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.
Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics.
Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. EPIC: inferring relevant cell types for complex traits by integrating genome-wide association studies and single-cell RNA sequencing More than a decade of genome-wide association studies (GWASs) have identified genetic risk variants that are significantly associated with complex traits. Emerging evidence suggests that the function of trait-associated variants likely acts in a tissue- or cell-type-specific fashion. Yet, it remains challenging to prioritize trait-relevant tissues or cell types to elucidate disease etiology. Here, we present EPIC (cEll tyPe enrIChment), a statistical framework that relates large-scale GWAS summary statistics to cell-type-specific gene expression measurements from single-cell RNA sequencing (scRNA-seq). We derive powerful gene-level test statistics for common and rare variants, separately and jointly, and adopt generalized least squares to prioritize trait-relevant cell types while accounting for the correlation structures both within and between genes. Using enrichment of loci associated with four lipid traits in the liver and enrichment of loci associated with three neurological disorders in the brain as ground truths, we show that EPIC outperforms existing methods. We apply our framework to multiple scRNA-seq datasets from different platforms and identify cell types underlying type 2 diabetes and schizophrenia. The enrichment is replicated using independent GWAS and scRNA-seq datasets and further validated using PubMed search and existing bulk case-control testing results. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers BackgroundStress-related neurobehavioral and metabolic disorders are associated with altered circulating adrenal-derived hormones and hyperglycemia. Temporal assessment of glucose and these hormones is critical for insights on an individuals health.

ObjectivesHere we used implantable-telemetry in rats to assess real-time changes in circulating glucose during and after exposure to the air pollutant ozone, and link responses to circulating neuroendocrine stress and metabolic hormones. We also compared rodent glucose and corticosterone (cortisol in humans) responses to humans exposed to ozone.

MethodsFirst, using a cross-over design, we monitored glucose levels during single or repeated ozone exposures (0.0, 0.2, 0.4 and 0.8-ppm) and non-exposure periods in male Wistar-Kyoto-rats implanted with glucose-telemeters. A second cohort of un-implanted rats was exposed to ozone (0.0, 0.4 or 0.8-ppm) for 30-min, 1-hour, 2-hour, or 4-hour with hormones measured immediately after exposure. Then we assessed glucose metabolism in sham and adrenalectomized rats with or without pharmacological interventions of adrenergic and glucocorticoid receptors. Finally, we assessed glucose and cortisol in serum samples from a clinical study involving exposure of human volunteers to air or 0.3 ppm ozone.

ResultsOzone (0.8-ppm) caused hyperglycemia and hypothermia beginning 90-min into exposure, with reversal of effects 4-6 hours post-exposure. Glucose monitoring during four daily 4-hour ozone exposures revealed duration of hyperglycemia, adaptation, and diurnal variations. Ozone-induced hyperglycemia was preceded by increased adrenocorticotropic hormone, corticosterone, and epinephrine, but depletion of thyroid-stimulating, prolactin, and luteinizing hormones. Hyperglycemia was inhibited in rats that were adrenalectomized and/or treated with glucocorticoid inhibitor. The depletion of cortisol was dampened in humans exposed to ozone during intermittent exercise.

DiscussionWe demonstrate for the first time the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced metabolic dysfunction as exposure occurs. Real-time glucose monitoring with stress hormones assessment may be useful in identifying interactions among pollutants and stress-related illnesses. The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies. The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities, found that the alga employs all three known photoinhibition protection mechanisms: i) massive reduction of the PSII antenna size; ii) accumulation of protective carotenoids; and iii) very rapid repair of photo-damaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

