GUIdEStaR (G-quadruplex, uORF, IRES, Epigenetics, Small RNA, Repeats), the integrated matadatabase: transcript-indexed binary information creation for chaining with neural network analysis GUIdEStaR integrates existing databases of various types of G-quadruplex, upstream Open Reading Frame (uORF), Internal Ribosome Entry Site (IRES), methylation to RNA and histone protein, small RNA, and repeats. GUIdEStaR consists of approx. 40,000 genes and 320,000 transcripts. An mRNA transcript is divided into 5 regions (5UTR, 3UTR, exon, intron, and biological region) where each region contains presence-absence data of 169 different types of elements. Recently, artificial intelligence (AI) based analysis of sequencing data has been gaining popularity in the area of bioinformatics. GUIdEStaR generates datasets that can be used as inputs to AI methods. At the GUIdEStaR homepage, users submit gene symbols by clicking a "Send" button, and shortly result files in CSV format are available for download at the result website. Users have an option to send the result files to their email addresses. Additionally, the entire database and the example Java codes are also freely available for download. Here, we demonstrate the database usage with three neural network classification studies-1) small RNA study for classifying transcription factor (TF) genes into either one of TF mediated by small RNA originated from SARS-CoV-2 or by human microRNA (miRNA), 2) cell membrane receptor study for classifying receptor genes as either with virus interaction or without one, and 3) nonsense mediated mRNA decay (NMD) study for classifying cell membrane and nuclear receptors as either NMD target or non-target. GUIdEStaR is available for access to the easy-to-use web-based database at www.guidestar.kr and for download at https://sourceforge.net/projects/guidestar.
Gene co-expression networks identify novel candidate genes for moulting and development in the Atlantic salmon louse (Lepeophtheirus salmonis) BackgroundThe salmon louse (Lepeophtheirus salmonis) is an obligate ectoparasitic copepod, living on Atlantic salmon and other salmonids in the marine environment. Salmon lice cause a number of environmental problems and lead to large economical losses in aquaculture every year. In order to develop novel parasite control strategies, a better understanding of the mechanisms of moulting and development of the salmon louse at the transcriptional level is required.

MethodsThree weighted gene co-expression networks were constructed based on the pairwise correlations of salmon louse gene expression profiles at different life stages. Network-based approaches and gene annotation information were applied to identify genes that might be important for the moulting and development of the salmon louse. RNA interference was performed for validation. Regulatory impact factors were calculated for all the transcription factor genes by examining the changes in co-expression patterns between transcription factor genes and deferentially expressed genes in middle stages and moulting stages.

ResultsEight gene modules were predicted as important, and 10 genes from six of the eight modules have been found to show observable phenotypes in RNA interference experiments. We knocked down five hub genes from three modules and observed phenotypic consequences in all experiments. In the infection trial, no copepodids with the RAB1A-like gene knocked down were found on fish, while control samples developed to chalimus-1 larvae. Also, a FOXO-like gene obtained highest scores in the regulatory impact factor calculation.

ConclusionsWe propose a gene co-expression network-based approach to identify genes playing an important role in the moulting and development of salmon louse. The RNA interference experiments confirmed the effectiveness of our approach and demonstrated the indispensable role of RAB1A-like gene in the development of salmon louse. In addition to salmon louse, this approach could be generalized to identify important genes associated with a phenotype of interest in other organisms.
Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms. Population bottlenecks constrain host microbiome diversity and genetic variation impeding fitness It is becoming increasingly clear that microbial symbionts influence key aspects of their hosts fitness, and vice versa. This may fundamentally change our thinking about how microbes and hosts interact in influencing fitness and adaptation to changing environments. Here we explore how reductions in population size commonly experienced by threatened species influence microbiome diversity. Consequences of such reductions are normally interpreted in terms of a loss of genetic variation, increased inbreeding and associated inbreeding depression. However, fitness effects of population bottlenecks might also be mediated through microbiome diversity, such as through loss of functionally important microbes. Here we utilise 50 Drosophila melanogaster lines with different histories of population bottlenecks to explore these questions. The lines were phenotyped for egg-to-adult viability and their genomes sequenced to estimate genetic variation. The bacterial 16S rRNA gene was amplified in these lines to investigate microbial diversity. We found that 1) host population bottlenecks constrained microbiome richness and diversity, 2) core microbiomes of hosts with low genetic variation were constituted from subsets of microbiomes found in flies with higher genetic variation, 3) both microbiome diversity and host genetic variation contributed to host population fitness, 4) connectivity and robustness of bacterial networks was low in the inbred lines regardless of host genetic variation, 5) reduced microbial diversity was associated with weaker evolutionary responses of hosts in stressful environments, and 6) these effects were unrelated to Wolbachia density. These findings suggest that population bottlenecks reduce hologenomic variation (combined host and microbial genetic variation). Thus, while the current biodiversity crisis focuses on population sizes and genetic variation of eukaryotes, an additional focal point should be the microbial diversity carried by the eukaryotes, which in turn may influence host fitness and adaptability with consequences for the persistence of populations.

