Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics.
Development of an in-situ printing system with human platelet lysate-based bio-adhesive to treat corneal perforation PurposeCorneal perforation is a clinical emergency. Tissue glue to seal the perforation, and supplementary topical medication represents existing standard treatment. Previously, our group developed a transparent human platelet lysate (hPL)-based biomaterial that showed good cell compatibility and accelerated corneal epithelial cells healing in-vitro. This study aims to develop a novel treatment method for corneal perforation using this biomaterial.

MethodsRheometry was used to measure the hPL-based biomaterial behaviour at room and corneal surface temperatures. Its adhesiveness to porcine skin and burst pressure limit were also measured. Based on rheological behaviour, a hand-held biopen was developed to extrude it onto the cornea. An animal trial (5 New Zealand white rabbits) to compare impact of the biomaterial and cyanoacrylate glue (control group) on a 2mm perforation was conducted to evaluate safety and efficacy.

ResultsThe hPL-based biomaterial showed higher adhesiveness compared to commercial fibrin glue and withstood burst pressure approximately 6.4x higher than routine intraocular pressure. Treatment rabbits had lower pain scores and faster recovery, despite generating similar scar-forming structure compared to controls. No secondary corneal ulcer was generated in rabbits treated with the bio-adhesive.

ConclusionsThis study reports a novel in-situ printing system capable of delivering a hPL-based, transparent bio-adhesive and successfully treating small corneal perforations. Bio-adhesive-treated rabbits recovered faster and required no additional analgesia. Both groups showed scarred corneal tissue after healing, however no infection and inflammation was observed by 3 weeks. The delivery system was easy to use and may represent an alternative treatment for corneal perforation.

HighlightsO_LIThis study presents a novel in situ printing system to treat corneal perforation
C_LIO_LIThe system is comprised of a human platelet lysate-based bio-adhesive and a pen-like hand held delivery system
C_LIO_LIMechanical tests showed our transparent bio-adhesive has a higher adhesiveness compared to existing treatments and burst pressure threshold approximately 6.4 times higher than normal intraocular pressure.
C_LIO_LIIn vivo rabbit trial showed that compared to cyanoacrylate glue, the bio-adhesive was safer, faster healing and led to less pain in rabbits.
C_LI
Predicting the effects of multiple global change drivers onmicrobial communities remains challenging Microbial communities in many ecosystems are facing a broad range of global change drivers, such as nutrient enrichment, chemical pollution, and temperature change. These drivers can cause changes in the abundance of taxa, the composition of communities, and the properties of ecosystems. While the influence of single drivers is already described in numerous studies, the effect and predictability of multiple drivers changing simultaneously is still poorly understood. In this study, we used 240 highly replicable oxic/anoxic aquatic lab micro-ecosystems and four drivers (fertilizer, glyphosate, metal pollution, antibiotics) in all possible combinations at three different temperatures (20 {degrees}C, 24 {degrees}C, and 28 {degrees}C) to shed light into consequences of multiple drivers on different levels of organization, ranging from species abundance to community and ecosystem parameters. We found (i) that at all levels of ecological organisation, combinations of drivers can change the biological consequence and direction of effect compared to single drivers (ii), that effects of combinations are further modified by temperature, (iii) that a larger number of drivers occurring simultaneously is often quite closely related to their effect size, and (iv) that there is little evidence that any of these effects are associated with the level of ecological organisation of the state variable. These findings suggest that, at least in this experimental ecosystem approximating a stratified aquatic ecosystem, there may be relatively little scope for predicting the effects of combinations of drivers from the effects of individual drivers, or by accounting for the level of ecological organisation in question, though there may be some scope for prediction based on the number of drivers that are occurring simultaneous. A priority, though also a considerable challenge, is to extend such research to consider continuous variation in the magnitude of multiple drivers acting together. Transcription disrupts DNA-scaffolded bacteriophage repressor complexes DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage {lambda} CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or {lambda} CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription. Genomic regions associate with major axes of variation driven by gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.) Stomata and leaf veins play an essential role in transpiration and the movement of water throughout leaves. These traits are thus thought to play a key role in the adaptation of plants to drought and a better understanding of the genetic basis of their variation and coordination could inform efforts to improve drought tolerance. Here, we explore patterns of variation and covariation in leaf anatomical traits and analyze their genetic architecture via genome-wide association (GWA) analyses in cultivated sunflower (Helianthus annuus L.). Traits related to stomatal density and morphology as well as lower order veins were manually measured from digital images while the density of minor veins was estimated using a novel deep learning approach. Leaf, stomatal, and vein traits exhibited numerous significant correlations that generally followed expectations based on functional relationships. Correlated suites of traits could further be separated along three major principal component (PC) axes that were heavily influenced by variation in traits related to gas exchange, leaf hydraulics, and leaf construction. While there was limited evidence of colocalization when individual traits were subjected to GWA analyses, major multivariate PC axes that were most strongly influenced by several traits related to gas exchange or leaf construction did exhibit significant genomic associations. These results provide insight into the genetic basis of leaf trait covariation and showcase potential targets for future efforts aimed at modifying leaf anatomical traits in sunflower.

