The interplay of chromatin phase separation and lamina interactions in nuclear organization The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin which are reminiscent of active multi-phase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. In order to dissect the organizational and dynamical implications of chromatins liquid behavior, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Employing the liquid chromatin model of Drosophila nucleus, we have carried out an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Our simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. We also quantitatively reproduce coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of our model by phase separation of chromatin types operating within constrained intra-chromosomal and inter-chromosomal boundaries. Finally, we illuminate the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets.

SIGNIFICANCE STATEMENTThe latest super-resolution imaging experiments have revealed a surprisingly dynamic and stochastic nature of chromatin in eukaryotic nuclei which is reminiscent of multi-phase fluid behavior. As a result, the concepts from the theory of complex fluids such as phase separation, viscoelasticity, and droplet nucleation have found widespread utility in understanding salient features of nuclear organization. In order to understand and disentangle the complex interplay of forces that contribute to the emergent patterns of organization and dynamics, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Armed with a mesoscopic model of nuclear chromatin, we have shed light on the distinct dynamical and structural contributions of chromatin type interactions intermingling of chromosomal territories and lamina binding. We also shed light on the dynamical heterogeneity and coherent motions of chromatin domains which are fully captured by an interplay of micro-phase separation of chromatin types and lamina binding.
Improving Martini 3 for disordered and multidomain proteins Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of proteins. Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of intrinsically disordered proteins (IDPs) and multidomain proteins when compared with small angle X-ray scattering (SAXS) data, and that increasing the strength of protein-water interactions favours more expanded conformations. We find that increasing the strength of interactions between protein and water by ca. 10% results in improved agreement with the SAXS data for IDPs and multi-domain proteins. We also show that this correction results in a more accurate description of self-association of IDPs and folded proteins and better agreement with paramagnetic relaxation enhancement data for most IDPs. While simulations with this revised force field still show deviations to experiments for some systems our results suggest that it is overall a substantial improvement for coarse-grained simulations of soluble proteins.
Highly scaled measurements of C. elegans development suggest that physical constraints guide growth trajectories and animal shape Growth control establishes organism size, requiring mechanisms to sense and adjust growth during development. Studies of single cells revealed that size homeostasis uses distinct control methods. In multicellular organisms, mechanisms that regulate single cell growth must integrate control across organs and tissues during development to generate adult size and shape. We leveraged the roundworm Caenorhabditis elegans as a scalable and tractable model to collect precise growth measurements of thousands of individuals, measure feeding behavior, and quantify changes in animal size and shape during a densely sampled developmental time course. As animals transitioned from one developmental stage to the next, we observed changes in body aspect ratio while body volume remained constant. Then, we modeled a physical mechanism by which constraints on cuticle stretch could cause changes in C. elegans body shape. The model-predicted shape changes are consistent with those observed in the data. Theoretically, cuticle stretch could be sensed by the animal to initiate larval-stage transitions, providing a means for physical constraints to influence developmental timing and growth rate in C. elegans.

HighlightsO_LIBody size measurements of thousands of animals in a dense developmental time course
C_LIO_LIGrowth rate exhibits nonlinear dynamics in both length and width
C_LIO_LIChanges in body shape but not volume occur during periods of increased quiescence
C_LIO_LIDynamics of animal shape consistent with a length-based threshold in cuticle stretch
C_LIO_LIModeling of cuticle stretch dynamics suggests a novel mode for growth control
C_LI Engineered bacteria detect tumor DNA Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site. An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner. 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. Humidity Reduces Rapid and Distant Airborne Dispersal of Viable Viral Particles in Classroom Settings The transmission of airborne pathogens via aerosols is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. It is therefore essential to quantify airborne transmission in closed spaces and determine the recommendations that should be implemented to minimize exposure to pathogens in built environments. We have developed a method to detect viable virus particles from aerosols by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. Based on this method we present two striking results. (1) We consistently detected viable phage particles at distances of up to 18 feet away from the source within 15-minutes of exposure in a classroom equipped with a state of the art HVAC system. (2) Increasing the relative humidity beyond 40% significantly reduces dispersal. Our method can be used to quantify the exposure to pathogens at various distances from the source for different amounts of time, data which can be used to set safety standards for room capacity and to ascertain the efficacy of interventions that aim to reduce pathogen levels in closed spaces of specified sizes and intended uses.

