The Alzheimer's gene SORL1 is a key regulator of endosomal traffic and recycling in human neurons BackgroundLoss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimers disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, ADs cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).

MethodsWe used isogenic hiPSCs engineered to have SORL1 depleted or to have enhanced SORL1 expression. We differentiated neurons from these cell lines and mapped the trafficking of APP, TRKB and GLUA1 within the endosomal network using confocal microscopy. We also performed cell surface recycling and lysosomal degradation assays to assess the functionality of the endosomal network in both SORL1 depleted and overexpressing neurons. Finally, we analyzed alterations in gene expression in SORL1 depleted neurons using RNA-sequencing.

ResultsWe find that as with APP, endosomal trafficking of GLUA1 and TRKB is impaired by loss of SORL1. We show that trafficking of all three cargo to late endosomes and lysosomes is affected by manipulating SORL1 expression. We also show that depletion of SORL1 significantly impacts the endosomal recycling pathway for APP and GLUA1 at the level of the recycling endosome and trafficking to the cell surface. This has a functional effect on neuronal activity as shown by multi-electrode array (MEA). Conversely, increased SORL1 expression enhances endosomal recycling for APP and GLUA1. Our unbiased transcriptomic data further support SORL1s role in endosomal recycling. We observe altered expression networks that regulate cell surface trafficking and neurotrophic signaling in SORL1 depleted neurons.

ConclusionCollectively, and together with other recent observations, these findings suggest that SORL1 is a broad regulator of retromer-dependent endosomal recycling in neurons, a conclusion that has both pathogenic and therapeutic implications for Alzheimers disease.
Basal Gp78 mitophagy promotes mitochondrial health and limits mitochondrial ROS production Mitochondria are major sources of cytotoxic reactive oxygen species (ROS) that contribute to cancer progression. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. Gp78 E3 ubiquitin ligase is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Basal Gp78-dependent mitophagic flux is therefore selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population and limiting ROS production.
Structural basis for Nirmatrelvir in vitro efficacy against SARS-CoV-2 variants The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). We evaluated the in vitro catalytic activity and in vitro potency of nirmatrelvir against the main protease (Mpro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (, B.1.1.7), Beta ({beta}, B.1.351), Delta ({delta}, B1.617.2), Gamma ({gamma}, P.1), Lambda ({lambda}, B.1.1.1.37/C37), Omicron (o, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the Mpro specifically for: , {beta},{gamma} (K90R), {lambda} (G15S) and o (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrate that they are catalytically comparable to wildtype. Nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the o, {lambda} and {beta} Mpro at 1.63 - 2.09 [A] resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells (1). Text has been revised, image definition has been revised. Maize is a staple food all over the world. Models for climate change suggest that, in the future, cloud formation might be reduced in the tropics increasing the exposure to Ultraviolet-B (UV-B) radiation, a DNA-damaging agent. UV-B (290 to 320 nm) has been shown to affect yield in maize. In this project we have determined the differences in DNA repair efficiencies between U.S. inbred lines B73 and Mo17, and Central American purple landraces from Guatemala and Costa Rica. Our results from single cell electrophoresis experiments (Comet Assay) suggest that the landrace Pujagua Santa Cruz (P1, Costa Rican) was resistant to damage caused by the radiomimetic agent zeocin (24h/100 micrograms per mL), while landrace Pujagua La Cruz (P2, Costa Rican) was able to repair DNA damage after one hour. On the other hand, line Mo17 (Missouri, USA) was unable to repair the damage, while B73 (Iowa, USA) and the landraces Jocopilas (Guatemalan), Orotina Congo (Costa Rican) and Talamanca (Costa Rican) were partially able to repair DNA damage. High Resolution Melting (HRM) curve analysis of putative homologous DNA repair gene ZeaATM1 showed that both P1 and P2 had differences in the melting temperatures for this gene compared to B73 and Mo17, while P1 showed additional differences in ZeaSOG1, ZeaRAD51 and ZeaBRCA1, suggesting that in this landrace the presence of polymorphisms may be common among key genes for this pathway. Taken together our results suggest that key adaptive differences in DNA repair efficiencies exist between inbred lines and landraces of maize and that some Central America landraces could be used as a valuable pool of alleles for plant breeding aiming to increase tolerance to radiation. Combinatorial drug-microenvironment interaction mapping reveals cell-extrinsic drug resistance mechanisms and clinically relevant patient subgroups in CLL The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited to small scale studies and systematic analyses are lacking. We chose Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in adults, as a model disease to study this complex interplay systematically. We performed a combinatorial assay using 12 drugs individually co-applied with each of 17 microenvironmental stimuli in 192 primary CLL samples, generating a comprehensive map of drug-microenvironment interactions in CLL. This data was combined with whole-exome sequencing, DNA-methylation, RNA-sequencing and copy number variant annotation. Our assay identified four distinct CLL subgroups that differed in their responses to the panel of microenvironmental stimuli. These subgroups were characterized by distinct clinical outcomes independently of known prognostic markers. We investigated the effect of CLL- specific recurrent genetic alterations on microenvironmental responses and identified trisomy 12 as an amplifier of multiple microenvironmental stimuli. We further quantified the impact of microenvironmental stimuli on drug response, confirmed known interactions such as Interleukin (IL) 4 mediated resistance to B cell receptor (BCR) inhibitors, and identified new interactions such as Interferon-{gamma} induced resistance to BCR inhibitors. Finally, we identified interactions which were limited to genetic subgroups. Resistance to chemotherapeutics, such as Fludarabine, induced by Toll-Like Receptor (TLR) agonists could be observed in IGHV unmutated patient samples and IGHV mutated samples with trisomy 12. In-vivo relevance was investigated in CLL-infiltrated lymph nodes, which showed increased IL4 and TLR signalling activity compared to healthy samples (p<0.001). High IL4 activity in lymph nodes correlated with faster disease progression (p=0.038).