Significance StatementUsing traditional and automated/high-throughput (using a novel deep learning approach) phenotyping methods we studied leaf anatomical variation in sunflower. Genome-wide association (GWA) analyses identified numerous genomic regions underlying individual trait variation and regions underlying major multivariate axes of phenotypic variation. These results illustrate the value of employing a multivariate approach to GWA analyses and shed light on the extent to which leaf trait (co-)variation can be genetically decoupled to explore novel phenotypic space. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Sequence-dependent model of genes with dual σ factor preference Escherichia coli uses {sigma} factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single {sigma} factor, approximately 5% of them have dual {sigma} factor preference. The most common are those responsive to both {sigma}70, which controls housekeeping genes, and {sigma}38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that  {sigma}70+38 genes are nearly as upregulated in stationary growth as  {sigma}38 genes. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in {sigma}38 levels. We then propose and validate a sequence dependent model of {sigma}70+38 genes, with dual sensitivity to {sigma}38 and {sigma}70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and {sigma} factor combinations. Given this, promoters controlling {sigma}70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases. What can cold-induced transcriptomes of Arctic Brassicaceae tell us about the evolution of cold tolerance? O_LILittle is known about the evolution of cold tolerance in polar plant species and how they differ from their temperate relatives. To gain insight into their biology and the evolution of cold tolerance, we compared the molecular basis of cold response in three Arctic Brassicaceae species.
C_LIO_LIWe conducted a comparative time series experiment to examine transcriptional responses to low temperature. RNA was sampled at 22 {degrees}C, and after 3h, 6h, and 24h at 2 {degrees}C. We then identified sets of genes that were differentially expressed in response to cold and compared them between species, as well as to published data from the temperate Arabidopsis thaliana.
C_LIO_LIMost differentially expressed genes were species-specific, but a significant portion of the cold response was also shared among species. Among thousands of differentially expressed genes, [~]200 were shared among the three Arctic species and A. thaliana, while [~]100 were exclusively shared among the three Arctic species.
C_LIO_LIOur results show that cold response differs markedly between Arctic Brassicaceae species, but likely builds on a conserved basis found across the family. They also confirm that highly polygenic traits such as cold tolerance may show little repeatability in their patterns of adaptation.
C_LI Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML - a model pre-mRNA substrate - guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome. The kidney protects against sepsis by producing systemic uromodulin Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. While electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We previously demonstrated that systemic Tamm-Horsfall Protein (THP, uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we show that the kidney increases circulating THP by enhancing basolateral release of THP from medullary thick ascending limb cells. In sepsis patients, changes in circulating THP are positively associated with critical illness. THP is also found de novo in injured lungs. Genetic ablation of THP in mice leads to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo leads macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis and chemotaxis and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improves survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function and systemic THP has therapeutic potential in sepsis.

Significance StatementSepsis is a significant contributor to kidney injury as well as morbidity and mortality worldwide. Specific therapies to improve outcomes in sepsis with kidney injury have largely been limited to symptom management and infectious agent control, in part because it is unclear how kidney injury increases sepsis mortality. This paper describes the identification of Tamm-Horsfall protein, previously known to protect in ischemic models of AKI, as protective in preclinical models of sepsis. It demonstrates how the loss of THP leads to decreased mononuclear phagocyte function and diversity, increased pathogen burden and decreased survival. THP also increases in sepsis without severe kidney injury and concentrates in injured organs. Further study of THP in sepsis could lead to novel sepsis therapeutics. Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy. Forecasting the effects of water regulation on the population viability of a threatened amphibian The regulation of river systems alters hydrodynamics and often reduces lateral connectivity between river channels and floodplains. For taxa such as frogs that rely on floodplain wetlands to complete their lifecycle, decreasing inundation frequency can reduce recruitment and increase the probability of local extinction. We virtually reconstructed the inundation patterns of wetlands under natural and regulated flow conditions and built stochastic population models to quantify the probability of local extinction under different inundation scenarios. Specifically, we explored the interplay of habitat size, inundation frequency, and successive dry years on the local extinction probability of the threatened southern bell frog Litoria raniformis in the Murray River floodplains of South Australia. We hypothesised that the changes to wetland inundation resulting from river regulation are a principal driver of L. raniformis declines in this semi-arid system.