SummaryWe present a method to experimentally determine the exposure to airborne pathogens in closed spaces. Extended Graphical Lasso for Multiple Interaction Networks for High Dimensional Omics Data There has been a spate of interest in association networks in biological and medical research, for example, genetic interaction networks. In this paper, we propose a novel method, the extended joint hub graphical lasso (EDOHA), to estimate multiple related interaction networks for high dimensional omics data across multiple distinct classes. To be specific, we construct a convex penalized log likelihood optimization problem and solve it with an alternating direction method of multipliers (ADMM) algorithm. The proposed method can also be adapted to estimate interaction networks for high dimensional compositional data such as microbial interaction networks. The performance of the proposed method in the simulated studies shows that EDOHA has remarkable advantages in recognizing class-specific hubs than the existing comparable methods. We also present three applications of real datasets. Biological interpretations of our results confirm those of previous studies and offer a more comprehensive understanding of the underlying mechanism in disease.

Author summaryReconstruction of multiple association networks from high dimensional omics data is an important topic, especially in biology. Previous studies focused on estimating different networks and detecting common hubs among all classes. Integration of information over different classes of data while allowing difference in the hub nodes is also biologically plausible. Therefore, we propose a method, EDOHA, to jointly construct multiple interaction networks with capacity in finding different hub networks for each class of data. Simulation studies show the better performance over conventional methods. The method has been demonstrated in three real world data. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. Genome editing in the unicellular holozoan Capsaspora owczarzaki suggests a premetazoan role for the Hippo pathway in multicellular morphogenesis Animal development is mediated by a surprisingly small set of canonical signaling pathways such as Wnt, Hedgehog, TGF-beta, Notch and Hippo pathways. Although once thought to be present only in animals, recent genome sequencing has revealed components of these pathways in the closest unicellular relatives of animals. These findings raise questions about the ancestral functions of these developmental pathways and their potential role in the emergence of animal multicellularity. Here, we provide the first functional characterization of any of these developmental pathways in unicellular organisms by developing techniques for genetic manipulation in Capsaspora owczarzaki, a close unicellular relative of animals that displays aggregative multicellularity. We then use these tools to characterize the Capsaspora ortholog of the Hippo signaling nuclear effector YAP/TAZ/Yorkie (coYki), a key regulator of tissue size in animals. In contrast to what might be expected based on studies in animals, we show that coYki is dispensable for cell proliferation but regulates cytoskeletal dynamics and the three-dimensional (3D) shape of multicellular structures. We further demonstrate that the cytoskeletal abnormalities of individual coYki mutant cells underlie the abnormal 3D shape of coYki mutant aggregates. Taken together, these findings implicate an ancestral role for the Hippo pathway in cytoskeletal dynamics and multicellular morphogenesis predating the origin of animal multicellularity, which was co-opted during evolution to regulate cell proliferation. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI In search of a core cellular network with single cell transcriptome data in the fruit fly Along with specialized functions, cells of multicellular organisms also perform essential functions. Whether diverse cells do this by using the same set of genes, interacting in a fixed coordinated fashion, remains a central question in biology. Single-cell RNA-sequencing (scRNA-seq) measures gene expression of individual cells, enabling researchers to discover gene expression patterns that contribute to the diversity of cell functions. Current efforts focus primarily on identifying differentially expressed genes. However, patterns of co-expression between genes are more indicative of biological processes than are the expression of individual genes. Here, we constructed cell type-specific gene co-expression networks using the fly brain transcriptome atlas to search for a core cellular network. We detected a set of highly coordinated genes preserved across cell types and defined this set as the core network. This network was further validated through two additional datasets profiling fly brains or heads. This core has a small size and shows dense connectivity. Modules within this core are enriched for basic cellular functions and gene members of these modules have distinct evolutionary signatures. Overall, we demonstrated that a core cellular network exists in diverse cell types of fly brains and this core exhibits unique topological, structural, functional and evolutionary properties. A nuclear import pathway exploited by pathogenic noncoding RNAs The prevailing view regarding intracellular RNA trafficking in eukaryotic cells describes that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope entering the cytoplasm for function. Interestingly, emerging evidence illustrates numerous functional RNAs trafficking in the reverse direction from the cytoplasm to the nucleus. However, the mechanism underlying the RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using nuclear-replicating viroids as a model, we showed that cellular Importin alpha-4 is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (Virp1) that binds both Importin alpha-4 and viroids. Furthermore, a conserved C-loop in nuclear-replicating viroids is critical for Virp1 binding. Disrupting C-loop impairs Virp1 binding, viroid nuclear accumulation and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on Virp1 for nuclear import. These results have significant implications for understanding