The Digital 3D-Atlas MAKER (DAMAKER): a dynamic and expandable digital 3D-tool for monitoring the temporal changes in tissue growth during hindbrain morphogenesis Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a digital 3D-atlas maker (DAMAKER) and built a digital 3D-temporal atlas to monitor the changes in the growth of the tissue and neuronal differentiation domain in the zebrafish hindbrain. DAMAKER integrates spatial and temporal data from cell populations, neuronal differentiation and brain morphogenesis, through in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a 3D-reference from several imaged hindbrains and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation growth patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain vs. the progenitor domain, and by in vivo neuronal birthdating experiments we generated a digital 3D-temporal map of the neuronal growth in the whole hindbrain, revealing the spatiotemporal dynamics of neuronal differentiation upon morphogenesis. Last, we applied it to glutamatergic and GABAergic neurons, as proof-of-concept that the digital 3D-temporal map could be used as a proxy to infer neuronal birthdate. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction.
The role of Kabuki Syndrome genes KMT2D and KDM6A in development: Analysis in Human sequencing data and compared to mice and zebrafish KMT2D and KDM6A are epigenetic regulators that have been implicated in Kabuki Syndrome, a rare congenital birth defect with multiple tissue and organ abnormalities, including craniofacial and heart defects. Our previous study identified human families with mutations in the epigenetic modifiers KMT2D and KDM6A, which is implicated in 32% and 10% of Kabuki Syndrome patients respectively. To understand the connection to Kabuki syndrome patients, and the transcriptional targets of KMT2D and KDM6A in humans, we performed RNA sequencing (seq) of lymphoblastoid cells from Kabuki Syndrome patients carrying mutations in KMT2D and KDM6A. We identified 1995 significant changes in transcriptional targets for KMT2D and 1917 for KDM6A, as compared to control. When compared with RNA-seq datasets obtained from other mouse and zebrafish studies, our analysis revealed KMT2D mutations affect the expression of 76 orthologous genes across all three datasets. Similarliy, KDM6A afftects the expprssion of 7 orthologous genes across three datasets. Despite the differences in cell types, stages, and species in the comparison between the transcriptomic datasets, there are common gene expression changes associated with KMT2D and KDM6A mutations. qPCR on novel zebrafish mutants confirmed the differentially expression in KMT2D or KDM6A mutant backgrounds. Taken together, our results show that KMT2D and KDM6A regulate common and unique genes across humans, mice, and zebrafish for early craniofacial and cardiac development and this information contributes to the understanding of epigenetic dysregulation during development of Kabuki syndrome.
Plant inositol-phosphate-glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection In mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms. Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungus AO_SCPLOWBSTRACTC_SCPLOWChromosomal rearrangements have been largely described among eukaryotes, and may have important consequences on evolution of species. High genome plasticity has been often reported in Fungi, which may explain their apparent ability to quickly adapt to new environments. Cryphonectria parasitica, causing the Chestnut blight disease, is an invasive fungal pathogen species associated with several recent host shifts during its successive introductions from Asia to North America and Europe. Previous cytological karyotyping and genomic studies suggested several chromosomal rearrangements which remains to be described in detail for this species. A serious limitation for valid genome comparisons is the access to robust genome assemblies that usually contain genomic regions of low complexity. We present a new de novo whole-genome assembly obtained from a new method of DNA extraction and long-reads sequencing Nanopore technology obtained from a Japanese isolate sampled in the native area of the species. The comparison with a recently published reference genome revealed stable gene and transposable elements (TEs) repertoires. We also showed that the C. parasitica genome is lowly compartmentalized, with a poor association between TEs and genes, such as those potentially involved in host interactions (i.e., genes coding for small secreted proteins or for secondary metabolites). This genome comparison, however, detected several large chromosomal rearrangements that may have important consequences in gene regulations and sexual mating in this invasive species. This study opens the way for more comparisons of high-quality assembled genomes, and questions the role of structural variations in the invasive success of this fungal pathogen species. Higher phage virulence accelerates the evolution of host resistance Parasites and pathogens vary strikingly in their virulence and the resulting selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and as of now mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if resistance costs are outweighed by the benefits of avoiding infection. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of the filamentous phage, VALG{Phi}8, that differ in their virulence. The bacterial host exhibited two alternative defence strategies against future phage infection: (1) super infection exclusion (SIE) whereby phage-infected cells were immune to subsequent infection at a cost of reduced growth, and (2) surface receptor mutations (SRM) in genes encoding the MSHA type-IV pilus providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high virulence compared to the low virulence phage. Using a mathematical model of our system we show that increasing virulence strengthens selection for SRM due to the higher costs of infection suffered by SIE immune hosts. In both the experiments and the model, higher levels of SRM in the host population drove more rapid phage extinction. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics. Task-independent metrics of computational hardness predict human cognitive performance The survival of human organisms depends on our ability to solve complex tasks in the face of limited cognitive resources. However, little is known about the factors that drive the complexity of those tasks. Here, building on insights from computational complexity theory, we quantify the computational hardness of cognitive tasks using a set of task-independent metrics related to the computational resource requirements of individual instances of a task. We then examine the relation between those metrics and human behavior and find that they predict both time spent on a task as well as accuracy in three canonical cognitive tasks. Our findings demonstrate that performance in cognitive tasks can be predicted based on generic metrics of their inherent computational hardness.

