Excitation and emission spectra of in silico red-shift predicted iLOV mutants iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to have reduced phototoxicity and better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOVL470T/Q489K and iLOVV392K/F410V/A426S, which were previously computationally proposed by Khrenova et al. (DOI: 10.1021/acs.jpcb.7b07533) to have red-shifted excitation and emission spectra. We found that mutants bearing the V392K mutation lost the ability to bind FMN. While iLOVL470T/Q489K is about 20% brighter compared to WT iLOV in vitro, it exhibits a blue shift in contrast to QM/MM predictions. Additionally, both mutants are expressed at low levels and have undetectable fluorescence in living cells, which prevents their utilization in imaging applications. Our results suggest that rational fluorescent protein design efforts can benefit from in silico protein stability and ligand affinity calculations.
PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microcopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized.

HighlightsO_LIWe present PySTACHIO, a refined version of our spot tracking algorithm
C_LIO_LIWe demonstrate highly improved performance over previous MATLAB versions
C_LIO_LIPySTACHIO can accurately estimate stoichiometries and 2D diffusion coefficients
C_LIO_LIPerformance is comparable to state-of-the-art packages on challenge data
C_LIO_LIPySTACHIO has both GUI and command line interfaces and can be hosted as a web app
C_LI

Graphical abstract

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Systematic monitoring of photosynthetically-derived oxidative signals by a peroxiredoxin-based biosensor Transmission of reductive cues from the photosynthetic electron transport chain to redox-regulated proteins plays a crucial role in activating chloroplast metabolism. However, deciphering the role of their counterbalanced oxidative signals is challenging due to monitoring difficulties. Here, we demonstrate the light-depended redox modification of chloroplast-targeted 2-Cys peroxiredoxins and introduce peroxiredoxin-based biosensors to monitor photosynthetically-derived oxidative signals. By employing a set of genetically encoded biosensors, we show the induction of oxidative signals under habitual light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast metabolism. A faster increase in carbon assimilation rates during photosynthesis induction phase was measured in plants deficient in 2-Cys peroxiredoxins compared to wild-type, suggesting the involvement of oxidative signals in attenuating photosynthesis under variable light environments. We suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system.

One-Sentence SummaryA genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions. 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. The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. The mutation effect reaction norm (Mu-RN) highlights environmentally dependent mutation effects and genetic interactions Since the modern synthesis, the fitness effects of mutations and epistasis have been central yet provocative concepts in evolutionary and population genetics. Studies of how the interactions between parcels of genetic information can change as a function of environmental context have added a layer of complexity to these discussions. Here I introduce the "mutation effect reaction norm" (Mu-RN), a new instrument through which one can analyze the phenotypic consequences of mutations and interactions across environmental contexts. It embodies the fusion of measurements of genetic interactions with the reaction norm, a classic depiction of the performance of genotypes across environments. I demonstrate the utility of the Mu-RN through the signature of a "compensatory ratchet" mutation that undermines reverse evolution of antimicrobial resistance. More broadly, I argue that the mutation effect reaction norm may help us resolve the dynamism and unpredictability of evolution, with implications for theoretical biology, genetic modification technology, and public health. LCK Regulates Homologous Recombination DNA Repair Identifying a New Therapeutic Target for Sensitizing PARP Inhibitors in HR Proficient Ovarian Cancer Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success in treatment of HR repair deficient cancers has not translated to HR-proficient cancer. Our studies identify the mechanism of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in HR repair in endometrioid epithelial ovarian cancer (eEOC). LCK expression is induced and activation in the nucleus in response to DNA damage insult. LCK inhibition attenuates expression of RAD51, BRCA1, and BRCA2 proteins necessary for HR-mediated DNA repair, sufficient to suppress RAD51 foci formation, and augments {gamma}H2AX foci formation. Mechanistically, DNA damage leads to direct interaction of LCK with RAD51 and BRCA1 in a kinase dependent manner. Attenuation of LCK sensitized HR-proficient eEOC cells to PARP inhibitor in cell culture and pre-clinical mouse studies. These findings identify a new mechanism for expanding utility of PARP inhibitors in HR proficient ovarian cancer.