the infection process of subviral agents. In addition, our data outline a cellular pathway responsible for the nuclear import of RNAs and uncover a 3-dimensional RNA motif-based regulation over RNA nuclear import. X chromosomes show relaxed selection and complete somatic dosage compensation across Timema stick insect species Sex chromosomes have evolved repeatedly across the tree of life. As they are present in different copy numbers in males and females, they are expected to experience different selection pressures than the autosomes, with consequences including a faster rate of evolution, increased accumulation of sexually antagonistic alleles, and the evolution of dosage compensation. Whether these consequences are general or linked to idiosyncrasies of specific taxa is not clear as relatively few taxa have been studied thus far. Here we use wholegenome sequencing to identify and characterize the evolution of the X chromosome in five species of Timema stick insects with XX:X0 sex determination. The X chromosome had a similar size (approximately 11% of the genome) and gene content across all five species, suggesting that the X chromosome originated prior to the diversification of the genus. Genes on the X showed evidence of relaxed selection (elevated dN/dS) and a slower evolutionary rate (dN + dS) than genes on the autosomes, likely due to sex-biased mutation rates. Genes on the X also showed almost complete dosage compensation in somatic tissues (heads and legs), but dosage compensation was absent in the reproductive tracts. Contrary to prediction, sex-biased genes showed little enrichment on the X, suggesting that the advantage X-linkage provides to the accumulation of sexually antagonistic alleles is weak. Overall, we found the consequences of X-linkage on gene sequences and expression to be similar across Timema species, showing the characteristics of the X chromosome are surprisingly consistent over 30 million years of evolution. Deficits in seizure threshold and other behaviors in adult mice without gross neuroanatomic injury after late gestation transient prenatal hypoxia Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain. GLOBAL LAND-USE AND LAND-COVER DATA: HISTORICAL, CURRENT AND FUTURE SCENARIOS Land-use land-cover (LULC) data are important predictors of species occurrence and biodiversity threat. Although there are LULC datasets available under current conditions, there is a lack of such data under historical and future climatic conditions. This hinders, for example, projecting niche and distribution models under global change scenarios at different time scenarios. The Land Use Harmonization Project (LUH2) is a global terrestrial dataset at 0.25{degrees} spatial resolution that provides LULC data from 850 to 2300 for 12 LULC state classes. The dataset, however, is compressed in a file format (NetCDF) that is incompatible for many analyses and requires layer extractions and transformations that are intractable for most researchers. Here we selected and transformed the LUH2 in a standard GIS format data to make it more user-friendly. We provide LULC for every year from 850 to 2100, and from 2015 on, the LULC dataset is provided under two Shared Socioeconomic Pathways (SSP2-4.5 and SSP5-8.5). We provide two types of files for each year: separate files with continuous values for each of the 12 LULC state classes, and a single categorical file with all state classes combined. To create the categorical layer, we assigned the state with the highest value in a given pixel among the 12 continuous data. LUH2 predicts a pronounced decrease in primary forest, particularly noticeable in the Amazon, the Brazilian Atlantic Forest, the Congo Basin and the boreal forests, an equally pronounced increase in secondary forest and non-forest lands, and in croplands in the Brazilian Atlantic Forest and sub-Saharan Africa. The final dataset provides LULC data for 1251 years that will be of interest for macroecology, ecological niche modeling, global change analysis, and other applications in ecology and conservation. Deep Learning-Inferred Multiplex ImmunoFluorescence for IHC Image Quantification Reporting biomarkers assessed by routine immunohistochemical (IHC) staining of tissue is broadly used in diagnostic pathology laboratories for patient care. To date, clinical reporting is predominantly qualitative or semi-quantitative. By creating a multitask deep learning framework referred to as DeepLIIF, we present a single-step solution to stain deconvolution/separation, cell segmentation, and quantitative single-cell IHC scoring. Leveraging a unique de novo dataset of co-registered IHC and multiplex immunofluorescence (mpIF) staining of the same slides, we segment and translate low-cost and prevalent IHC slides to more expensive-yet-informative mpIF images, while simultaneously providing the essential ground truth for the superimposed brightfield IHC channels. Moreover, a new nuclear-envelop stain, LAP2beta, with high (>95%) cell coverage is introduced to improve cell delineation/segmentation and protein expression quantification on IHC slides. By simultaneously translating input IHC images to clean/separated mpIF channels and performing cell segmentation/classification, we show that our model trained on clean IHC Ki67 data can generalize to more noisy and artifact-ridden images as well as other nuclear and non-nuclear markers such as CD3, CD8, BCL2, BCL6, MYC, MUM1, CD10, and TP53. We thoroughly evaluate our method on publicly available benchmark datasets as well as against pathologists semi-quantitative scoring. The code, the pre-trained models, along with easy-to-run containerized docker files as well as Google CoLab project are available at https://github.com/nadeemlab/deepliif. Deficiency of gluconeogenic enzyme PCK1 promotes murine NASH progression and fibrosis by activation PI3K/AKT/PDGF axis Background and AimsNonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. However, the pathomechanisms underlying NASH are incompletely explored. Phosphoenolpyruvate carboxykinase 1 (PCK1) catalyzes the first rate-limiting step of gluconeogenesis. This study was designed to determine the role of PCK1 in regulating NASH progression.