One Sentence HighlightAnalysis of the photosynthetic properties of a desert algae that thrives at extreme high light intensities revealed protection from photoinhibition driven by the remarkable enhancement of three protection mechanisms. Visualization of lake succession as indicated by macrophyte species composition and trophic levels in lakes subdivided by volcanic eruption at Akan Caldera, Hokkaido, Japan It is not feasible to continuously observe ecological succession in lakes because of the long time-scales generally involved. Thus, the process has been inductively deduced by comparing many lakes with different succession states, or indirectly simulated by tracking studies of smaller water bodies, experiments using microcosms or mesocosms, and reconstruction of lake history by sediment analysis. However, the reality of succession processes in large lakes with slow succession is not well understood, and new approaches are needed. Theoretically, in a group of large and small lakes of similar ages and with similar initial and watershed environments, the rate of nutrient accumulation in each lake depends on the ratio of watershed area to lake size, and the lakes are predicted to evolve to different trophic levels over time. Here, we tested this hypothesis on the 10 lakes of varying sizes in Akan Caldera, Japan, which were formed thousands of years ago by fragmentation due to volcanic eruptions within the caldera. Topographic and water quality studies showed that the ratio of accumulated watershed area to lake size (area and volume), expressed logarithmically, had a positive linear regression with the total phosphorus concentration, an indicator of trophic level. The trophic levels of the lakes were diverse, including oligotrophic, mesotrophic, and eutrophic types in the traditional "lake type" classification based on total phosphorus and chlorophyll-a concentrations. Furthermore, 21 species of aquatic macrophytes were observed by a diving survey, and the plant species composition was classified into five groups corresponding to the trophic status of the lakes, indicating a conventional "hydrarch succession". The diversity of water quality and aquatic vegetation in a group of lakes with similar origins paves the way for new comparative studies of lakes, including large lakes. Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. 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. Vesalius: high-resolution in silico anatomization of Spatial Transcriptomic data using Image Analysis Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain. Reuse of a wing venation gene-regulatory network in patterning the eyespot rings of butterflies Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we propose that a conserved venation gene network, used in patterning insect wings, has been recruited to pattern the sharp and distinct rings of color in butterfly eyespot development. This network involves Decapentaplegic (Dpp), acting as a central morphogen, activating optix and spalt at different concentration thresholds. It also involves Spalt cross-regulating optix resulting in the formation of a sharp boundary between the two genes. First, using in situ hybridizations, we show that dpp is expressed in the eyespot centers during the early pupal stage and that CRISPR-Cas9 embryonic knockouts of dpp, optix, and spalt result in the loss of eyespots, loss of the orange ring, and loss of the black scales, respectively. Furthermore, using RNAi during the pupal stage, we show that dpp down-regulation results in the complete or partial loss of eyespots. Using CRISPR disruptions of optix or spalt, followed by immunostainings, we show that Spalt represses optix in the central region of the eyespot, limiting optix expression to a more peripheral ring. We further illustrate that optix disruptions alter scale pigmentation and scale ultrastructure as they transform orange to brown scales. Altogether, the regulatory interactions between dpp, spalt and optix mimic those found in earlier larval developmental stages, in the anterior compartment of an insects wing, where these genes play a role in venation patterning. The network similarities suggest that the venation GRN was co-opted to aid in the differentiation of the eyespot rings. Discovery of archaeal Fusexins homologous to eukaryotic HAP2/GCS1 gamete fusion proteins Sexual reproduction consists of genome reduction by meiosis and subsequent gamete fusion. Presence of meiotic genes in prokaryotes suggests that DNA repair mechanisms evolved toward meiotic recombination; however, fusogenic proteins resembling those found in eukaryotes were not identified in prokaryotes. Here, we identify archaeal proteins that are homologs of fusexins, a superfamily of fusogens that mediate eukaryotic gamete and somatic cell fusion, as well as virus entry. The crystal structure of a trimeric archaeal Fusexin1 reveals novel features such as a six-helix bundle and an additional globular domain. Ectopically expressed Fusexin1 can fuse mammalian cells, and this process involves the additional domain and a conserved fusion loop. Archaeal fusexin genes exist within integrated mobile elements, potentially linking ancient archaeal gene exchanges and eukaryotic sex.

One-Sentence SummaryCell membrane fusion proteins of viruses and eukaryotes are also present in archaea. Reticular Dysgenesis-associated Adenylate Kinase 2 deficiency causes failure of myelopoiesis through disordered purine metabolism Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect.

This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. 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. Glycolysis/gluconeogenesis specialization in microbes is driven by biochemical constraints of flux sensing Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic re-organization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida. We combined growth dynamics and dynamic changes of intracellular metabolite levels with a coarse-grained model that integrates fluxes, regulation, protein synthesis and growth and uncovered fundamental limitations of the regulatory network: after nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade-offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.

Graphical synopsis O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Standfirst textMicrobes face a series of fundamental trade-offs that limit their ability to optimize simultaneously for both glycolytic and gluconeogenic growth.

