Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics.
Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.
Hydrophobic mismatch is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes. Disruption of miR-18a alters proliferation, photoreceptor replacement kinetics, inflammatory signaling and microglia/macrophage numbers during retinal regeneration in zebrafish In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Muller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration. A noble TGF beta biogenesis inhibitor exhibits both potent anti-fibrotic and anti-inflammatory capabilities AbstractsIdiopathy pulmonary fibrosis (IPF) is an intractable and fatal human disorder. Our previous study showed that eupatilin exerted a potent anti-fibrotic effect on both in vitro fibrogenesis and bleomycin-induced lung fibrosis model (BLM). Subsequently, an analog called ONG41008 had been identified as a more potent anti-fibrotic than eupatilin and also showed a potent anti-inflammatory capability. Orally administered ONG41008 significantly improved onset of BLM in both prophylactic and therapeutic model and its therapeutic efficacy was similarly compared to or better than pirfenidone by measuring production of collagen and hydroxyproline. Staining collagen or SMA corroborated these results.

As in vitro fibrogenesis models, DHLF (Diseased Human Lung Fibroblasts from IPF patients) and HSC (hepatic stellate cells) were used for direct effects of ONG41008 on pivotal cellular and molecular functions associated with pathogenic myofibroblasts; ONG41008 dismantled latent TGFb complex (LTC), generating inactive forms of TGF{beta}, likely limiting TGF{beta} to TGF{beta} receptor via depolymerization of F-actin and this blunted SMAD2/SMAD3 phosphorylation, thereby reprogramming EMT. A set of cell imaging studies and transcriptomic analysis were conducted to explore how ONG41008 elicited both anti-fibrotic and anti-inflammatory capabilities. Elastin (ELN) seemed to be a pioneering pharmacodynamic marker. It was also found that NOX4 played an important role in anti- fibrosis because it was functionally connected to major central nod proteins such as lysyl- oxidase (LOX) and numerous collagen family members in an ONG41008-specific fibrogenic interactome. Human NOX4 was significantly induced by TGF{beta} and completely knocked down by ONG41008. It has been shown that production of reactive oxygen species (ROS) led to activation of inflammasome. ONG41008 may be likely related to anti-inflammation, leading to a key protective effect on fibrogenesis. Concomitant with downregulation of NOX4, expression of macrophages homing chemokines, CCL2 and CCL7 were significantly attenuated by ONG41008. In vitro anti-inflammatory activities of ONG41008 were investigated in RAW264.7 cells, a mouse monocytic cell line stimulated with LPS. ONG41008 substantially attenuated TNF, CXCL10, CCL2 and CCL7, which are proinflammatory cytokine and important chemokines influencing T cells or macrophages. TNF was situated at the central nod in LPS-treated macrophages via an ONG41008-specific interactome analysis.

Taken together, ONG41008 is a TGF{beta} biogenesis inhibitor, being a potent drug for a broad range of fibrotic diseases and could antagonize inflammatory diseases as well. High resolution, serial imaging of early mouse and human liver bud morphogenesis in three dimensions BackgroundLiver organogenesis has thus far served as a paradigm for solid organ formation. The developing liver bud is a well established model of organogenesis, and murine genetic studies demonstrate key molecules involved in key morphogenetic changes. However, the analysis of the liver bud is typically limited to 2D tissue sections, which precludes extensive visualization, quantitation, and analysis. Further, the lack of human liver bud data has further hindered our understanding of human liver organogenesis. Therefore, new analytical and visualization approaches are needed to elicit further morphogenetic details of liver organogenesis and to elucidate differences between mouse and human liver bud growth.

ResultsTo address this need, we focused on high resolution imaging, visualization, and analysis of early liver growth by using available online databases for both mouse (EMAP, Toronto Phenogenomics center) and human (3D Atlas of Human Embryology), noninvasive multimodality imaging studies of the murine embryo, and mouse/human liver weight data. First, we performed three-dimensional (3D reconstructions) of stacked, digital tissue sections that had been initially segmented for the liver epithelium and the septum transversum mesenchyme (STM). 3D reconstruction of both mouse and human data sets enabled visualization and analysis of the dynamics of liver bud morphogenesis, including hepatic cord formation and remodeling, mechanisms of growth, and liver-epithelial STM interactions. These studies demonstrated potentially under-appreciated mechanisms of growth, including rapid exponential growth that is matched at the earliest stages by STM growth, and unique differences between mouse and human liver bud growth. To gain further insight into the exponential liver bud growth that was observed, we plotted volumetric data from 3D reconstruction together with fetal liver growth data from multimodality (optical projection tomography, magnetic resonance imaging, micro-CT) and liver weight data to compose complete growth curves during mouse (E8.5-E18) and human (day 25-300) liver development. For further analysis, we performed curve fitting and parameter estimation, using Gompertzian models, which enables the comparison between mouse and human liver bud growth, as well as comparisons to processes like liver regeneration. To demonstrate the importance of mesenchyme in rapid liver bud growth and morphogenesis in the human liver bud, we performed functional analysis in which human pluripotent stem cell (hPSC)-derived hepatic organoids were used to model collective migration that occurs in vivo, demonstrating that migration is strongly dependent upon mesenchyme.