MethodsLiver metabolism, hepatic steatosis, and fibrosis were evaluated at 24 weeks in liver-specific Pck1-knockout mice fed with NASH diet or chow diet. Gain- or loss-of-function approaches were used to explore the underlying mechanism in vitro. AKT and RhoA inhibitors were evaluated for NASH treatment in vivo.

ResultsHepatic PCK1 was downregulated in patients with NASH and mouse models of NASH. Mice with liver Pck1 deficiency displayed hepatic lipid disorder and liver injury fed with normal diet, while showed aggravated fibrosis and inflammation when fed NASH diet. Mechanistically, PCK1 deficiency upregulated genes involved in fatty acid transport and lipid droplet formation. Moreover, metabolomics analysis showed the accumulation of glycerol 3-phosphate, the substrate of triglyceride synthesis. Furthermore, the loss of hepatic PCK1 could activate the RhoA/PI3K/AKT pathway, which leads to increased secretion of PDGF-AA and promotes the activation of hepatic stellate cells. Accordingly, treatment with RhoA and AKT inhibitors alleviated NASH progression in the presence of Pck1 deletion in vivo.

ConclusionsPCK1 deficiency plays a key role in the development of hepatic steatosis and fibrosis by facilitating the RhoA/PI3K/AKT/PDGF-AA axis. These findings provide a novel insight into therapeutic approaches for the treatment of NASH. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI Force profile of the two-handed hardstyle kettlebell swing performed by an RKC-certified Instructor BackgroundThe effects of hardstyle kettlebell training are increasingly cited in strength and conditioning research, yet reference data from a proficient swing is scarce. The aim of this exploratory study was to investigate the force profile of a two-handed hardstyle swing performed by a Russian Kettlebell Challenge (RKC) instructor.

MethodsThe subject is a 44-year-old male, body mass 75.6 kg, height 173.5 cm, with six years of regular hardstyle training experience. Two-handed hardstyle swings were performed with a series of incremental mass kettlebells (8-68 kg). Ground reaction force (GRFs) was obtained from a floor-mounted force platform. Force-time curves (FTCs), peak force, forward force, rate of force development (RFD) and swing cadence were investigated.

ResultsData revealed the FTC of a proficient swing is highly consistent and dominated by a single force peak (mean SD = 47 N), with a profile that remained largely unchanged to 24 kg. Pearson correlation analysis revealed a very strong positive correlation in peak force with kettlebell mass (r = 0.95), which increased disproportionately from the lightest to heaviest kettlebells; net peak force increased from 8.36 {+/-} 0.75 N.kg-1 (0.85 x BW) to 12.82 {+/-} 0.39 N.kg-1 (1.3x BW). There was a strong negative correlation between RFD and kettlebell mass (r = 0.82) that decreased from 39.2 N.s-1.kg-1 to 21.5 N.s-1.kg-1. There was a very strong positive correlation in forward ground reaction force with kettlebell mass (r = 0.99), expressed as a ratio of vertical ground reaction, that increased from 0.092 (9.2%) to 0.205 (20.5%). Swing cadence exceeded 40 swings per minute (SPM) with all kettlebells.