Bullet pointsO_LILag times between glycolysis and gluconeogenesis show asymmetry in many microbes: A long lag in one direction, but a short lag in the other.
C_LIO_LILong lag times are caused by an inability to sense fluxes after nutrient shifts.
C_LIO_LIWith existing regulation, lag time asymmetry can only be overcome by reducing either growth rate or increasing futile cycling in metabolism.
C_LI Discovery of Aminoglycosides as First in Class, Nanomolar Inhibitors of Heptosyltransferase I A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis. Oncogenic protein condensates suppress growth factor perception and modulate drug tolerance Drug resistance remains a central challenge towards durable cancer therapy, including for cancers driven by the EML4-ALK oncogene. EML4-ALK and related fusion oncogenes form cytoplasmic protein condensates that transmit oncogenic signals through the Ras/Erk pathway. However, whether such condensates play a role in drug response is unclear. Here, we used optogenetics to find that condensates suppress signaling through endogenous RTKs including EGFR. Notably, ALK inhibition hypersensitized RTK signals, which are known to drive resistance. Suppression of RTKs occurred because condensates sequestered downstream adapter proteins that are required for RTK signal transmission. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation, which originated from neighboring apoptotic cells. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals suppressed drug tolerance. Our results uncover a regulatory role for RTK fusion condensates in cancer drug response and demonstrate the potential of optogenetics for uncovering functional biomarkers of cancer cells. SARS-CoV-2 promotes microglial synapse elimination in human brain organoids Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection causes an extensive cell death and loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synaptic stripping. To a large extent, SARS-CoV-2 exposed microglia display a transcriptomic profile previously observed in neurodegenerative disorders characterized by early a synapse loss and an increased incident risk after a Covid-19 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from Covid-19. Investigation of Chordwise Functionally Graded Flexural Rigidity in Flapping Wings Using a Two-Dimensional Pitch-Plunge Model Insect wings are heterogeneous structures, with flexural rigidity varying one to two orders of magnitude over the wing surface. This heterogeneity influences the deformation the flapping wing experiences during flight. However, it is not well understood how this flexural rigidity gradient affects wing performance. Here, we develop a simplified 2D model of a flapping wing as a pitching, plunging airfoil using the assumed mode method and unsteady vortex lattice method to model the structural and fluid dynamics, respectively. We conduct parameter studies to explore how variable flexural rigidity affects mean lift production, power consumption and the forces required to flap the wing. We find that there is an optimal flexural rigidity distribution that maximizes lift production; this distribution generally corresponds to a 3:1 ratio between the wings flapping and natural frequencies, though the ratio is sensitive to flapping kinematics. For hovering flight, the optimized flexible wing produces 20% more lift and requires 15% less power compared to a rigid wing but needs 20% higher forces to flap. Even when flapping kinematics deviate from those observed during hover, the flexible wing outperforms the rigid wing in terms of aerodynamic force generation and power across a wide range of flexural rigidity gradients. Peak force requirements and power consumption are inversely proportional with respect to flexural rigidity gradient, which may present a trade-off between insect muscle size and energy storage requirements. The model developed in this work can be used to efficiently investigate other spatially variant morphological or material wing features moving forward. Foraging with friends, moving with anyone: effects of the social environment on movement-integrated habitat selection O_LIMovement links the distribution of habitats with the social environment of animals using those habitats; yet integrating movement, habitat selection, and socioecology remains an opportunity for further study.
C_LIO_LIHere, our objective was to disentangle the roles of habitat selection and social association as drivers of collective movement in a gregarious ungulate. To accomplish this objective, we (1) assessed whether socially familiar individuals form discrete social communities and whether social communities have high spatial, but not necessarily temporal, overlap; and (2) we modelled the relationship between collective movement and selection of foraging habitats using socially informed integrated step selection analysis.
C_LIO_LIWe used social network analysis to assign individuals to social communities and determine short and long-term social preference among individuals. Using integrated step selection functions (iSSF), we then modelled the effect of social processes, i.e., nearest neighbour distance and social preference, and movement behaviour on patterns of habitat selection.
C_LIO_LIBased on assignment of individuals to social communities and home range overlap analyses, individuals assorted into discrete social communities, and these communities had high spatial overlap. By unifying social network analysis with iSSF, we identified movement-dependent social association, where individuals foraged with more familiar individuals, but moved collectively with any between foraging patches.
C_LIO_LIOur study demonstrates that social behaviour and space use are inter-related based on spatial overlap of social communities and movement-dependent habitat selection. Movement, habitat selection, and social behaviour are linked in theory. Here, we put these concepts into practice to demonstrate that movement is the glue connecting individual habitat selection to the social environment.
C_LI Immune complex solubility and size govern Fc-gamma receptor responses: A scalable cell-based reporter system Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. The Achilles' heel of coronaviruses: targeting the 5' Polyuridines tract of the antigenome to inhibit Mouse coronavirus-induced cell death The current coronavirus pandemic situation is worsened by the rapidly-spreading SARS-CoV-2 virus variants. Identification of viral targets that are indispensable for the virus can be targeted to inhibit mutation-based new escape variant development. The 5-polyU tract of the antigenome offers such a target. Host cells do not harbor 5-polyU tracts on any of their transcripts, making the tract an attractive, virus-specific target. Inhibiting the 5-polyU can limit the use of the tract as template to generate 3 polyA tails of +RNAs of coronaviruses. Here, a modified DNA oligo with 3 polyAs is used to target the 5-polyU tract in mouse coronavirus (MHV-A59). The oligo treatment in mouse 17CL-1 cells infected with MHV-A59 significantly prevented virus-induced cell deaths. This proof-of-concept result shows a unique mode of action against mouse coronavirus without affecting host cells, and can be used for the development of novel classes of drugs that inhibit coronavirus infection in host cells, specifically by the COVID-19-causing virus SARS-CoV-2. In addition, as the 5-polyU tract is immediately generated upon infection, the tag can also be targeted for reliable early detection of viral infection.