Challenges in modelling the sediment retention ecosystem service for an ecosystem account, with examples from the Mitchell catchment If soil resources and the benefits derived from water quality are to be maintained, the on- and off-site effects of soil erosion must be adequately represented and communicated to decision makers so that appropriate management responses can be identified. The System of Environmental-Economic Accounting - Ecosystem Accounting (SEEA-EA), is one approach to quantify both the contributions that ecosystems make to the economy, and the impacts of economic activity on ecosystems. However, due to the difficulty of obtaining empirical data on ecosystem service flows, in many cases such quantification is informed by ecosystem service models. Previous research on the Mitchell catchment, Queensland Australia allowed us to explore the implications of using a model of hillslope erosion and sediment delivery in isolation (as represented in one of the most frequently used ecosystem service models - InVEST) by comparing such estimates against multiple lines of local empirical data, and a more comprehensive representation of locally important erosion and deposition processes through a sediment budget model. Estimates of the magnitude of hillslope erosion modelled using an approach similar to InVEST and the calibrated sediment budget differed by an order of magnitude. If an uncalibrated InVEST-type model was used to inform the relative distribution of erosion magnitude, findings suggest the incorrect erosion process would be identified as the dominant contributor to suspended sediment loads. However, the sediment budget model could only be calibrated using data on sediment sources and sinks that had been collected in the catchment through a sustained research effort. A comparable level of research investment may not be available to inform ecosystem service assessments elsewhere. The results summarised here for the Mitchell catchment demonstrate that practitioners must exercise caution when using estimates of the sediment retention ecosystem service flow which have not been calibrated and validated against locally collected empirical data.
Extracting spatial networks from capture-recapture data reveal structured habitat use within a marine mammal's spatial range O_LIEstimating the impacts of anthropogenic disturbances requires an understanding of the habitat use patterns of individuals within a population. This is especially the case when disturbances are localized within a populations spatial range, as variation in habitat-use within a population can drastically alter the distribution of impacts.
C_LIO_LIHere, we illustrate the potential for multilevel multinomial models to generate spatial networks from capture-recapture data, a common data source use in wildlife studies to monitor population dynamics and habitat use. These spatial networks capture which regions of a populations spatial distribution share similar/dissimilar individual usage patterns, and can be especially useful for detecting structured habitat use within the populations spatial range.
C_LIO_LIUsing simulations and 18 years of capture-recapture data from St. Lawrence Estuary (SLE) beluga, we show that this approach can successfully estimate the magnitude of similarities/dissimilarities in individual usage patterns across sectors, and identify sectors that share similar individual usage patterns that differ from other sectors, i.e., structured habitat use. In the case of SLE beluga, this method identified multiple clusters of individuals, each preferentially using restricted areas within their summer range of the SLE.
C_LIO_LISynthesis and applications. Multilevel multinomial models can be effective at estimating spatial structure in habitat use within wildlife populations sampled by capture-recapture of individuals. Our finding of a structured habitat use within the SLE beluga summer range has direct implications for estimating individual exposures to localized stressors, such as underwater noise from shipping or other activities.
C_LI
Activity-based cell sorting reveals resistance of functionally degenerate Nitrospira during a press disturbance in nitrifying activated sludge Managing and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/L. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed-out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiations within activated sludge and inform strategies to engineer and control microbiome function.

ImportanceNitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash-out nitrite oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of an applied press disturbance to the activated sludge process. Substrate analog probing corroborated the time-series reactor sampling, showing that coexisting and functionally redundant Nitrospira provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management. Synergy of chemotherapy and macrophage depletion leads to B cell-mediated T cell memory activation and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis. Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change. Living with high potassium: how plants balance nutrient acquisition during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI Neutralization potential of Covishield vaccinated individuals sera against B.1.617.1 Covishield comprises the larger proportion in the vaccination program in India. Hence, it is of utmost importance to understand neutralizing capability of vaccine against the B.1.617.1 variant which is considered to responsible for surge of the cases in India. The neutralizing-antibody (NAb) titer against B.1.167.1 and prototype B.1 variant (D614G) was determined of the vaccine sera (4 weeks after second dose) of COVID-19 naive subjects (n=43) and COVID-19 recovered subjects (n=18). The results demonstrated that sera of COVID-19 recovered subjects (n=18) who received two doses of Covishield have higher NAb response compared to the COVID-19 naive with a significant difference (p<0.0001) in NAb titer against B.1 and B.1.617.1 In-spite of reduction in the neutralizing titer against B.1.617.1 variant; Covishield vaccine-induced antibodies are likely to be protective to limit the severity and mortality of the disease in the vaccinated individuals. Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes Bacterial specialized metabolites are a proven source of antibiotics and cancer therapies, but whether we have sampled all the secondary metabolite chemical diversity of cultivated bacteria is not known. We analysed ~ 170,000 bacterial genomes and ~ 47,000 metagenome assembled genomes (MAGs) using a modified BiG-SLiCE and the new clust-o-matic algorithm. We found that only 3% of the natural products potentially encoded in bacterial genomes have been experimentally characterized. We show that the variation of secondary metabolite biosynthetic diversity drops significantly at the genus level, identifying it as an appropriate taxonomic rank for comparison. Equal comparison of genera based on Relative Evolutionary Distance revealed that Streptomyces bacteria encode the largest biosynthetic diversity by far, with Amycolatopsis, Kutzneria and Micromonospora also encoding substantial diversity. Finally we find that several less-well-studied taxa, such as Weeksellaceae (Bacteroidota), Myxococcaceae (Myxococcota), Pleurocapsa and Nostocaceae (Cyanobacteria), have potential to produce highly diverse sets of secondary metabolites that warrant further investigation. Whole-Brain opto-fMRI Map of Mouse VTA Dopaminergic Activation Reflects Structural Projections with Small but Significant Deviations Ascending dopaminergic projections from neurons located in the Ventral Tegmental Area (VTA) are key to the etiology, dysfunction, and control of motivation, learning, and addiction. Due to evolutionary conservation of this nucleus and the extensive use of mice as disease models, establishing an assay for VTA dopaminergic signalling in the mouse brain is crucial for the translational investigation of motivational control as well as of neuronal function phenotypes for diseases and interventions. In this article we use optogenetic stimulation directed at VTA dopaminergic neurons in combination with functional Magnetic Resonance Imaging (fMRI), a method widely used in human deep brain imaging. We present a comprehensive assay producing the first whole-brain opto-fMRI map of dopaminergic activation in the mouse, and show that VTA dopaminergic system function is consistent with its structural VTA projections, diverging only in a few key aspects. While the activation map predominantly highlights target areas according to their relative projection densities (e.g. strong activation of the nucleus accumbens and low activation of the hippocampus), it also includes areas for which a structural connection is not well established (such as the dorsomedial striatum). We further detail the variability of the assay with regard to multiple experimental parameters, including stimulation protocol and implant position, and provide evidence-based recommendations for assay reuse, publishing both reference results and a reference analysis workflow implementation. Supergenes origin and maintenance in Atlantic cod Supergenes are sets of genes that are inherited as a single marker and encode complex phenotypes through their joint action. They are identified in an increasing number of organisms, yet their origins and evolution remain enigmatic. In Atlantic cod, four megabase-scale supergenes have been identified and linked to migratory lifestyle and environmental adaptations. Here, we investigate the origin and maintenance of these four supergenes through analysis of whole-genome-sequencing data, including a new long-read-based genome assembly for a non-migratory Atlantic cod individual. We corroborate that chromosomal inversions underlie all four supergenes, and show that they originated at different times between 0.40 and 1.66 million years ago. While we found no evidence for a role of introgression in the origin of the four supergenes, we reveal gene flux between supergene haplotypes with derived and ancestral arrangements, occurring both through gene conversion and double crossover. Our results suggest that supergenes can be maintained over long timescales in the same way as hybridizing species, through the purging of introduced genetic variation. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries two powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. In this study, we identified, phased, and reconstructed the two haplotypes of each resistance-associated locus. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat (NLR) genes. Structural variations also impacted the domain composition of some NLRs. These findings emphasize the need of generating haplotype-resolved sequences instead of using consensus sequences for identifying haplotype-specific candidate genes. Comparison of the NLRs in the Run1.2 and Run2.2 loci indicated that the two loci are composed of a different number and classes of NLR genes. We provide a list of candidate NLR genes from the Run1.2b and Run2.2 loci, whose expression suggests a role in powdery mildew resistance in Trayshed. These first complete and haplotype-resolved resistance-associated loci, and their candidate NLR genes, represent new resources to develop powdery mildew-resistant grape cultivars. Outer hair cell electro-mechanical power conversion Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function requires expression of the protein prestin in the OHC lateral membrane and mechano-electrical transduction in the apical hair bundle. In the present report, electrical power consumption and mechanical power output are determined