Author summaryIt is becoming increasingly clear that organisms and the microbes that live on or in them - their microbiome - affect each other in profound ways that we are just beginning to understand. For instance, a diverse microbiome can help maintain metabolic functions or fight pathogens causing diseases. A disrupted microbiome may be especially critical for animals and plants that occur in low numbers because of threats from e.g. human exploitation or climate change, as they may already suffer from genetic challenges such as inbreeding and reduced evolutionary potential. The importance of such a reduction in population size, called a bottleneck, on the microbial diversity and the potential interactive effects on host health remains unexplored. Here we experimentally test these associations by investigating the microbiomes of 50 inbred or non-inbred populations of vinegar flies. We found that restricting the population size constrain the hosts genetic variation and simultaneously decreases the diversity of the microbiome that they harbor, and that both effects were detrimental to host fitness. The microbial communities in inbred host populations were less robust than in their non-inbred counterparts, suggesting that we should increasingly consider the microbiome diversity, which may ultimately influence the health and persistence of threatened species. 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. Chromosome breaks in breast cancers occur near herpes tumor virus sequences and explain why cancer drugs stop working One explanation for why breast cancer has a relentless tendency to come back after treatment is that breast cancer phenomes are fluid, so it is difficult for therapy to target new phenotypes that arise. In breast cancer, viral interactions cause chromosome breakage and fusion, producing anaphase bridges containing two centromeres. Cell division then leads to more chromosome breaks, more mutations, and new cancer phenotypes. Analyses of public data from about 2100 breast cancers implicate Epstein-Barr virus (EBV or Human Herpes Virus 4) in the cycles that break chromosomes and produce anaphase bridges. EBV generates breakage cycles with highly fragmented chromosomes (chromothripsis) as an inherent feature. These episodes continually produce new cancer driver mutations. DNA at chromothripsis boundaries in breast cancers consistently matches EBV tumor variants. Breakpoints are often near known EBV binding sites, and breast cancer shares an EBV methylation signature with other known EBV cancers. Breast cancer breakpoints on all chromosomes cluster around the same positions as in cancers definitively associated with EBV infection (nasopharyngeal cancer and Burkitts lymphoma). The status of EBV in the microbiome and the presence of anaphase bridges are phenotypic markers of breast cancer that are not explicitly targeted by chemotherapy. The breast cancer phenome continually supplies new cancer driver genes that foster breast cancer recurrence.

SummaryBreaks in breast cancer chromosomes and in fragments of anaphase bridges occur mainly near regions that resemble Epstein-Barr virus (EBV) tumor variants

Extensive breaks are an inherent outcome of anaphase bridges and the reassembly of highly fragmented chromosomes continually generates new cancer driver mutations and new cancer phenotypes.

Treatment does not explicitly target the underlying breakage and reassembly cycles so breast cancer often comes back.

The breast cancer phenome is not fixed and can continuously generate new phenotypes

Graphical Abstract

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org.highwire.dtl.DTLVardef@6537cforg.highwire.dtl.DTLVardef@132fa50org.highwire.dtl.DTLVardef@1cfcb04org.highwire.dtl.DTLVardef@11979aa_HPS_FORMAT_FIGEXP  M_FIG C_FIG Failure to account for behavioral variability significantly compromises accuracy in indirect population monitoring Indirect wildlife population surveying largely depends upon counts of artefacts of behavior (e.g., nests or dung). Likelihood to encounter these artefacts is derived from both artefact production and decay, and variability in artefact production behavior is considered to contribute minimally to inaccuracy in wildlife estimation. Here we demonstrate how ignoring behavioral variability contributes to significant population misestimation, using an example of an endangered ape, the bonobo (Pan paniscus). Until now, a single estimate of (i.e., nest construction) has been used to extrapolate bonobo densities, assumed to be broadly representative of bonobo sign production behavior. We estimated nest construction rate across seasons and social groups at the Kokolopori Bonobo Reserve, DRC and find that nest construction rates in bonobos to be highly variable across populations as well as seasonal. Failure to account for this variability led to potentially severe degradation in accuracy of bonobo population estimates of abundance, accounting for a likely overestimation of bonobo numbers by 34%, and at worst as high as 80%. With this example, we demonstrate that failure to account for inter- and intra-population behavioral variation compromises the ability to estimate both relative and absolute wildlife abundances. We argue that variation in sign production is but one of several potential ways that behavioral variability can affect conservation monitoring, should be measured across contexts whenever possible, and must be considered in population estimation confidence intervals. With increasing attention to behavioral variability as a potential tool for conservation, conservationists must also account for the impact that behavioral variability can play upon wildlife population estimation. Our results underline the importance of observational research to wildlife monitoring schemes as a critical but overlooked component of conservation management. We extensively discuss the various avenues through which behavioral variability is likely to impact wildlife monitoring accuracy and precision and propose potential avenues for accounting for behavioral variability in wildlife monitoring. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. Transcripts switched off at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit. Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronized according to their individual growth patterns. The net rates of water, sugars and K+ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidized at low rate. Re-synchronized individual berries displayed negligible variations in gene expression among triplicates. RNA-Seq studies revealed sharp reprogramming of cell wall enzymes and structural proteins at the stop of phloem unloading, associated with an 80% repression of multiple sugar transporters and aquaporins on the plasma or tonoplast membranes, with the noticeable exception of H+/sugar symporters, that were rather weakly and constitutively expressed. This was verified in three genotypes placed in contrasted thermo-hydric conditions. The prevalence of SWEET suggests that electrogenic transporters would play a minor role on the plasma membranes of SE/CC complex and the one of the flesh, while sucrose/H+ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall related genes. Together with simple thermodynamic considerations, these results lead to propose that H+/sugar exchangers at the tonoplast, associated with a considerably acidic vacuolar pH, may exhaust cytosolic sugars in the flesh and alleviate the need for supplementary energization of sugar transport at the plasma membrane. Dynamic Phosphorylation of RGS Provides Spatial Regulation of MAP Kinase And Promotes Completion of Cytokinesis during the Yeast Pheromone Response Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS. 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. 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. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type virus and Delta variant Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches. Prediction of survival after partial hepatectomy using a physiologically based pharmacokinetic model of indocyanine green liver function tests The evaluation of hepatic function and functional capacity of the liver are essential tasks in hepatology as well as in hepatobiliary surgery. Indocyanine green (ICG) is a widely applied test compound that is used in clinical routine to evaluate hepatic function. Important questions for the functional evaluation with ICG in the context of hepatectomy are how liver disease such as cirrhosis alters ICG elimination, and if postoperative survival can be predicted from preoperative ICG measurements. Within this work a physiologically based pharmacokinetic (PBPK) model of ICG was developed and applied to the prediction of the effects of a liver resection under various degrees of cirrhosis. For the parametrization of the computational model and validation of model predictions a database of ICG pharmacokinetic data was established. The model was applied (i) to study the effect of liver cirrhosis and liver resection on ICG pharmacokinetics; and (ii) to evaluate the model-based prediction of postoperative ICG-R15 as a measure for postoperative outcome. Key results are the accurate prediction of changes in ICG pharmacokinetics caused by liver cirrhosis and postoperative changes of ICG-elimination after liver resection, as validated with a wide range of data sets. Based on the PBPK model, individual survival after liver resection could be classified, demonstrating its potential value as a clinical tool. In vitro synthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly Membrane proteins perform numerous important functions in cells and tissues. Approximately 20% of the human genome encodes for membrane proteins, which represent the majority of targets for clinically relevant small molecules. Consequently, understanding their structure and structure-function relationships is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional membrane proteins in supported lipid bilayers. Despite having shown the directional reconstitution of CFE-synthesized FL inner nuclear membrane SUN proteins (i.e. SUN1 and SUN2), which directly interact with outer nuclear membrane KASH proteins within the nuclear envelope lumen to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that mechanically couple the cytoskeleton and nucleus, the mechanism underlying regulated LINC complex assembly remains unclear. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment (e.g. calcium ions and redox state) on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and hetero-typic interactions that can occur between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies of membrane protein biochemistry and function, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research. 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. wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Vesalius: high-resolution in silico anatomization of Spatial Transcriptomic data using Image Analysis Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain. Hypertrophic Chondrocytes Serve as a Reservoir for Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage. 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. BlenderPhotonics - an integrated open-source software environment for 3-D meshing and photon simulations in complex tissues SignificanceRapid advances in biophotonics techniques require quantitative, model-based computational approaches to obtain functional and structural information from increasingly complex and multi-scaled anatomies. The lack of efficient tools to accurately model tissue structures and subsequently perform quantitative multi-physics modeling greatly impedes the clinical translation of these modalities.

AimWhile the mesh-based Monte Carlo (MMC) method expands our capabilities in simulating complex tissues by using tetrahedral meshes, the generation of such domains often requires specialized meshing tools such as Iso2Mesh. Creating a simplified and intuitive interface for tissue anatomical modeling and optical simulations is essential towards making these advanced modeling techniques broadly accessible to the user community.

ApproachWe responded to the above challenge by combining the powerful, open-source 3-D modeling software, Blender, with state-of-the-art 3-D mesh generation and MC simulation tools, utilizing the interactive graphical user interface (GUI) in Blender as the front-end to allow users to create complex tissue mesh models, and subsequently launch MMC light simulations.

ResultsHere we present a tutorial to our newly developed Python-based Blender add-on - BlenderPhotonics - to interface with Iso2Mesh and MMC, allowing users to create, configure and refine complex simulation domains and run hardware-accelerated 3-D light simulations with only a few clicks. We provide a comprehensive introduction to this new tool and walk readers through 5 examples, ranging from simple shapes to sophisticated realistic tissue models.

ConclusionBlenderPhotonics is user-friendly and open-source, leveraging the vastly rich ecosystem of Blender. It wraps advanced modeling capabilities within an easy-to-use and interactive interface. The latest software can be downloaded at http://mcx.space/bp. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low.