Regulation has reduced the inundation frequency of essential habitats below critical thresholds for the persistence of many fresh water-dependent species. Successive dry years raise the probability of local extinction, and these effects are strongest in smaller wetlands. Larger wetlands and those with more frequent average inundation are less susceptible to these effects.

Elucidating these trends informs the prioritisation of treatment sites and the frequency of conservation interventions. Environmental water provision (through pumping or the operation of flow-regulating structures) is a promising tool to reduce the probability of breeding failure and local extinction. Our modelling approach can be used to prioritise the delivery of environmental water to L. raniformis and potentially many other frog species. 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. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. Sgs1BLM independent role of Dna2DNA2 nuclease at DNA double strand break is inhibited by Nej1XLF The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. The regulation of 5 DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and more recently, it was shown to be important for DNA end-bridging and inhibiting 5 resection mediated by Dna2-Sgs1. Nej1 interacts with Sae2 and this impacts DSB repair in three ways. First, Nej1 inhibits MRX-Sae2 interactions and Sae2 localization to a DSB. Second, Nej1 inhibits Sae2-dependent recruitment of Dna2 in the absence of Sgs1. Third, NEJ1 and SAE2 showed an epistatic relationship for DNA end-bridging, an event that restrains the broken ends and reduces the frequency of genomic deletions from developing at the DSB. Deletion of NEJ1 suppressed the synthetic lethality of sae2{Delta} sgs1{Delta} and was dependent on the nuclease activity of Dna2. These Nej1 functions promote end-joining DSB repair, but could also be relevant for controlling resection initiation during HR repair.

HighlightsO_LINej1 physically interacts with Sae2 and inhibits end-resection at a DSB.
C_LIO_LINej1 inhibits Sae2 interactions with the MRX complex.
C_LIO_LINej1 inhibits Sae2-dependent recruitment of Dna2 to a DSB.
C_LIO_LINEJ1 and SAE2 are epistatic for DNA end-bridging.
C_LIO_LIDeletion of NEJ1 suppresses the synthetic lethality of sae2{Delta} sgs1{Delta}, which is dependent on Dna2 nuclease activity.
C_LI Assessment and Optimization of the Interpretability of Machine Learning Models Applied to Transcriptomic Data Explainable artificial intelligence aims to interpret how the machine learning models make decisions, and many model explainers have been developed in the computer vision field. However, the understandings of the applicability of these model explainers to biological data are still lacking. In this study, we comprehensively evaluated multiple explainers by interpreting pretrained models of predicting tissue types from transcriptomic data, and by identifying top contributing genes from each sample with the greatest impacts on model prediction. To improve the reproducibility and interpretability of results generated by model explainers, we proposed a series of optimization strategies for each explainer on two different model architectures of Multilayer Perceptron (MLP) and Convolutional Neural Network (CNN). We observed three groups of explainer and model architecture combinations with high reproducibility. Group II, which contains three model explainers on aggregated MLP models, identified top contributing genes in different tissues that exhibited tissue-specific manifestation and were potential cancer biomarkers. In summary, our work provides novel insights and guidance for exploring biological mechanisms using explainable machine learning models. Spontaneous differentiation leads to emergence of hybrid cell states relate to poor prognosis in oral cancer PurposeCellular dynamics between phenotypically heterogeneous subpopulations of cancer cells within individual tumor is shown to be responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Therefore, this study was undertaken to describe the dynamic phenotypic states among oral cancer cells, its influence on transcriptomic heterogeneity as well as its clinical significance.

Experimental DesignWe multiplexed phenotypic markers of putative oral-stem-like cancer cells (SLCCs) and characterized diversity among CD44-positive oral cancer cell subpopulations with respect to distinct expression of CD24 and aldehyde dehydrogenase (ALDH)-activity in multiple cell lines. Population trajectories were characterized by Markov model and cell states were defined based on the population specific RNA sequencing (RNAseq). ssGSEA based gene expression signatures were explore for prognostic significance.