using previously published voltage clamp data from isolated OHCs and membrane patches. Results reveal that power output by the prestin-motor complex peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90{degrees} shift in the phase of prestin-dependent electrical charge displacement, manifested electrically as emergence of imaginary-valued nonlinear capacitance. Behavioral effects of ethanol in the Red Swamp Crayfish (Procambarus clarkii) Alcohol abuse and dependence remains one of the primary preventable sources of human mortality in the United States. Model laboratory species can be used to evaluate behavioral, neuropharmacological and other biological changes associated with alcohol exposure and to identify novel treatment modalities. This report describes methods for evaluating the behavioral effects of ethyl alcohol (ethanol; EtOH) in a crayfish model. Crayfish (Procambarus clarkii) were immersed in ethanol baths with concentrations ranging from 0.1 to 1.0 molar, and for durations of 10-30 minutes. Studies evaluated hemolymph alcohol concentration, locomotor behavior in an open field and anxiety-like behavior using a Light/Dark transfer approach. EtOH immersion produced dose dependent increases in hemolymph EtOH concentration (up to 249 mg/dL) and reductions in open field locomotor behavior that depended on EtOH concentration or exposure duration. Under baseline conditions, crayfish exhibit avoidance of the open parts of the locomotor arena and a preference for the covered portion, when available. Acute EtOH immersion decreased time spent in the covered portion of the Light/Dark arena, consistent with a decrease in anxiety-like behavior. EtOH immersion once per day for 5 days did not alter the acute locomotor behavioral response, however increased activity was observed 3 days after the repeated EtOH regimen. Overall, this study shows that this inexpensive, easily maintained species can be used for behavioral pharmacological experiments designed to assess the acute and repeated effects of EtOH. Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold. Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV.
C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain.
C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery.
C_LI 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. Rattus Economicus: A neural model for rational regulation of water-seeking effort in rodents In the laboratory, animals motivation to work tends to be positively correlated with reward magnitude. But in nature, rewards earned by work are essential to survival (e.g., working to find water), and the payoff of that work can vary on long timescales (e.g., seasonally). Under these constraints, the strategy of working less when rewards are small could be fatal. We found that instead, rats in a closed economy did more work for water rewards when the rewards were stably smaller, a phenomenon also observed in human labor supply curves. Like human consumers, rats also showed elasticity of demand, consuming far more water per day when its price in effort was lower. The neural mechanisms underlying such "rational" market behaviors remain largely unexplored. We propose a dynamic utility maximization model that can account for the dependence of rat labor supply (trials/day) on the wage rate (ml/trial), and also predict the temporal dynamics of when rats work. Based on data from mice, we hypothesize that SFOGLUT neurons in lamina terminalis continuously compute the instantaneous marginal utility of voluntary work for water reward, and causally determine the amount and timing of work. An Extended Motif in the SARS-CoV-2 Spike Modulates Binding and Release of Host Coatomer in Retrograde Trafficking {beta}-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for COPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. COPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking. A Neurogenic Signature Involving Monoamine Oxidase-A controls Human Thermogenic Adipose Tissue Development Mechanisms that control "beige/brite" thermogenic adipose tissue development may be harnessed to improve human metabolic health. To define these mechanisms, we developed a species-hybrid model in which human mesenchymal progenitor cells were used to develop white or thermogenic/beige adipose tissue in mice. The hybrid adipose tissue developed distinctive features of human adipose tissue, such as larger adipocyte size, despite its neurovascular architecture being entirely of murine origin. Thermogenic adipose tissue recruited a denser, qualitatively distinct vascular network, differing in genes mapping to circadian rhythm pathways, and denser sympathetic innervation. The enhanced thermogenic neurovascular network was associated with human adipocyte expression of THBS4, TNC, NTRK3 and SPARCL1, which enhance neurogenesis, and decreased expression of MAOA and ACHE, which control neurotransmitter tone. Systemic inhibition of MAOA, which is present in human but absent in mouse adipocytes, induced browning of human but not mouse adipose tissue, revealing the physiological relevance of this pathway. Our results reveal species-specific cell type dependencies controlling the development of thermogenic adipose tissue and point to human adipocyte MAOA as a potential target for metabolic disease therapy. Itaconate and its derivatives repress C2C12 myogenesis A Krebs cycle intermediate metabolite, itaconate, has gained attention as a potential antimicrobial and autoimmune disease treatment due to its anti-inflammatory effects. While itaconate and its derivatives pose an attractive therapeutic option for the treatment of inflammatory diseases, the effects outside the immune system still remain limited, particularly in the muscle. Therefore, we endeavored to determine if itaconate signaling impacts muscle differentiation. Utilizing the well-established C2C12 model of in vitro myogenesis, we evaluated the effects of itaconate and its derivatives on transcriptional and protein markers of muscle differentiation as well as mitochondrial function. We found itaconate and the derivatives dimethyl itaconate and 4-octyl itaconate disrupt differentiation media-induced myogenesis. A primary biological effect of itaconate is a succinate dehydrogenase (SDH) inhibitor. We find the SDH inhibitors dimethyl malonate and harzianopyridone phenocopie the anti-myogenic effects of itaconate. Furthermore, we find treatment with exogenous succinate results in blunted myogenesis. Together our data indicate itaconate and its derivatives interfere with in vitro myogenesis, potentially through inhibition of SDH and subsequent succinate accumulation. More importantly, our findings suggest the therapeutic potential of itaconate and its derivatives could be limited due to deleterious effects on myogenesis. Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Dynamic tracing using ultra-bright labelling and multi-photon microscopy identifies endothelial uptake of poloxamer-188 coated poly(lactic-co-glycolic acid)nano-carriers in vivo Poly(lactic-co-glycolic acid) (PLGA)-based drug formulations are approved for the use in humans, however, the potential of PLGA to design nanoparticles (NPs) and target the central nervous system remains to be exploited.

