Towards a unified model of naive T cell dynamics across the lifespan Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we employ multiple experimental systems to identify a unified model of naive CD4 and CD8 T cell population dynamics across the mouse lifespan. We infer that both subsets divide rarely and progressively increase their survival capacity with cell age. Strikingly, this simple model captures naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics from birth into old age.
Epithelial and neutrophil interactions and coordinated response to Shigella in a human intestinal enteroid-neutrophil co-culture model Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Herein, we report the development and the characterization of an ex vivo tissue co-culture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN intercalated within the epithelial cells and moved paracellularly toward the apical side. Co-cultured PMN also increased basal secretion of IL-8. Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, NET formation as well as bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the co-culture; apical MCP-1, TNF-, and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation, mobilization, and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN co-culture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools.

ImportanceStudies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which render non-physiological and often unreliable results. Herein we report the development and characterization of an ex vivo enteroid-PMN co-culture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.
Actomyosin activity-dependent apical targeting of Rab11 vesicles reinforces apical constriction During tissue morphogenesis, cell shape changes resulting from cell-generated forces often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin dependent mechanism involving Rab11- mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding. 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 Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. Simple mechanisms of plant reproductive benefits yield different dynamics in pollination and seed dispersal mutualisms Pollination and seed dispersal mutualisms are critical for biodiversity and ecosystem services yet face mounting threats from anthropogenic perturbations that cause their populations to decline. Characterizing the dynamics of these mutualisms when populations are at low density is important to anticipate consequences of these perturbations. We developed simple population dynamic models detailed enough to distinguish different mechanisms by which plant populations benefit from animal pollination or seed dispersal. We modeled benefits as functions of foraging rate by animals on plant rewards and specified whether they affected plant seed set, germination, or negative density dependence during recruitment. We found that pollination and seed dispersal mutualisms are stable at high density but exhibit different dynamics at low density, depending on plant carrying capacity, animal foraging efficiency, and whether populations are obligate upon their partners for persistence. Under certain conditions, all mutualisms experience destabilizing thresholds in which one population declines because its partner is too rare. Plants additionally experience Allee effects when obligate upon pollinators. Finally, pollination mutualisms can exhibit bistable coexistence at low or high density when plants are facultative upon pollinators. Insights from our models can inform conservation efforts, as mutualist populations continue to decline globally. Misoprostol Treatment Prevents Hypoxia-Induced Cardiac Dysfunction Through a 14-3-3 and PKA regulatory motif on Bnip3 Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analogue misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury. A platform of patient-derived microtumors identifies individual treatment responses and therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment. Elucidation of SARS-CoV-2 budding mechanisms through molecular dynamics simulations of M and E protein complexes SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding. Evaluation of methods for estimating coalescence times using ancestral recombination graphs The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices. Comparative analysis of molecular representations in prediction of drug combination effects AO_SCPLOWBSTRACTC_SCPLOWApplication of machine and deep learning methods in drug discovery and cancer research has gained a considerable amount of attention in the past years. As the field grows, it becomes crucial to systematically evaluate the performance of novel computational solutions in relation to established techniques. To this end we compare rule-based and data-driven molecular representations in prediction of drug combination sensitivity and drug synergy scores using standardized results of 14 throughput screening studies, comprising 64 200 unique combinations of 4 153 molecules tested in 112 cancer cell lines. We evaluate the clustering performance of molecular representations and quantify their similarity by adapting the Centered Kernel Alignment metric. Our work demonstrates that to identify an optimal molecular representation type it is necessary to supplement quantitative benchmark results with qualitative considerations, such as model interpretability and robustness, which may vary between and throughout preclinical drug development projects.



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org.highwire.dtl.DTLVardef@1bc4509org.highwire.dtl.DTLVardef@1586640org.highwire.dtl.DTLVardef@a11129org.highwire.dtl.DTLVardef@6dc93b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Advanced Functional Biotextiles for a Circular Materials Economy A circular materials economy can disrupt linear production models and mitigate its environmental threats. We harness extracellular biosynthesis of microbial nanocellulose (MC), coupled to a protocol that represents a serendipitous intersection between ancient textile techniques and green processing as a route to high-performance bioleather with a circular life cycle. Specifically, a plant-based lecithin phosphocholine treatment modulates cellulose cross-linking through phosphate and methylene groups, to yield a biodegradable material with superior mechanical and flame-retardant properties. A broad color palette and controlled color modulation was achieved with cultural heritage dyes and waste-to-resource strategies. Life cycle impact assessment shows up to an order of magnitude reduction in environmental impacts relative to conventional textiles, making MC relevant for widespread application in fashion, interiors, construction and insulating materials. The translational potential of this approach is tremendous, as use of microbes and green chemistry to engineer regenerative, high-performance products can accelerate a paradigm shift to a circular bioeconomy. Force profile of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

MethodsThirty-five community-dwelling males and females (59-79 years) were recruited, from applicants to participate in the BELL trial. Two-handed hardstyle swings were performed with 8-16 kg. Deadlifts were performed with 8-24 kg and 8-32 kg for females and males, respectively. Ground reaction force was obtained from a floor-mounted force platform. Pairwise comparisons of peak force, forward force, rate of force development, swing cadence, sex, and kettlebell mass, were investigated for the kettlebell swing, with representative force-time curves described. Pairwise comparisons of peak force, sex and kettlebell mass were investigated for the deadlift, with comparisons of peak force, kettlebell mass, and sex, between swings and deadlifts.