TeaserThe ability of humans to solve cognitive tasks is affected by generic mathematical properties of problems related to their computational complexity. Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes - a commonly used approach - is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.

HighlightsGenExpA - next-generation software for normalizer selection and validation

The GenExpA tool defines how low the stability value of the reference should be

The GenExpA tool determines the level of gene expression analysis robustness

The GenExpA tool increases the speed of analysis and reduces cost Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

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

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Luminance texture boundaries and luminance step boundaries are segmented using different mechanisms In natural scenes, two adjacent surfaces may differ in mean luminance without any sharp change in luminance at their boundary, but rather due to different relative proportions of light and dark regions within each surface. We refer to such boundaries as luminance texture boundaries (LTBs), and in this study we investigate whether LTBs are segmented using different mechanisms than luminance step boundaries (LSBs). We develop a novel method to generate luminance texture boundaries from natural uniform textures, and using these natural LTB stimuli in a boundary segmentation task, we find that observers are much more sensitive to identical luminance differences which are defined by textures (LTBs) than by uniform luminance steps (LSBs), consistent with the possibility of different mechanisms. In a second and third set of experiments, we characterize observer performance segmenting natural LTBs in the presence of masking LSBs which observers are instructed to ignore. We show that although there may be some masking of LTB segmentation by LSBs, it is far less than that observed in a control experiment where both the masker and target are LSBs, and far less than that predicted by a model assuming identical mechanisms. Finally, we perform a fourth set of boundary segmentation experiments using artificial LTB stimuli comprised of differing proportions of white and black dots on opposite sides of the boundary. We find that these stimuli are also highly robust to masking by supra-threshold LSBs, consistent with our results using natural stimuli, and with our earlier studies using similar stimuli. Taken as a whole, these results suggest that the visual system contains mechanisms well suited to detecting surface boundaries that are robust to interference from luminance differences arising from luminance steps like those formed by cast shadows. Compression for population genetic data through finite-state entropy We improve the efficiency of population genetic file formats and GWAS computation by leveraging the distribution of sample ordering in population-level genetic data. We identify conditional exchangeability of these data, recommending finite state entropy algorithms as an arithmetic code naturally suited to population genetic data. We show between 10% and 40% speed and size improvements over dictionary compression methods for population genetic data such as Zstd and Zlib in computation and and decompression tasks. We provide a prototype for genome-wide association study with finite state entropy compression demonstrating significant space saving and speed comparable to the state-of-the-art. Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands. Dietary palmitic acid induces innate immune memory via ceramide production that enhances severity of acute septic shock and clearance of infection Trained immunity is an innate immune memory response that is induced by primary microbial or sterile stimuli that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Nutritional components, such as dietary fatty acids, can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli in the context of innate immune memory. Here we find mice fed diets enriched in saturated fatty acids (SFAs) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and glycemic modulation. Ex vivo, we show monocytes and splenocytes from mice fed enriched SFAs do not have altered baseline inflammation, but enhanced responses to a secondary inflammatory challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in SFA-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA alone reprograms macrophages to induce a hyper-inflammatory response to secondary challenge with LPS. This response was found to be dependent on the synthesis of ceramide, and reversible when treated with oleic acid, a mono-unsaturated FA that depletes intracellular ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality during an acute septic response to systemic LPS, which was not reversible for up to 7 days post-PA-exposure. While PA-treatment is harmful for acute septic shock outcome, we