DiscussionOur data demonstrates improved visualization with 3D images, under-appreciated and potentially new mechanisms of growth, complete liver growth curves with quantitative analysis through embryonic and fetal stages, and a new functional human stem cell-derived liver organoid assay demonstrating mesenchyme-driven collective migration. These data enhance our understanding of liver organogenesis. High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. 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. Identification and analysis of novel variants in SARS-COV-2 genomes isolated from the Kingdom of Bahrain The challenges imposed by the ongoing outbreak of severe acute respiratory syndrome coronavirus-2 affects every aspect of our modern world, ranging from our health to our socio-economic needs. Our existence highly depends on the vaccines availability, which demands in-depth research of the available strains and their mutations. In this work, we have analyzed all the available SERS-CoV2 genomes isolated from the Kingdom of Bahrain in terms of their variance and origin analysis. We have predicted various known and unique mutations in the SERS-CoV2 isolated from Bahrain. The complexity of the phylogenetic tree and dot plot representation of the strains mentioned above with other isolates of Asia indicates the versatility and multiple origins of Bahrains SERS-CoV2 isolates. We have also identified two high impact spike mutations from these strains which increase the virulence of SARS-CoV2. Our research could have a high impact on vaccine development and distinguishes the source of SERS-CoV2 in the Kingdom of Bahrain. ALLOPURINOL BLOCKS THE AORTIC ANEURYSM IN A MOUSE MODEL OF MARFAN SYNDROME VIA REDUCING AORTIC OXIDATIVE STRESS BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Micro topographical instruction of bacterial attachment, biofilm formation and in vivo host response Bio-instructive materials that prevent bacterial biofilm formation and drive an appropriate host immune response have the potential to significantly reduce the burden of medical device-associated infections. As bacterial surface attachment is sensitive to surface topography, we experimentally surveyed 2,176 combinatorially generated shapes using an unbiased high-throughput micro topographical polystyrene polymer chip screen. This identified topographies that reduced colonization in vitro by up to 15-fold compared with a flat surface for both motile and non-motile bacterial pathogens. Equivalent reductions were achieved on polyurethane, a polymer commonly used in medical devices. Using machine learning methods, a set of design rules based on generalisable topo-descriptors was established for predicting bacteria-resistant micro topographies. In a murine foreign body infection model, anti-attachment topographies were shown to be refractory to P. aeruginosa colonization and serendipitously, the fibrotic response to the implant was reduced, offering control of foreign body response using simple topographical patterning of non-eluting implants. Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades. In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes. Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98. Large Bi-ethnic Study of Plasma Proteome Leads to Comprehensive Mapping of cis-pQTL and Models for Proteome-wide Association Studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Structure of the IL-27 quaternary receptor signaling complex Interleukin 27 (IL-27) is a heterodimeric cytokine that functions to constrain T cell-mediated inflammation and plays an important role in immune homeostasis. Binding of IL-27 to cell surface receptors IL-27R and gp130 results in activation of receptor-associated Janus Kinases and nuclear translocation of STAT1 and STAT3 transcription factors. Despite the emerging therapeutic importance of this cytokine axis in cancer and autoimmunity, a molecular blueprint of the IL-27 receptor signaling complex, and its relation to other gp130/IL-12 family cytokines, is currently unclear. We used cryogenic-electron microscopy (cryo-EM) to determine the quaternary structure of IL-27 (p28/Ebi3) bound to receptor subunits, IL-27R and gp130. The resulting 3.47 [A] resolution structure revealed a three-site assembly mechanism nucleated by the central p28 subunit of the cytokine. The overall topology and molecular details of this binding are reminiscent of IL-6 but distinct from related heterodimeric cytokines IL-12 and IL-23. These results indicate distinct receptor assembly mechanisms used by heterodimeric cytokines with important consequences for targeted agonism and antagonism of IL-27 signaling. RadGenNets: Deep Learning-Based Radiogenomics Model For Gene Mutation Prediction In Lung Cancer AO_SCPLOWBSTRACTC_SCPLOWIn this paper, we present our methodology that can be used for predicting gene mutation in patients with non-small cell lung cancer (NSCLC). There are three major types of gene mutations that a NSCLC patients gene structure can change to: epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), and Anaplastic lymphoma kinase (ALK). We worked with the clinical and genomics data for each patient as well CT scans. We preprocessed all of the data and then built a novel pipeline to integrate both the image and tabular data. We built a novel pipeline that used a fusion of Convolutional Neural Networks and Dense Neural Networks. Also, using a search approach, we pick an ensemble of deep learning models to classify the separate gene mutations. These models include EfficientNets, SENet, and ResNeXt WSL, among others. Our model achieved a high area under curve (AUC) score of 94% in detecting gene mutation. Virtual screening for small molecule pathway regulators by image profile matching Identifying chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual profile-based screening approach instead matches compounds to pathways based on phenotypic information in public cell image data, created using the Cell Painting assay. Our simple correlation-based computational strategy efficiently uncovered the expected small molecule regulators for 32% of positive control gene queries. In discovery mode, we identified compounds related to three of seven tested query genes, validated in subsequent gene-relevant assays. This image profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