ResultsFor kettlebells up to 16 kg, paired samples T-tests show a large exercise effect ({delta} > 1.4) with peak force higher for swings than deadlifts. Data shows: (i) higher peak force during swings than deadlifts ({delta} = 1.77), reaching 4.5 (1.0) N.kg-1, (ii) peak force during an 8 kg swing was greater than a 32kg deadlift, (iii) negligible difference in normalised peak force between males and females performing kettlebell swings, but a moderately large effect size during deadlifts (males > females, {delta} = 0.69), (iv) mean rate of force development of 19.9 (4.7) N.s-1.kg-1 with a very weak, positive correlation with kettlebell mass (y = 14.4 + 0.32x), and trivial effect of sex, (v) mean forward force equal to 5.5% of vertical force during swings, increasing from 3.8 (1.6) % with 8 kg to 7.1 (2.6) % with 16 kg.

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults. CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7 V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal  helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity. Somatic uniparental disomy mitigates the most damaging EFL1 allele combination in Shwachman-Diamond syndrome Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy. 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. Methylome dynamic upon proteasome inhibition by the Pseudomonas syringae virulence factor Syringolin A DNA methylation is an important epigenetic mark required for proper gene expression and silencing of transposable elements. DNA methylation patterns can be modified by environmental factors such as pathogen infection, where modification of DNA methylation can be associated with plant resistance. To counter the plant defense pathways, pathogens produce effectors molecule, several of which act as proteasome inhibitors. Here we investigated the effect of proteasome inhibition by the bacterial virulence factor Syringolin A on genome-wide DNA methylation. We show that Syringolin A treatment results in an increase of DNA methylation at centromeric and pericentromeric regions of Arabidopsis chromosomes. We identify several CHH DMRs that are enriched in the proximity of transcriptional start sites. Syringolin A treatment does not result in significant changes in small RNA composition. However, significant changes in genome transcriptional activity can be observed, including a strong upregulation of resistance genes that are located on chromosomal arms. We hypothesize that DNA methylation changes could be linked to the upregulation of some atypical members of the de novo DNA methylation pathway: AGO3, AGO9 and DRM1. Our data suggests that modification of genome-wide DNA methylation resulting from an inhibition of the proteasome by bacterial effectors could be part of a epi-genomic arms race against pathogens. Substantial genetic mixing among sexual and androgenetic lineages within the clam genus Corbicula AO_SCPLOWBSTRACTC_SCPLOW"Occasional" sexuality occurs when a species combines clonal reproduction and genetic mixing. This strategy is predicted to combine the advantages of both asexuality and sexuality, but its actual consequences on the genetic diversity and species longevity are poorly understood. Androgenesis, a reproductive mode in which the offspring inherits its entire nuclear genome from the father, is often reported as a strictly clonal reproductive mode. Androgenesis is the predominant reproductive mode within the hermaphroditic, invasive lineages of the mollusk genus Corbicula. Their ability to reproduce clonally through androgenesis has been determinant in their invasive success, having colonized during the 20th century American and European freshwater systems, where they became notorious invaders with a widespread, global distribution. However, in androgenetic Corbicula clams, occasional genetic mixing between distinct lineages has also been observed when the sperm of one lineage fertilizes the oocyte of another one. Because of these occasional introgressions, the genetic relationships between Corbicula species remained unclear, and the biogeographic origins of the invasive androgenetic lineages have been challenging to identify. To address these issues, we analyzed the patterns of allele sharing for several nuclear and mitochondrial molecular markers among Corbicula individuals collected across both the native and invasive range. Our results show the occurrence of an allelic pool encompassing all Corbicula freshwater species worldwide, including sexual and androgenetic ones, which highlights the substantial genetic mixing within this genus. However, the differences in allele sharing patterns between invasive lineages, and the low diversity within each lineage, suggest recent, distinct biogeographic origins of invasive Corbicula androgenetic lineages. Finally, the polyploidy, high heterozygosity, and hybrid phenotypes and genotypes found in our study probably originated from hybridization events following egg parasitism between distinct Corbicula lineages. This extensive cross-lineage mixing found in Corbicula may generate nuclear diversity in an otherwise asexually reproducing species. Intron-mediated induction of phenotypic heterogeneity Introns are universally present in the nuclear genomes of eukaryotes1. The budding yeast, an otherwise intron-poor species, preserves two sets of ribosomal protein (RP) genes differing primarily in their introns2-4. Despite recent findings on the role of RP introns under stress and starvation5-7, understanding the contribution of introns to ribosome regulation remains challenging. Here, combining isogrowth profiling8 with single-cell protein measurements9, we found that introns can mediate inducible phenotypic heterogeneity conferring a clear fitness advantage. Osmotic stress leads to bimodal expression of the small ribosomal subunit protein Rps22B, mediated by an intron in the 5 untranslated region of its transcript. The two resulting yeast subpopulations differ in their ability to cope with starvation. Low Rps22B protein levels resulted in prolonged survival under sustained starvation, while high Rps22B levels enabled cells to grow faster after transient starvation. Further, yeast growing at high sugar concentrations - similar to those in ripe grapes - exhibit bimodal Rps22B expression when approaching stationary phase. Differential intron-mediated regulation of RP genes thus provides a way to diversify the population when starvation looms in natural environments. Our findings reveal a new role for introns in inducing phenotypic heterogeneity in changing environments and suggest that duplicated RP genes in yeast contribute to resolving the evolutionary conflict between precise expression control and environmental responsiveness10. Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP  M_FIG C_FIG Temporal responses of bumblebee gustatoryneurons to sugars The sense of taste permits the recognition of valuable nutrients and the avoidance of potential toxins. Models of gustatory coding propose that within modalities (e.g. sweet, bitter, etc.), taste ligands are not distinct stimuli. However, these models are based on data from mice or flies that have omnivorous, non-specialist diets. A specialist feeder might, however, be expected to have acuity within modality if stimulus resolution was critical to survival. Previously, we found that bumblebees have a specialized mechanism for sensing sugars whereby two gustatory receptor neurons (GRNs) within the galeal sensilla of the bees mouthparts exhibit burst of spikes. Here, we show that the temporal firing patterns of these GRNs separate sugars into four distinct groups that correlate with sugar nutritional value and palatability. We also identified a third GRN that responded to stimulation with relatively high concentrations of fructose, sucrose, and maltose. Sugars that were non-metabolizable or toxic suppressed the responses of bursting GRNs to sucrose. These abilities to encode information about sugar value are a refinement to the bumblebees sense of sweet taste that could be an adaptation that enables precise calculations of the nature and nutritional value of floral nectar. Greater spear nosed bats in Panama do not use social proximity to improve foraging efficiency Group foraging animals may change their spatial relationships in response to the resource landscape. The strength of social network ties across foraging groups could then reflect the energy balance of individual group members through their effect on foraging efficiency. Greater spear-nosed bats (Phyllostomus hastatus) live in social groups where unrelated females develop group-specific social calls, defend each others offspring, and are hypothesized to coordinate social foraging to feed on balsa flowers. To test if social groups forage together, and if this increases foraging efficiency, we tracked 18 individuals from three social groups of P. hastatus on Isla Colon, Panama using high frequency GPS for 1 - 3 days each. We found that individuals commuted alone but then joined group members in small foraging patches with high densities of flowering balsas, 25 km from their roost on the mainland. Proximity-based social network measures did not predict foraging efficiency or energy expenditure, but individuals with higher social associations spent more time outside of the cave. Individuals did not coordinate movement, but close proximity among individuals increased resting bout duration and synchrony, particularly when in the same patch. These results show that stronger social proximity associations were more closely tied to resting behavior, and indicate that factors other than increased feeding efficiency may structure network relationships of group members while foraging.