Graphical Abstract

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org.highwire.dtl.DTLVardef@1715c60org.highwire.dtl.DTLVardef@167cb5forg.highwire.dtl.DTLVardef@158d807org.highwire.dtl.DTLVardef@1ced201_HPS_FORMAT_FIGEXP  M_FIG C_FIG Tunable Cellular Localization and Extensive Cytoskeleton-Interplay of Reflectins Reflectin proteins are nature copolymers consist of repeated canonical domains, locating in a biophotonic system called Bragg lamellae and manipulating the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. In this study, reflectin A1, A2, B1 and C were found to present distinguished cyto-/nucleoplasmic localization preferences. Comparable intracellular localization can be reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. Secondly, the size-dependent access of reflectin variants into nucleus demonstrate a potential model of how reflectins get into Bragg lamellae. Thirdly, RfA1 was found to extensively interact with cytoskeleton, including its binding to actin and microtubule minus-end-directed movement. This implies cytoskeleton system plays fundamental role during the organization and transportation of reflectin proteins. Findings presented here recommend reflectins as programmable biomaterials which can be used to decipher their evolution processes, to delineate their biological mechanism, and to achieve tunable intracellular targeting as editable tags. Atrazine induced In vivo immunotoxicity in Bivalves Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves. Non-invasive imaging of gene expression and protein secretion dynamics in living mice: identification of ectopic prothrombin expression as driver of thrombosis in cancer The topology of gene expression and protein localization is a crucial characteristic of life, where the spatiotemporal dynamic of secretory proteins instruct higher order organization, including the orchestration of developmental and adaptive programs. However tools to non-invasively interrogate the fate of secretory proteins in vivo are scarce. Here we introduce a genetic tagging strategy for in vivo imaging of the secretion and expression dynamics of secretory proteins in living animals. Applying this to a prototypical liver-derived secretory protein, we demonstrate that this approach, combined with optical in-vivo imaging, uncovers extrahepatic prothrombin expression in multiple novel anatomical sites (including testes, placenta, brain, kidney, heart and lymphatic system) and in emerging tumors, resulting in significant amounts of tumor-derived prothrombin in the blood with procoagulant properties. Syngeneic cell lines from this mouse model enable unravelling regulatory mechanisms in high resolution, and in a scalable format ex vivo. Beyond discovering new functions of proteins in a targeted manner, this model allows identifying rheostats in the cross-talk between gene expression and availability of a secretory protein. It is also a valuable resource for uncovering novel (tissue-specific) therapeutic vulnerabilities. Improved radiation expression profiling in blood by sequential application of sensitive and specific gene signatures PurposeCombinations of expressed genes can discriminate radiation-exposed from normal control blood samples by machine learning based signatures (with 8 to 20% misclassification rates). These signatures can quantify therapeutically-relevant as well as accidental radiation exposures. The prodromal symptoms of Acute Radiation Syndrome (ARS) overlap those present in Influenza and Dengue Fever infections. Surprisingly, these human radiation signatures misclassified gene expression profiles of virally infected samples as false positive exposures. The present study investigates these and other confounders, and then mitigates their impact on signature accuracy.

MethodsThis study investigated recall by previous and novel radiation signatures independently derived from multiple Gene Expression Omnibus datasets on common and rare non-malignant blood disorders and blood-borne infections (thromboembolism, S. aureus bacteremia, malaria, sickle cell disease, polycythemia vera, and aplastic anemia). Normalized expression levels of signature genes are used as input to machine learning-based classifiers to predict radiation exposure in other hematological conditions.

ResultsExcept for aplastic anemia, these blood-borne disorders modify the normal baseline expression values of genes present in radiation signatures, leading to false-positive misclassification of radiation exposures in 8 to 54% of individuals. Shared changes, predominantly in DNA damage response and apoptosis-related gene transcripts in radiation and confounding hematological conditions, compromise the utility of these signatures for radiation assessment. These confounding conditions (sickle cell disease, thromboembolism, S. aureus bacteremia, malaria) induce neutrophil extracellular traps, initiated by chromatin decondensation, DNA damage response and fragmentation followed by programmed cell death. Riboviral infections (for example, Influenza or Dengue fever) have been proposed to bind and deplete host RNA binding proteins, inducing R-loops in chromatin. R-loops that collide with incoming replication forks can result in incompletely repaired DNA damage, inducing apoptosis and releasing mature virus. To mitigate the effects of confounders, we evaluated predicted radiation-positive samples with novel gene expression signatures derived from radiation-responsive transcripts encoding secreted blood plasma proteins whose expression levels are unperturbed by these conditions.

