteen motile appendages (cirri). We found that cellular walking entails highly regulated transitions between a discrete set of gait states. The set of observed transitions decomposes into a small group of high-probability, temporally irreversible transitions forming a cycle and a large group of low-probability time-symmetric transitions, thus revealing stereotypy in sequential patterns of state transitions. Taken together, these findings implicate a finite state machine-like process. Cirri are connected by microtubule bundles, and we found that the dynamics of cirri involved in different state transitions are associated with the structure of the microtubule bundle system. Perturbative experiments revealed that the fibers mediate gait coordination, suggesting a mechanical basis of gait control.

One sentence summaryThe ciliate Euplotes walks across surfaces with an unusual gait, which involves modular subcellular structure and combines elements of stereotypy and variability according to a computational process actively coordinated by a microtubule fiber system. 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated. The female pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster Specific mate communication and recognition underlies reproduction and hence speciation. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues.

Our study provides new insights in Drosophila melanogaster premating olfactory communication, showing that female pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone cVA interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males.

Twin variants of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons, and feed into the same glomerulus in the antennal lobe. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. That Z4-11Al mediates courtship in experienced males, not (R)- linalool, is probably due to courtship learning. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds.

Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in to contrast to Z4-11Al. A blend of cVA and the food odour vinegar attracted females, while a blend of female pheromone Z4-11Al and vinegar attracted males instead.

Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.

SummaryThe female-produced, species-specific volatile pheromone of D. melanogaster attracts both sexes from a distance, alone and in concert with food odorants, and elicits courtship in experienced males. Spatial control of cell division by GA-OsGRF7/8 module in a leaf explains the leaf length variation between cultivated and wild rice O_LICellular and genetic understanding of rice leaf size regulation is limited, despite rice being the staple food of more than half of the global population. We investigated the mechanism controlling the rice leaf length using cultivated and wild rice accessions that remarkably differed for leaf size.
C_LIO_LIComparative transcriptomics, Gibberellic Acid (GA) quantification, and leaf kinematics of the contrasting accessions suggested the involvement of GA, cell cycle, and Growth-Regulating Factors (GRFs) in the rice leaf size regulation. Zone-specific expression analysis and VIGS established the functions of specific GRFs in the process.
C_LIO_LIThe leaf length of the selected accessions was strongly correlated with GA levels. Higher GA content in wild rice accessions with longer leaves and GA-induced increase in the leaf length via an increase in cell division confirmed a GA-mediated regulation of division zone in rice. Downstream to GA, OsGRF7 and OsGRF8 function for controlling cell division to determine the rice leaf length.
C_LIO_LISpatial control of cell division to determine the division zone size mediated by GA and downstream OsGRF7 and OsGRF8 explains the leaf length differences between the cultivated and wild rice. This mechanism to control rice leaf length might have contributed to optimizing leaf size during domestication.
C_LI Immune complex solubility and size govern individual Fc-gamma receptor responses: Novel biomarkers for autoimmune diseases Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent minimal MET receptor agonist. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent on MET activation. An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner. TNFa-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cellsan arterial endothelial cells Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNF) in modulating low density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNF promoted up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNF could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), receptor associated protein (RAP), or apolipoprotein (apoE) depletion. Moreover, TNF-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. Effects of TNF included LDLR cell surface increase by 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNF-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNF on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-{kappa}B, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on transwell inserts, TNF did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNF promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation. Active enhancers strengthen insulation by RNA-mediated CTCF binding at TAD boundaries Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.