We provide a publicly available resource (www.dietrichlab.de/CLL_Microenvironment/) which uncovers tumour cell extrinsic influences on drug response and disease progression in CLL, and how these interactions are modulated by cell intrinsic molecular features. 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 A chromosome-level assembly of the "Cascade" hop (Humulus lupulus) genome uncovers signatures of molecular evolution and improves time of divergence estimates for the Cannabaceae family O_LIWe present a chromosome-level assembly of the Cascade hop (Humulus lupulus L. var. lupulus) genome. The hop genome is large (2.8 Gb) and complex, and early attempts at assembly resulted in fragmented assemblies. Recent advances have made assembly of the hop genome more tractable, transforming the extent of investigation that can occur.
C_LIO_LIThe chromosome-level assembly of Cascade was developed by scaffolding the previously-reported Cascade assembly generated with PacBio long-read sequencing, and polishing with Illumina short-read DNA sequencing. We developed gene models and repeat annotations, and used a controlled bi-parental mapping population to identify significant sex-associated markers. We assess molecular evolution in gene sequences, gene family expansion and contraction, and time divergence using Bayesian inference.
C_LIO_LIWe identified the putative sex chromosome in the female genome based on significant sex-associated markers from the bi-parental mapping population. While the estimate of repeat content (~64%) is similar to the hemp genome, syntenic blocks in hop contain a greater percentage of LTRs. Hop is enriched for disease resistance-associated genes in syntenic gene blocks and expanded gene families.
C_LIO_LIThe Cascade chromosome-level assembly will inform cultivation strategies and serve to deepen our understanding of the hop genomic landscape, benefiting hop researchers and the Cannabaceae genomics community.
C_LI Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates. Low heritability and high phenotypic plasticity of salivary cortisol in response to environmental heterogeneity in a wild pinniped Individuals are unique in how they interact with and respond to their environment. Correspondingly, unpredictable challenges or environmental stressors often produce an individualized response of the hypothalamic-pituitary-adrenal axis and its downstream effector cortisol. We used a fully crossed, repeated measures design to investigate the factors shaping individual variation in baseline cortisol in Antarctic fur seal pups and their mothers. Saliva samples were collected from focal individuals at two breeding colonies, one with low and the other with high density, during two consecutive years of contrasting food availability. Mothers and pups were sampled concurrently at birth and shortly before weaning, while pups were additionally sampled every 20 days. We found that heritability was low for baseline cortisol, while within-individual repeatability and among-individual variability were high. A substantial proportion of the variation in baseline cortisol could be explained in pups and mothers by a combination of intrinsic and extrinsic factors including sex, weight, day, season, and colony of birth. Our findings provide detailed insights into the individualization of endocrine phenotypes and their genetic and environmental drivers in a wild pinniped. Furthermore, the strong associations between cortisol and life history traits that we report in fur seals could have important implications for understanding the population dynamics of species impacted by environmental change. Substrate traits shape the structure of microbial community engaged in metabolic division of labor Metabolic division of labor (MDOL) is widespread in nature, whereby a complex metabolic pathway is shared between different strains within a community for mutual benefit. However, little is known about how the mutual interactions in the microbial community engaged in MDOL are regulated. We hypothesized that when degradation of an organic compound is carried out via MDOL, the substrate traits (i.e., concentration and its toxicity) modulate the benefit allocation between the two microbial populations, thus affecting the structure of this community. We tested this hypothesis by combining mathematical modelling with experiments using engineered synthetic microbial consortia. Numerous modelling analyses suggested that the proportion of the population executing the first metabolic step can be simply estimated by Monod-like formulas governed by substrate traits. The model and the proposed formula quantitatively predicted the structure of our synthetic consortia composed of two strains degrading salicylate through MDOL. Individual-based modelling and colony pattern formation assays further indicated that our rule is also applicable to estimating community structure in spatially structured environments. Our results demonstrate that the structure of the microbial communities can be quantitatively predicted from simple environmental factors, such as substrate concentration and its toxicity, which provides novel perspectives on understanding the assembly of natural communities, as well as insights into how to manage artificial microbial systems. 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. Accounting for stimulations that do not elicit motor-evoked potentials when mapping cortical representations of multiple muscles The representation of muscles in the cortex can be mapped using navigated transcranial magnetic stimulation. The mapping can be quantified via measures like the centre of gravity or the size of the region of excitability. Determining these measures typically relies only on stimulation points that yield motor-evoked potentials (MEPs); stimulations that do not elicit an MEP, i.e., non-MEP points, are ignored entirely. In this study, we show how incorporating non-MEP points may affect the estimates of the size and centroid of the excitable area in eight hand and forearm muscles after mono-phasic single-pulse TMS. We performed test-retest assessments in twenty participants and estimated the reliability of centroids and sizes of the corresponding areas using inter-class correlation coefficients. For most muscles, the reliability turned out good, if not excellent. As expected, removing the non-MEP points significantly decreased area sizes and area weights, suggesting that conventional approaches that do not account for non-MEP points are likely to overestimate the regions of excitability. Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel. The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues. Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit. The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. The "dark matter" of protein variants carries a distinct DNA signature and predicts damaging variant effects AO_SCPLOWBSTRACTC_SCPLOWSignatures of DNA motifs associated with distinct mutagenic exposures have been defined for somatic variants, but little is known about the consequences different mutational processes pose to the cell, especially how mutagens exert damage on specific proteins and their three-dimensional structures. Here we identify a DNA mutational signature which corresponds to damaging protein variants. We show that this mutational signature is under-sampled in sequencing data from tumour cohorts, constituting the "dark matter" of the mutational landscape which could only be accessed using deep mutational scanning (DMS) data. By training a set of gradient boosting classifiers, we illustrate that DMS data from only a handful ({approx} 10) of experiments can accurately predict variant impact, and that DNA mutational signatures embed information about the protein-level impact of variants. We bridge the gap between DNA sequence variations and protein-level consequences, discuss the significance of this signature in informing protein design and molecular principles of protein stability, and clarify the relationship between disease association and the true impact mutations bring to protein function. Effects of fine-scale population structure on the distribution of heterozygosity in a long-term study of (Antirrhinum majus) Many studies have quantified the distribution of heterozygosity and relatedness in natural populations, but surprisingly few have examined the demographic processes driving these patterns. In this study we take a novel approach by studying how population structure affects both pairwise identity and the distribution of heterozygosity in a natural population of a self-incompatible plant Antirrhinum majus. We look at a measure of the variance in heterozygosity within a population, identity disequilibrium (g2), together with FST using a panel of 91 SNPs in 22,353 individuals collected over 11 years. We find that pairwise relatedness (FST) declines rapidly over short spatial scales, and the excess variance in heterozygosity between individuals (g2) reflects significant variation in inbreeding. Additionally, we detect an excess of individuals with around half the average heterozygosity, indicating that some are due to selfing or matings between close relatives. We use two types of simulation to ask whether variation in heterozygosity is consistent with fine-scale spatial population structure. First, by simulating offspring using parents drawn from a range of spatial scales, we show that the known pollen dispersal kernel explains g2. Second, we simulate a 1000-generation pedigree using the known dispersal and spatial distribution and find that the resulting g2 is consistent with that observed from the field data. In contrast, a simulated population with uniform density underestimates g2, indicating that heterogeneous density promotes identity disequilibrium. Our study shows that heterogeneous density and leptokurtic dispersal can together explain the distribution of heterozygosity. Furthermore, our study highlights the limitations of making theoretical predictions from simulations that only assume simple density and dispersal distributions. Diverse classes of constraints enable broader applicability of a linear programming-based dynamic metabolic modeling framework Current metabolic modeling tools suffer from a variety of limitations, from scalability to simplifying assumptions, that preclude their use in many applications. We recently created a modeling framework, LK-DFBA, that addresses a key gap: capturing metabolite dynamics and regulation while retaining a potentially scalable linear programming structure. Key to this frameworks success are the linear kinetics and regulatory constraints imposed on the system. However, while the linearity of these constraints reduces computational complexity, it may not accurately capture the behavior of many biochemical systems. Here, we developed three new classes of LK-DFBA constraints to better model interactions between metabolites and the reactions they regulate. We tested these new approaches on several synthetic and biological systems, and also performed the first-ever comparison of LK-DFBA predictions to experimental data. We found that no single constraint approach was optimal across all systems examined, and systems with the same topological structure but different parameters were often best modeled by different types of constraints. However, we did find that the optimal constraint approach was generally robust to local perturbations of the system, indicating that just a single wild-type dataset could allow identification of the ideal constraint for a given system. These results suggest that the availability of multiple constraint approaches will allow LK-DFBA to model a wider range of metabolic systems. A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information. Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation at many enhancers, distal regulatory elements that control spatio-temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific, code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.

ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that is able to predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Then, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity and liver enhancers associated with liver regeneration have a stronger tendency than the general population to have predicted lineage-specific open chromatin.

ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements. 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. Theory of condensin mediated loop extrusion in genomes Condensation of hundreds of mega-base-pair-long human chromosomes in a small nuclear volume is a spectacular biological phenomenon. This process is driven by the formation of chromosome loops. The ATP consuming motor, condensin, interacts with chromatin segments to actively extrude loops. Motivated by real-time imaging of loop extrusion (LE), we created an analytically solvable model, predicting the LE velocity and step size distribution as a function of external load. The theory fits the available experimental data quantitatively, and suggests that condensin must undergo a large conformational change, induced by ATP binding, bringing distant parts of the motor to proximity. Simulations using a simple model confirm that the motor transitions between an open and a closed state in order to extrude loops by a scrunching mechanism, similar to that proposed in DNA bubble formation during bacterial transcription. Changes in the orientation of the motor domains are transmitted over ~ 50 nm, connecting the motor head and the hinge, thus providing an allosteric basis for LE. Groping in the fog: soaring migrants exhibit wider scatter in flight directions and respond differently to wind under low visibility conditions Atmospheric conditions are known to affect flight propensity, behaviour during flight, and migration route in birds. Yet, the effects of fog have only rarely been studied although they could disrupt orientation and hamper flight. Fog could limit the visibility of migrating birds such that they might not be able to detect landmarks that guide them during their journey. Soaring migrants modulate their flight speed and direction in relation to the wind vector to optimize the cost of transport. Consequently, landmark-based orientation, as well as adjustments of flight speed and direction in relation to wind conditions, could be jeopardized when flying in fog. Using a radar system operated in a migration bottleneck (Strait of Messina, Italy), we studied the behaviour of soaring birds under variable wind and fog conditions over two consecutive springs (2016 and 2017), discovering that migrating birds exhibited a wider scatter of flight directions and responded differently to wind under fog conditions. Birds flying through fog deviated more from the mean migration direction and increased their speed with increasing crosswinds. In addition, airspeed and groundspeed increased in the direction of the crosswind, causing the individuals to drift laterally. Our findings represent the first quantitative empirical evidence of flight behaviour changes when birds migrate through fog and explain why low visibility conditions could risk their migration journey. 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. Blocking antibodies against integrin-a3, integrin-aM, and integrin-aMb2 de-differentiate myofibroblasts and reverse lung and kidney fibroses in a mouse model Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of the disease. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins 3, M, and M{beta}2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and reverse lung and kidney fibrosis in vivo. Decorins collagen-binding peptide directs blocking antibodies (against integrins-3, -M, -M{beta}2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to reverse fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment and reversal of fibrosis.

SummaryBlocking antibodies against integrins-3, -M, and -M{beta}2 can be targeted to sites of fibrosis, reverse lung and kidney fibroses, and offer the potential to bring immunotherapy to fibrosis