ConclusionsThis approach identifies and eliminates misclassified samples with underlying hematological or infectious conditions, leaving only samples with true radiation exposures. Diagnostic accuracy is significantly improved by selecting genes that maximize both sensitivity and specificity in the appropriate tissue using combinations of the best signatures for each of these classes of signatures. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. A New 'Comprehensive' Annotation of Leucine-Rich Repeat-Containing Receptors in Rice Rice plays an essential food security role for more than half of the worlds population. Obtaining crops with high levels of disease resistance is a major challenge for breeders, especially today given the urgent need for agriculture to be more sustainable. Plant resistance genes are mainly encoded by three large Leucine-Rich Repeat (LRR)-containing receptor (LRR-CR) subfamilies: the LRR-Receptor-Like Kinase (RLK), LRR-Receptor-Like Protein (RLP) and Nucleotide-binding LRR Receptor (NLR) subfamilies. Using LRRprofiler, a pipeline we developed to annotate and classify those proteins, we compared three publicly available annotations of the rice Nipponbare reference genome. The extended discrepancies we observed for LRR-CR gene models led us to perform in-depth manual curation of their annotations while paying special attention to nonsense mutations. We then transferred this manually curated annotation to Kitaake, a Nipponbare closely related cultivar, using an optimised strategy. Here we discuss the breakthrough achieved by manual curation when comparing genomes and, in addition to  functional and  structural annotations, we propose the community to adopt this new approach, which we call  comprehensive annotation. The resulting data are crucial for further studies on the natural variability and evolution of LRR-CR in order to promote their use in breeding future resilient varieties. A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs. Meiotic DSB-independent role of protein phosphatase 4 in Hop1 assembly to promote meiotic chromosome axis formation in budding yeast Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among these structures, meiosis-specific chromosomal axis-loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis-loop construction is not well understood. Here, we showed that in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of chromosomal axis components Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, in contrast, did not affect Rec8 assembly. Notably, unlike the previously known function of PP4, this novel function of PP4 was independent of meiotic DSB-dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis. This suggests that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate that phosphorylation/dephosphorylation-mediated regulation of Hop1 recruitment onto chromatin occurs during chromosome axis construction before meiotic double-strand break formation. Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 sixteen-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of E. coli resistant to fluoroquinolones, tetracycline, amoxicillin, and streptomycin, but not to cefalexin or cefotaxime. Whole genome sequencing of 30 fluoroquinolone-resistant (FQ-R), 22 cefotaxime-resistant (CTX-R) and seven dual FQ-R/CTX-R E. coli isolates showed a wide range of sequence types (STs), an approximately 50:50 split of CTX-M:AmpC-mediated CTX-R, and almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 x 50 km region with those isolated from human urinary tract and bloodstream infections (isolated in parallel in the same region) identified a clone of ST963 E. coli carrying chromosomal blaCMY.2 in two puppies and causing two urinary tract infections and one bloodstream infection. Furthermore, an ST744 FQ-R clone was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at sixteen weeks of age and that bacteria carried by dogs are shared with humans. Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