The aim of the current study was design PLGA NPs, loading them with bulky fluorophores thereby increasing single particle fluorescence to a level visible by in vivo microscopy, and investigate their brain biodistribution. We developed, highly fluorescent 70 nm PLGA NPs significantly brighter then quantum dots enabling their visualization by intravital real-time 2-photon microscopy. We found that PLGA NPs coated with pluronic F-68 (PF-68) had a substantially longer plasma half-life than uncoated NPs and were taken up by cerebro-vascular endothelial cells. High resolution confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs.

The current data suggest that PF-68 coated PLGA NPs are taken up by mouse cerebral and peripheral endothelial cells in vivo. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers. SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19 IntroductionCoagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis.

MethodsTo understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer.

ResultsThe investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease.

ConclusionsIn summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity. PrISM: precision for integrative structural models SummaryA single precision value is currently reported for an integrative model. However, precision may vary for different regions of an integrative model owing to varying amounts of input information. We develop PrISM (Precision for Integrative Structural Models), to efficiently identify high and low-precision regions for integrative models.

Availability and ImplementationPrISM is available at https://github.com/isblab/prism; benchmark data used in this paper is available at doi:10.5281/zenodo.6241200

Contactshruthiv@ncbs.res.in

Supplementary informationThis article contains Supplementary data online. Cargo-mediated mechanisms reduce inter-motor mechanical interference, promote load-sharing and enhance processivity in teams of molecular motors In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work as teams is unclear. Here we explored the possibility that purely physical mechanisms may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a 3D dynamical simulation of cargo transport along microtubules by teams of canonically non-cooperative kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. We showed explicitly, for the first time, that surface fluidity leads to the reduction of negative mechanical interference between kinesins, enhancing load sharing thereby decreasing single motor off-rates and increasing processivity. Remarkably, we also showed that, independent of fluidity, increasing numbers of bound motors pull the cargo closer to the microtubule, increasing the on-rates of individual unbound motors, resulting in a cooperative increase in bound motor numbers that depends on 3D cargo geometry. At the cargo level, surface fluidity makes more motors available for binding, though this effect is significant only at low ATP or high motor density. Interestingly, we find that the fluidity induced reduction in mechanical interference dominates over the increased availability of motors for typical physiological and in vitro conditions, this allowing us to reconcile different experimental results in different regimes. Finally, we show that these effects can altogether result in enhanced mechanical efficiency and tunable cargo run-lengths for teams of molecular motors over physiological ranges of fluidity with implications for new experimental validation efforts, transport in vivo, artificial cargo design and multi-motor-driven mechanical processes in general.

Author summaryIn cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of protein filaments. How do intrinsically non-cooperative motors function as teams within cells? In this paper, we show, using computer simulations, that the fluid surfaces of cellular cargo reduce the mechanical interference between motors allowing better load sharing and decreasing their detachment rates, thereby increasing the distance over which they can carry cargo. We additionally found that motors pull cargo closer to the filament increasing the attachment rates of other unbound motors. These effects act synergistically with an increased availability of motors due to fluidity to further increase travel distances. Our simulation model accounted for the dynamics of the motors on the cargo surface and their load and ATP dependent kinetics, allowing us to connect single motor properties to overall transport. Our work on understanding how teamwork arises in mechanically coupled motors sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving mechanical efficiency and transport of artificial cargo powered by motor teams.