ResultsOral cancer cells followed two distinct patterns of spontaneous repopulation dynamics with stochastic inter-conversions on  ALDH-axis, however a strict non-interconvertible transition on  CD24-axis. Interestingly, plastic  ALDH-axis was harnessed to enrich ALDHHigh subpopulations in response to Cisplatin treatment, to adapt a drug tolerant state. Phenotype-specific RNAseq results suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate states of stemness within the differentiating oral cancer cells. Further, survival analysis with each subpopulation-specific gene signature strongly suggested that the cell-state dynamics may act as possible mechanism to drive ITH, resulting in poor prognosis in patient.

ConclusionsOur results emphasized the prognostic power of the population dynamics in oral cancer. Importantly, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behaviour in oral cancer; which is a prerequisite knowledge important for precision treatment, however largely lacking for most solid tumors.

Graphical AbstractWe have characterized diversity among CD44-positive oral cancer cells lines with respect to distinct expression of CD24 and ALDH-activity. Subpopulations showed stochastic inter-conversions on ALDH-axis but a strict non-interconvertible transition of CD24Low to CD24High phenotype, even in response to chemotherapy-induced stress. RNAseq study suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate alternate states of stemness within the differentiating oral cancer cells. The described population dynamics demonstrtaed influence tumor behaviour possibly by increasing intratumoral heterogeneity in aggressive oral tumors.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/457509v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1dc6a7org.highwire.dtl.DTLVardef@dd0948org.highwire.dtl.DTLVardef@18c9603org.highwire.dtl.DTLVardef@cca8fb_HPS_FORMAT_FIGEXP  M_FIG C_FIG Translational relevanceIntratumoral heterogeneity (ITH) has been the clinically important factor, impacting aggressive cancer behaviour, drug tolerance and overall poor prognosis. Recent high-throughput studies have provided better cellular and molecular resolution of ITH; however, the prerequisite knowledge which defines the composition of subpopulations critical for global tumor behaviour is majorly lacking for most of the solid tumors. By combining phenotypic markers, we have defined four subpopulations of oral cancer cells. These subpopulations showed stochastic inter-conversions as well as a strict non-interconvertible transition among them to acheive heterogeneity. Importantly, transcriptional states of each subpopulations indicated a clinically relevant signatures for patient prognosis. Also, we observed interconversions of these subpopulations in response to Cisplatin to accumulate drug-tolerant cell state, as rapid and reversible strategy to respond to chemotherapy induced stress. Thus, the characteristics of described phenotypic subgroups may be translated to the clinic for estimating the extent of intratumoral heterogeneity in oral cancer patients. The cell growth cycle: resource allocation and subcellular pattern formation in budding yeast. Metabolic oscillation percolate throughout cellular physiology. The transcriptome oscillates between gene cohorts encoding for biosynthesis and growth, and for catabolism and stress response. However, long protein half-lives dampen effects on protein abundance, and the function of periodic transcription remains elusive. To test three prevailing hypotheses, we performed RNA-seq analysis during a dynamic state of the system, just preceding a bifurcation of system dynamics to a longer period. A few enzymes with short protein half-live and high transcript abundance amplitude could play a direct role in metabolic dynamics (hypothesis H1), e.g., carbonic anhydrase, sulfate uptake and the glyoxylate cycle. Substantial changes in the relative duration of expression of the antagonistic cohorts precede the bifurcation, supporting the idea of a function in cellular resource allocation (H2). The pulse-width modulation model, a mathematical formulation of this idea, explains the dependence of the cycle period on the culture growth rate. This pulse-like model of cell growth provides a first theoretical framework, where the phenomenon is understood as a mechanism of cellular resource allocation and protein homeostasis. The universal temporal program of transcription encodes the spatial structure of the cell, and we further suggest a function in pattern formation of the growing cell (H3). Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Embracing curiosity eliminates the exploration-exploitation dilemma Balancing exploration with exploitation is seen as a mathematically intractable dilemma that all animals face. In this paper, we provide an alternative view of this classic problem that does not depend on exploring to optimize for reward. We argue that the goal of exploration should be pure curiosity, or learning for learnings sake. Through theory and simulations we prove that explore-exploit problems based on this can be solved by a simple rule that yields optimal solutions: when information is more valuable than rewards, be curious, otherwise seek rewards. We show that this rule performs well and robustly under naturalistic constraints. We suggest three criteria can be used to distinguish our approach from other theories. Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ-9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. 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. Estrogen-related receptor alpha and Rplp1-dependent translation coordinately regulate starvation response and decrease NASH progression Although general translation declines during fasting, maintaining the translation of a subset or proteins is necessary for metabolic homeostasis and cell viability. Using unbiased proteome analysis of hepatic cells during starvation, we identified a novel pathway in which Esrra-mediated transcription of Rplp1-dependent translation of lysosomal proteins declined during early starvation and recovered after prolonged starvation to restore autophagy-lysosome function. Interestingly, hepatic Esrra-Rplp1-dependent translation rate of lysosomal proteins also was impaired in patients and mice with non-alcoholic steatohepatitis (NASH), and translational response to starvation was dysregulated in mice with NASH. Remarkably, activation of Esrra pharmacologically, genetically, or by alternate day fasting restored protein translation, increased expression of lysosomal proteins, induced autophagy, and reduced lipotoxicity, inflammation, and fibrosis in cell culture and in vivo models of NASH. Thus, hepatic Esrra is essential for ribosome-dependent translation of lysosomal proteins during starvation, and prevention of lipotoxicity and progression in NASH.