ConclusionOur findings challenge some of the popular beliefs of the hardstyle kettlebell swing. Consistent with hardstyle practice, and previous kinematic analysis of expert and novice, force-time curves show a characteristic single large force peak, differentiating passive from active shoulder flexion. Ground reaction force did not increase proportionate to kettlebell mass, with a magnitude of forward force smaller than described in practice. These results could be useful for coaches and trainers wanting to improve athletic performance, and healthcare providers using the kettlebell swing for therapeutic purposes. Findings from this study were used to inform the BELL Trial, a pragmatic controlled trial of kettlebell training with older adults. www.anzctr.org.au ACTRN12619001177145. Immunomodulatory Contribution of Mast Cells to the Regenerative Biomaterial Microenvironment Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials. Top-down generation of low-precision representations improves the perception and imagination of fine-scale visual information Predictive-coding perception or imagination requires top-down generated neural representations in the primary visual cortex (V1) to reconstruct or simulate the V1 representations bottom-up stimulated by the seen images. However, top-down generated representations have low coding precision in both perception and imagination tasks. Why and how does the brain use top-down generated low-precision representations to reconstruct or simulate bottom-up stimulated high-precision representations? How can information of fine spatial scale be perceived or imagined using the top-down generated low-precision representations? By modeling the visual system using variational auto-encoder, we reveal that training to generate low-precision representations facilitates high-order cortex to form representations smooth to shape morphing of stimuli. Fine-scale visual information, if sparse, can be more accurately and robustly perceived or creatively imagined by inferring from generated representations with low (instead of high) coding precision. Therefore, generating low-precision representations is not a shortcoming, but instead a strategy that the brain uses to improve the perception and imagination of fine-scale information. Creation of a single cell RNA seq meta-atlas to define human liver immune homeostasis The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine  human liver scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and [~]32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver. Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[-&gt;]4Gal (Gb3 and the P1 antigen) and Gal1[-&gt;]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. A General Biphasic-Bodyweight Model for Scaling Basal Metabolic Rate, Glomerular Filtration Rate and Drug Clearance from Birth to Adulthood Understanding the maturation process of human physiology and metabolism has broad medical and pharmaceutical implications. Age and bodyweight are frequently considered as separate variables in modeling the dynamical changes of human organ functions and of drug clearance from birth to adulthood. The objective of this study is to propose a unified, continuous and bodyweight-only equation to quantify the changes of human basal metabolic rate (BMR), glomerular filtration rate (GFR) and drug clearance (CL) from infancy to adulthood. The BMR datasets were retrieved from a comprehensive historical database of male and female subjects (0.02 to 64 years). The CL datasets for 17 drugs and the GFR dataset were generated from age-incorporated maturation-and-growth models with reported parameter values. The model used in the simulation is independent of the proposed model. A statistical approach was used to simulate the model generated CL and GFR data for a hypothetical population with 26 age groups (ranging from 0 to 20 years). Besides, individual CL data for one drug, and sparse PBPK-modeled CL values for two drugs were also included for analysis. A 4-parameter, mixed-allometry equation with two power-law functions of bodyweight was proposed and evaluated as a general model using nonlinear regression and dimensionless analysis. All datasets universally reveal biphasic curves with two distinct linear segments on log-log plots. Compared with simple allometry, the biphasic model fits satisfactorily to all datasets (based on Akaikes Information Criterion and residual plots). The biphasic equation consists of two reciprocal allometric terms that asymptotically determine the overall curvature. The fitting results show a superlinear scaling phase (slope >1; ca. 1.5 - 3.5) below the characteristic bodyweight at the phase transition; and above which, a sublinear scaling phase (slope <1; ca. 0.5 - 0.7) is evident. The phase-transition bodyweight is ranging from 5 to 20 kg (corresponding to 0.5 - 9 years) and the mean value is around 10 kg ([~]2 years) for all data sets. The dimensionless analysis generalizes, and offers quantitative realization of, the maturation and growth process. In conclusion, the proposed mixed-allometry equation is a generic model that quantitatively describes the phase transition occurring in the human maturation process of BMR, GFR and drug CL.