NEW AND NOTEWORTHYGenetic analysis of cardiac hypertrophy in Drosophila holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. However, this potential has been hindered by a lack of rapid and effective methods for analysis of heart size in flies. Here we demonstrate that analysis of the Drosophila heart with micro-computerized tomography yields accurate and highly reproducible heart size measurements that can be used to efficiently analyze heart growth and cardiac hypertrophy in Drosophila. 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. Is Pisum sativum a good model species for the study of root responses to neighbours and barriers in soil? A bayesian hierarchical meta-analysis Plant-plant competition is ubiquitous in nature. However, studying below ground behaviour of roots has always posed certain difficulties. Pea (Pisum sativum L.) has become a sort of model species for ecological studies about how plant roots respond to neighbouring plant roots and barriers in soil. However, published results point in several different directions. This has sometimes been interpreted as pea having sophisticated context dependent responses that can change in complex ways depending on its surroundings. To explore this further, here, we combine the result of five new experiments with published results to examine 18 unique experiments from 7 different studies for a total of 254 replicates. We used a Bayesian hierarchical meta-analysis approach to estimating the likely effect size from the available data, as well as quantify heterogeneity among different experiments, studies and cultivars. The posterior distributions show that, at the coarsest possible scale of total root production, it is unlikely that P. sativum root growth is influenced by either neighbours or barriers to root growth imposed by the walls of pots that vary in volume. We suggest that further work should consider repeating experiments that reported finer scale root plasticity in pea at the rhizosphere scale, and also consider alternative model species for the study of plant root responses to external cues. Neural Mechanism of Hunger Gated Food-seeking and Evaluating The physiological need for energy evokes motivated feeding behaviours that help to ensure survival. However, the neural mechanisms underlying the generation of food motivation remain poorly understood. We investigated these mechanisms by subdividing feeding-related motivated behaviours into food-seeking, evaluating, and swallowing. Micro-endoscopic results indicated that neurons containing leptin receptors (LepRs) in the lateral hypothalamus (LH) are the major food-specific subpopulation of LHGABA neurons. Optogenetic manipulation of LHLepR neurons bidirectionally regulated both food-seeking and evaluating. Furthermore, micro-endoscope data revealed that distinct LHLepR neurons encode seeking and evaluating. Computational modelling analysis demonstrated that LHLepR neurons encode motivation, whereas neurons containing agouti-related peptide and neuropeptide Y (AgRP/NPY) encode the need for food. Additionally, slice studies revealed that NPY decreases inhibitory input to LHLepR neurons via LHGABA interneurons. This mechanism explains the permissive gate role of hunger (food need) in seeking/evaluating motivation. Together, the present study provides a comprehensive neural mechanism of how physiological needs drive distinct motivated behaviours. SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal. Bayesian Additive Regression Trees for Genotype by Environment Interaction Models We propose a new class of models for the estimation of genotype by environment (GxE) interactions in plant-based genetics. Our approach, named AMBARTI, uses semi-parametric Bayesian additive regression trees to accurately capture marginal genotypic and environment effects along with their interaction in a fully Bayesian model. We demonstrate that our approach is competitive or superior to similar models widely used in the literature via both simulation and a real world data set. Furthermore, we introduce new types of visualisation to properly assess both the marginal and interactive predictions from the model. An R package that implements our approach is available at https://github.com/ebprado/ambarti. Physiological Substrates and Ontogeny-Specific Expression of the Ubiquitin Ligases MARCH1 and MARCH8 MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and "atypical" antigen presenting cells of hematopoietic origin, whereas MARCH8 only operated in non-hematopoietic cells. Our results reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated. They also establish MARCH1 and MARCH8 as specialized regulators of CD4+ T cell immunity in two ontogenically distinct cellular compartments. Effects of substrate on shrimp growth, water quality and bacterial community in the biofloc system nursing Penaeus vannamei postlarvae This study aimed to investigate the effects of substrate on water quality, shrimp growth and bacterial community in the biofloc system with a salinity of 5{per thousand}. Two treatments, biofloc system with (sB) or without (nB) addition of elastic solid packing filler (nylon) as substrate, were set up. Penaeus vannamei postlarvae (PL, [~] stage 15) were stocked at a density of 4000 PL m-3 in each treatment with triplicates for a 28-days culture experiment, taking glucose as carbon source (C:N 15:1). Results showed that the survival rate (96.3{+/-}3.6%), FCR (0.76{+/-}0.06) and productivity (1.54{+/-}0.12 kg m-3) in sB treatment were significantly better than those in nB treatment (81.0{+/-}7.1%, 0.98{+/-}0.08 and 1.14{+/-}0.09 kg m-3, P<0.05). All water parameters were in the recommended ranges. Substrate showed significant effect on TAN, TSS, turbidity, biofloc volume, pH and carbonate alkalinity (P < 0.05). Actinobacteria (4.0-22.7%), Bacteroidetes (10.4-33.5%), Firmicutes (0.2-11.2%), Planctomycetes (4.0-14.9%) and Proteobacteria (29.4-59.0%) were the most dominant phyla for both treatments. However, the bacterial community in sB treatment showed to be significantly different from that in nB treatment (Jaccard distance 0.94{+/-}0.01, P=0.001). Substrate showed significant effects on Shannon, Heip, Pielou and Simpson index, as well as relative abundances of Actinobacteria, Bacteroidetes and Planctomycetes (P < 0.05). The results suggested that addition of substrate affected the shrimp growth, water quality and bacterial community in the biofloc system nursing P. vannamei PL with a 5{per thousand} salinity.