Lay summaryFasting for weight loss improves non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH); however, the mechanism is not well understood. Our study shows that a nuclear protein, estrogen related receptor alpha (Esrra), increases ribosome-mediated translation of autophagy and lysosome proteins during chronic starvation to maintain essential metabolic pathways for cell survival. Surprisingly, this translational pathway is impaired during NASH with reduced lysosome-autophagy activity accompanied by increased inflammation and fibrosis gene expression in the liver. Pharmacologic, genetic and dietary activation of Esrra decreases lipid-mediated toxicity in liver cells as well as inflammation and fibrosis in livers from mice with NASH. These findings suggest that the Esrra-ribosome-lysosome pathway is important for liver response to fasting and NASH and thus may be a good therapeutic target for the treatment of NASH. A behavioral paradigm for cortical control of a robotic arm by freely moving rats in a one-dimensional two-target reaching task Controlling the trajectory of a neuroprosthetic device to reach multiple targets is a commonly used brain-machine interface (BMI) task in primates and has not been available for rodents yet. Here, we describe a novel experimental paradigm which enables this task for rats in one-dimensional space for reaching two distant targets depending on their limited cognitive and visual capabilities compared to primates. An online transform was used to convert the activity of a pair of primary motor cortex (M1) units into two robotic actions. The rats were shaped to adapt to the transform and direct the robotic actuator toward the selected target by modulating the activity of the M1 neurons. All three rats involved in the study were capable of achieving randomly selected targets with at least 78% accuracy. A total of 9 out of 16 pairs of units examined were eligible for exceeding this success criterion. Two out of three rats were capable of reversal learning, where the mapping between the activity of the unit pairs and the robotic actions were reversed. The present work is the first demonstration of trajectory-based control of a neuroprosthetic device by rodents to reach two distant targets using visual feedback. The paradigm introduced here may be used as a cost-effective platform for elucidating the information processing principles in the neural circuits related to neuroprosthetic control and for studying the performance of novel BMI technologies using freely moving rats. Revisiting regulatory decoherence: Accounting for temporal bias in a co-expression analysis reveals novel candidates controlling environmental response Environment can alter the degree of phenotypic variation and covariation, potentially influencing evolutionary trajectories. However, environment-driven changes in phenotypic variation remain understudied. In an effort to exploit the abundance of RNASequencing data now available, an increasing number of ecological studies rely on population-level correlation to characterize the plastic response of the entire transcriptome and to identify environmentally responsive molecular pathways. These studies are fundamentally interested in identifying groups of genes that respond in concert to environmental shifts. We show that population-level differential co-expression exhibits biases when capturing changes of regulatory activity and strength in rice plants responding to elevated temperature. One possible cause of this bias is regulatory saturation, the observation that detectable co-variance between a regulator and its target may be low as their transcript abundances are induced. This phenomenon appears to be particularly acute for rapid-onset environmental stressors. However, our results suggest that temporal correlations may be a reliable means to detect transient regulatory activity following rapid onset environmental perturbations such as temperature stress. Such temporal bias is likely to confound the studies of phenotypic integration, where high-order organismal traits are hypothesized to be more integrated with strong correlation under stressful conditions, while recent transcriptome studies exhibited weaker coexpression between genes under stressful conditions. Collectively, our results point to the need to account for the nuances of molecular interactions and the possibly confounding effects that these can introduce into conventional approaches to study transcriptome datasets.