Lay SummarySocial foraging can increase foraging efficiency on hard-to-find food sources. Miniaturized high-frequency GPS allowed us to test this on groups of greater spear-nosed bats. We estimated food intake rates, energy expenditure, and the strength of bat social relationships. In contrast to our expectations, we did not find a relationship between bat social relationships and energy expenditure or foraging efficiency. However, bats took longer resting breaks when group members were close. Why these bats commute 25 km individually across the ocean to then forage near each other thus requires further investigation. Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis. Morphogenesis and cell ordering in confined bacterial biofilms Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments, in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm-environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species, and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

Significance StatementBiofilms are microbial cities in which bacterial cells reside in a polymeric matrix. They are commonly found inside soft confining environments such as food matrices and host tissues, against which bacteria must push to proliferate. Here, by combining single-cell live imaging and mechanical characterization, we show that the confining environment determines the dynamics of biofilm shape and internal structure. This self-organized evolution of biofilm architecture is caused by force transmission between the environment and the biofilm, mediated by the extracellular matrix secreted by the cells. Our findings lead to new ways to understand how bacterial communities develop under mechanical constraints, and potentially to new strategies for preventing and controlling biofilm growth in three-dimensional 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 Non-targeted multimodal metabolomics and lipidomics data from ovine rumen fluid fractions BackgroundMetabolomics is a powerful and sensitive approach for investigating low molecular weight metabolite profiles present in rumen biofluids to identify potential roles of metabolites in rumen microbiome and understanding host-level regulatory mechanisms associated with animal production.

FindingsRumen samples from sheep grazed on a mixed ryegrass and clover pasture diet, were fractionated based on molecular weight and analysed using metabolomics and lipidomics to detect the small molecules present in ovine rumen fluid fractions.

ConclusionsUntargeted metabolomics provides a detailed snapshot of the ovine ruminal fluid metabolome that can be used as reference for future studies on ovine rumen fluid or as a comparator for other ruminant species. All data and metadata are available for download in the MetaboLights database.