find PA exposure enhanced clearance of Candida albicans in RAG-/- mice. These are the first data to implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory that is detrimental during an acute septic response, but beneficial for clearance of pathogens. Microtubule inhibitors identified through non-biased screening enhance DNA transfection efficiency by delaying p62-dependent ubiquitin recruitment Ectopic gene expression is an indispensable tool in biology and medicine, but is often limited by the low efficiency of DNA transfection. We previously reported that depletion of the autophagy receptor p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target for drugs to increase transfection efficiency. To identify such drugs, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the p62-dependent mechanisms, the time required for p62-dependent ubiquitination, which is required for autophagosome formation, was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. Microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405 was markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings demonstrate for the first time that microtubule inhibitors suppress p62 activation as a mechanism for increasing DNA transfection efficiency and provide solutions to increase efficiency. A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology. Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. Nascent alt-protein chemoproteomics reveals a repressor of ribosome biogenesis Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins. 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. Spatial organization of nuclear pores in Xenopus laevis oocytes Nuclear pores are protein assemblies inserted in the nuclear envelope of eukaryotic cells, acting as main gates for communication between nucleus and cytoplasm. So far, nuclear pores have been extensively studied to determine their structure and composition, yet their spatial organization and geometric arrangement on the nuclear surface are still poorly understood. Here, we analyze super-resolution images of the surface of Xenopus laevis oocyte nuclei during development, and characterize the arrangement of nuclear pores using tools commonly employed to study the atomic structural and topological features of soft matter. To interpret the experimental results, we hypothesize an effective interaction among nuclear pores and implemented it in extensive numerical simulations of octagonal clusters mimicking typical pore shapes. Thanks to our simple model, we find simulated spatial distributions of nuclear pores that are in excellent agreement with experiments, suggesting that an effective interaction among nuclear pores exists and could explain their geometrical arrangement. Furthermore, our results show that the statistical features of the geometric arrangement of nuclear pores do not depend on the type of pore-pore interaction, attractive or repulsive, but are mainly determined by the octagonal symmetry of each single pore. These results pave the way to further studies needed to determine the biological nature of pore-pore interactions. Risk modelling of single cell populations revealed the heterogeneity of immune infiltration in hepatocellular carcinoma Hepatocellular carcinoma (HCC) is one of the most common primary hepatic malignancies. E2F transcription factors play an important role in the tumorigenesis and progression of HCC, mainly through the RB/E2F pathway. Prognostic models for HCC based on gene signatures have been developed rapidly in recent years; however, their discriminating ability at the single cell level remains elusive, which could reflect the underlying mechanisms driving the sample bifurcation. In this study, we constructed and validated a predictive model based on E2F expression, successfully stratifying HCC patients into two groups with different survival risks. Then we used a single-cell dataset to test the discriminating ability of the predictive model on infiltrating T cells, demonstrating remarkable cellular heterogeneity as well as altered cell fates. We identified distinct cell subpopulations with diverse molecular characteristics. We also found that the distribution of cell subpopulations varied considerably across onset stages among patients, providing fundamental basis for patient-oriented precision evaluation. Moreover, single-sample gene set enrichment analysis revealed that subsets of CD8+ T cells with significantly different cell adhesion level could be associated with different patterns of tumor cell dissemination. Therefore, our findings linked the conventional prognostic gene signature to the immune microenvironment and cellular heterogeneity at the single-cell level, thereby providing deeper insights into the understanding of HCC tumorigenesis. Electrical Stimulated GLUT4 Signaling Attenuates Critical Illness-Associated Muscle Wasting BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.

MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.

ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.

ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM. Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons Symbiotic bacteria interact with their host through the release of symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically-defined holidic diets, we found that association with Lp improves the growth of larvae fed on amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its hosts growth through a molecular dialog that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the GCN2 kinase in Drosophilas enterocytes. Our data indicate that products of Lp r/tRNAs loci activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Our findings unravel a novel beneficial molecular dialog between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons. Pan-cancer HLA Gene-mediated Tumor Immunogenicity and Immune Evasion Human Leukocyte Antigen (HLA) expression contributes to the activation of anti-tumor immunity through interactions with T cell receptors. However, pan-cancer HLA-mediated immunogenicity and immunoediting mechanisms has not been systematically studied. In a retrospective analysis of 33 tumor types from the Cancer Genome Atlas, we uncovered HLA class I and class II differential expression, which outperformed traditional clinical metrics in predicting patient survival. We also characterized the distribution of HLA supertypes across cancers and showed that patients with high HLA allelic diversity and gene expression had better prognosis. Immune microenvironments with varied survival outcomes could be predicted using a neural network model trained on HLA expression data. Furthermore, we identified a subset of tumors which upregulated HLA class I but not class II genes and exploited HLA-mediated escape strategies. Our results suggest the potential of using HLA expression data to predict patient prognosis. Taken together, we emphasize the crucial role of HLA upregulation in shaping prolonged anti-tumor immunity.

SynopsisIn a retrospective analysis of 11080 patients of 33 TCGA cancer types, we showed HLA class I and class II differential expression shape various immune microenvironments and tumor immunoediting mechanisms, predicting tumor immunogenicity and patient survival. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. A novel 5 prime DNA binding site in RFC facilitates PCNA loading for gap DNA repair RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis. 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. How much does it take to be old? Modelling the time since the last harvesting to infer the distribution of overmature forests in France AimThe distribution of overmature forests in metropolitan France is poorly known, with only a few well-studied prominent sites, and has never been evaluated countrywide. Here, we modelled French forest reserves time since the last harvesting operation - a proxy for forest maturity - then inferred the current statistical distribution of overmature forests (i.e. forests over 50 years without harvesting) in France.

LocationMetropolitan France

MethodsWe used inventories from forest reserves and managed forests to calibrate a generalised linear mixed model explaining the time since the last harvesting with selected structural attributes and environmental variables. We then projected this model on the independent National Forest Inventory dataset. We thus obtained an updated estimation of the proportion and a rough distribution of overmature forest stands in metropolitan France.

ResultsWe found that high basal area of very large trees, high volumes of standing and downed deadwood, high diversity of tree-related microhabitats and more marginally diversity of decay stages best characterized the time since the last harvesting. Volumes of stumps and high density of coppices translating legacy of past forest management also distinguished more overmature plots. Our projection yielded an estimated 3% of French forests over 50 years without harvesting mostly located in more inaccessible areas (i.e. mountainous areas) and a promising proportion of future overmature forests if left unharvested.

Main conclusionsOur study showed that the time since the last harvesting is a good proxy for a combination of stand structure attributes key in characterising overmature temperate forests. It gives the first robust statistical estimate of the proportion of overmature forests and may serve to report on their status in metropolitan France. Our method could be implemented at a larger spatial scale, notably in countries with accessible National Forest Inventory and calibration data, to produce indicators at international level.