One sentence summaryIf a genetic perturbation impacts cell morphology, a computational query can reveal compounds whose morphology "matches". The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies. A pilot study on the home range and movement patterns of the Andean Fox Lycalopex culpaeus (Molina 1782) in Cotopaxi National Park, Ecuador This study reports movement patterns and home range estimates of an Andean fox (Lycalopex culpaeus) in Cotopaxi National Park in Ecuador, representing the first GPS-tagging of the species. The GPS functioned well during the 197-day tracking period. Home range sizes ranged between 4.9 - 8.1 km2, depending on the estimation method. Movement speeds averaged 0.17 km/hr at day vs. 0.87 km/hr. at night, and distance traveled averaged 0.23 km at day vs. 0.89 km at night. These preliminary results highlight the importance of collecting unbiased, high-quality data which enables an enhanced understanding on mammal behavior and human/animal interaction. Quantifying the Morphology and Mechanisms of Cancer Progression in 3D in-vitro environments: Integrating Experiments and Multiscale Models Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns. Its implementation enabled us to pursue two goals; first, the quantitative description of the morphology of cancer progression in 3D cultures, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration. We applied this framework to the study of the spatiotemporal progression of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that cancer cells exhibited chemotactic migration and accumulation, as well as random motion during the examined time period of development. To our knowledge, this is the first time that a multiscale model is used to quantify the relationship between the spatial patterns and the underlying mechanisms of cancer growth in 3D environments. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Scaling effects of temperature on parasitism from individuals to host-parasite systems Parasitism is expected to change in a warmer future, but whether warming leads to substantial increases in parasitism remains unclear. Understanding how warming effects on parasitism in individual hosts (e.g., parasite load) translate to effects on population-level parasitism (e.g., prevalence, R0) remains a major knowledge gap. We analyzed the temperature dependence of parasitism at both host and population levels in thirteen empirical vector-borne host-parasite systems and found a strong, significant positive correlation between the thermal optima of individual- and population-level parasitism. We also found a significant, positive correlation in eleven environmentally-transmitted parasite systems, though several of these systems exhibited thermal optima >5{o}C apart between individual and population levels. Similarly, parasitism thermal optima were close to host performance thermal optima in vector-borne systems but not in environmentally-transmitted systems. We then adapted and simulated simple models for both transmission modes and found a similar pattern to the empirical systems: thermal optima in vector-borne systems were more strongly correlated across scales compared to environmentally-transmitted systems. Generally, our results suggest that information on the temperature-dependence, and specifically the thermal optimum, at either the individual- or population-level should provide a useful--though not quantitatively exact--baseline for predicting temperature dependence at the other level, especially in vector-borne parasite systems. Environmentally-transmitted parasitism may operate by a different set of rules, in which temperature-dependence is decoupled in some systems, requiring the need for trait-based studies of temperature-dependence at individual and population levels. Full-likelihood genomic analysis clarifies a complex history of species divergence and introgression: the example of the erato-sara group of Heliconius butterflies AO_SCPLOWBSTRACTC_SCPLOWIntrogressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species makes estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood analysis methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ full-likelihood methods under the multispecies coalescent (MSC) model with and without gene flow to analyze the genomic data from six members of the erato-sara clade of Heliconius butterflies and infer the species phylogeny and cross-species introgression events. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of H. telesiphe, a likely hybrid speciation origin for H. hecalesia, and gene flow between sister species H. erato and H. himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the multispecies coalescent makes it possible to extract rich historical information of species divergence and gene flow from genomic data. Chromatin-contact atlas reveals disorder-mediated protein interactions RNA-binding proteins (RBPs) play diverse roles in regulating co-transcriptional RNA-processing and chromatin functions, but our knowledge of the repertoire of chromatin-associated RBPs (caRBPs) and their interactions with chromatin remains limited. Here, we developed SPACE (Silica Particle Assisted Chromatin Enrichment) to isolate global and regional chromatin components with high specificity and sensitivity, and SPACEmap to identify the chromatin-contact regions in proteins. Applied to mouse embryonic stem cells, SPACE identified 1,459 chromatin-associated proteins, [~]48% of which are annotated as RBPs, indicating their dual roles in chromatin and RNA-binding. Additionally, SPACEmap stringently verified chromatin-binding of 404 RBPs and identified their chromatin-contact regions. Notably, SPACEmap showed that about half of the caRBPs bind to chromatin by intrinsically disordered regions (IDRs). Studying SPACE and total proteome dynamics from mES cells grown in 2iL and serum medium indicates significant correlation (R = 0.62). One of the most dynamic caRBPs is Dazl, which we find co-localized with PRC2 at transcription start sites of genes that are distinct from Dazl mRNA binding. Dazl and other PRC2-colocalised caRBPs are rich in intrinsically disordered regions (IDRs), which could contribute to the formation and regulation of phase-separated PRC condensates. Together, our approach provides an unprecedented insight into IDR-mediated interactions and caRBPs with moonlighting functions in native chromatin. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs.