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Liver cancer is the fourth leading cause of cancer-related death. Hepatocellular carcinoma (HCC) is a primary liver cancer that results from chronic hepatitis caused by multiple predisposing factors such as viral infection, alcohol consumption, and non-alcoholic fatty liver disease. Accumulating studies have indicated that dysfunction of the gut epithelial barrier and hepatic translocation of gut microbes may be implicated in the pathogenesis of HCC. However, the translocated bacteria in HCC patients remains unclear. Here, we characterised tumour-associated microbiota in patients with liver cancer and focused on HCC. We observed that the number of amplicon sequence variants in tumour-associated microbiota was significantly higher compared with that in non-tumour regions of the liver. The tumour-associated microbiota consisted of Bacteroidetes, Firmicutes, and Proteobacteria as the dominant phyla. We identified an unclassified genus that belonged to the Bacteroides, Romboutsia, uncultured bacterium of Lachnospiraceae as a signature taxon for primary liver cancer. Additionally, we identified Ruminococcus gnavus as a signature taxon for HCC patients infected with hepatitis B and/or hepatitis C viruses. This study suggests that tumour microbiota may contribute to the pathology of HCC.

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a ‘sandbox’ for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

In assisted reproductive technology (ART), embryos produced by in vitro fertilization (IVF) are graded according to their live birth potential, and high-grade embryos are preferentially transplanted. However, rates of live birth following clinical ART remain low worldwide. Grading is based on the embryo shape at a limited number of stages and does not consider the shape of embryos and intracellular structures, e.g., nuclei, at various stages important for normal embryogenesis. Here, we developed a Normalized Multi-View Attention Network (NVAN) that directly predicts live birth potential from the nuclear structure in live-cell fluorescence images of mouse embryos from zygote to across a wide range of stages. The input is morphological features of cell nuclei, which were extracted as multivariate time-series data by using the segmentation algorithm for mouse embryos. The classification accuracy of our method (83.87%) greatly exceeded that of existing machine-learning methods and that of visual inspection by embryo culture specialists. Our method also has a new attention mechanism that allows us to determine which values of multivariate time-series data, used to describe nuclear morphology, were the basis for the prediction. By visualizing the features that contributed most to the prediction of live birth potential, we found that the size and shape of the nucleus at the morula stage and at the time of cell division were important for live birth prediction. We anticipate that our method will help ART and developmental engineering as a new basic technology for IVF embryo selection.

Background Non-alcoholic liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, and it can progress to non-alcoholic steatohepatitis (NASH). Alterations in the gut microbiome have been implicated in the development of NAFLD/NASH, although the underlying mechanisms remain unclear. Results We found that the consumption of the prebiotic inulin markedly ameliorated the phenotype of NAFLD/NASH, including hepatic steatosis and fibrosis, in mice. Inulin consumption resulted in global changes in the gut microbiome, including concomitant enrichment of the genera Bacteroides and Blautia, and increased concentrations of short-chain fatty acids, particularly acetate, in the gut lumen and portal blood. The consumption of acetate-releasing resistant starch protected against NAFLD development. Colonisation by Bacteroides acidifaciens and Blautia producta in germ-free mice resulted in synergetic effects on acetate production from inulin. Furthermore, the absence of free fatty acid receptor 2 (FFAR2), an acetate receptor, abolished the protective effect of inulin, as indicated by the more severe liver hypertrophy, hypercholesterolaemia and inflammation. These effects can be attributed to an exacerbation of insulin resistance in the liver, but not in muscle or adipose tissue. Conclusion These findings demonstrated that the commensal microbiome–acetate–FFAR2 molecular circuit improves insulin sensitivity in the liver and prevents the development of NAFLD/NASH.

The field of bioengineering depends on technologies for stable cell culture. Conventionally, every process involved in cell culture has been performed manually, so the culture efficiency and stability can vary between trials or depending on the technician. Among these processes, cell counting is particularly important because cell density affects cell function. Conventional cell counting techniques for cell number estimation are inefficient and unstable because they involve the manual work of collecting a sample of the cell suspension. Thus, a cell counting method that is not susceptible to human error is needed. In this study, we present a novel cell counting method based on smartphone imaging and convolutional neural network-based image processing. Cells are aggregated by centrifuging in a tube and then imaged using a smartphone. The image is transferred to a server, and the cell number is predicted using convolutional neural networks on the server. All processes are performed by a custom-developed smartphone-compatible web app. Compared with the conventional method using a hemocytometer, our method yields more stable cell counting. Furthermore, the time and labor required for cell counting are significantly reduced. Our new method could potentially replace conventional cell counting techniques and thus enhance the stability and efficiency of bioengineering studies that require cell culture.

In assisted reproductive technology (ART), embryos produced by in vitro fertilization (IVF) are graded according to their live birth potential, and high-grade embryos are preferentially transplanted. However, the rate of live birth following clinical ART remains low worldwide, suggesting that grading is inaccurate. One explanation is that grading is classically based on the characteristic shape of embryos at a limited number of developmental stages and does not consider the shape of embryos and intracellular structures, e.g., nuclei, at various stages important for normal embryogenesis. Therefore, here we developed a Normalized Multi-View Attention Network (NVAN) that directly predicts live birth potential from nuclear structural features in live-cell fluorescence images taken of mouse embryos across a wide range of stages. The classification accuracy of our method was 83.87%, which greatly exceeded that of existing machine-learning methods and that of visual inspection by embryo culture specialists. By visualizing the features that contributed most to the prediction of live birth potential, we found that the size and shape of the cell nucleus at the morula stage and at the time of cell division were important for live birth prediction. We anticipate that our method will help ART and developmental engineering as a new basic technology for IVF embryo selection.

In systems biology, biological phenomena are often modeled by Ordinary Differential Equations (ODEs) and distributed in the de facto standard file format SBML. The primary analyses performed with such models are dynamic simulation, steady-state analysis, and parameter estimation. These methodologies are mathematically formalized, and libraries for such analyses have been published. Several tools exist to create, simulate, or visualize models encoded in SBML. However, setting up and establishing analysis environments is a crucial hurdle for non-modelers. Therefore, easy access to perform fundamental analyses of ODE models is a significant challenge. We developed SBMLWebApp, a web-based service to execute SBML-based simulation, steady-state analysis, and parameter estimation directly in the browser without the need for any setup or prior knowledge to address this issue. SBMLWebApp visualizes the result and numerical table of each analysis and provides a download of the results. SBMLWebApp allows users to select and analyze SBML models directly from the BioModels Database. Taken together, SBMLWebApp provides barrier-free access to an SBML analysis environment for simulation, steady-state analysis, and parameter estimation for SBML models. SBMLWebApp is implemented in Java™ based on an Apache Tomcat® web server using COPASI, the Systems Biology Simulation Core Library (SBSCL), and LibSBMLSim as simulation engines. SBMLWebApp is licensed under MIT with source code freely available. At the end of this article, the Data Availability Statement gives the internet links to the two websites to find the source code and run the program online.

Background Trichuris suis ova (TSO), with the potential to modulate the human immune system, have been tested for therapeutic application in autoimmune and allergic diseases such as inflammatory bowel disease (IBD). Previous clinical studies were limited to European and American participants, whereas Asian populations have not been well documented. In this study, a clinical trial was conducted to examine the safety and tolerability of TSO administration among a healthy Japanese population. Methods The study was a randomized, double-blind, placebo-controlled trial held at Jikei University Hospital, Tokyo. Twelve volunteers were stratified into three groups receiving different doses of TSO (TSO 1000, 2500, and 7500) and another into the control group. These cases were limited to healthy Japanese men aged over 20 years old. Single doses of medicinal TSO or placebo were given to three participants of each group. All participants were followed up to 56 days after ingestion. During the follow-up period, clinical practitioners checked each participant at the clinic at 7, 14, 28, and 56 days post-ingestion (dpi). Clinical symptoms were evaluated using questionnaire-based self-reporting, which participants filled at every visit. Blood samples were drawn at 7, 14, 28, and 56 dpi. Fecal samples were collected at 28 and 56 dpi. Results During the study period, twelve healthy Japanese male volunteers were enrolled. All participants completed the follow-up period. No severe adverse events were observed during the study period in all groups. Three participants in the TSO 1000, 2500, and 7500 groups had mild to moderate abdominal symptoms, diarrhea, bloating, and appetite loss during the observation period. One participant in the placebo group presented with mild diarrhea. Microscopic examination identified no parasite ova in any fecal samples. Blood sample examination indicated elevated eosinophil count in several cases, especially in the groups with the higher dose of TSO. No extra-abdominal symptoms were present in all cases. Conclusions Healthy Japanese people tolerated all doses of TSO without any severe adverse events. On the other hand, mild to moderate abdominal symptoms were observed in several participants. This study suggested that the medicinal use of TSO in Japan is relatively safe, and close follow-up is recommended for sustainable usage.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in the enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.; We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in the enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.

Tissue engineering is a promising technology for both its possible applications in the fields of regenerative medicine and medical research. One of its challenges is to create an environment which allows the cell cultures to develop correctly. This environment is far from static and normally is modified by the cells themselves. To this end, adaptable systems have to be developed to ensure a successful development of the in-vitro tissue. This research focuses on a system which is able to modify an external electrical stimulus on a culture chamber, during its cultivation. This system, through a variation of the stimulus direction, successfully improved the orientation of the developing myocytes.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin – calcineurin – NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin – calmodulin Kinase – CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose–induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

During embryogenesis, cells repeatedly divide and dynamically change their positions in three-dimensional (3D) space. A robust and accurate algorithm to acquire the 3D positions of the cells would help to reveal the mechanisms of embryogenesis. To acquire quantitative criteria of embryogenesis from time-series 3D microscopic images, image processing algorithms such as segmentation have been applied. Because the cells in embryos are considerably crowded, an algorithm to segment individual cells in detail and accurately is needed. To quantify the nuclear region of every cell from a time-series 3D fluorescence microscopic image of living cells, we developed QCANet, a convolutional neural network-based segmentation algorithm for 3D fluorescence bioimages. We demonstrated that QCANet outperformed 3D Mask R-CNN, which is currently considered as the best algorithm of instance segmentation. We showed that QCANet can be applied not only to developing mouse embryos but also to developing embryos of two other model species. Using QCANet, we were able to extract several quantitative criteria of embryogenesis from 11 early mouse embryos. We showed that the extracted criteria could be used to evaluate the differences between individual embryos. This study contributes to the development of fundamental approaches for assessing embryogenesis on the basis of extracted quantitative criteria.

Background Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. Methods We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. Findings Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. Interpretation Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. Funding This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).

Proper determination of the cell division axis is essential during development. Wnt3a is a known regulator of the cell division axis; however, the sensitivity of cells to Wnt3a signalling and its role in determining the cell division axis have not been measured to date. To address this gap, we took advantage of the asymmetric distribution of outer dense fibre 2 (ODF2/cenexin) proteins on centrosomes in dividing cells. To precisely quantify the sensitivity of cells to Wnt3a signalling, we developed a microfluidic cell culture device, which can produce a quantitative gradient of signalling molecules. We confirmed that mitotic SH-SY5Y neuroblastoma cells could detect a 2.5 ~ 5 × 10-3 nm·μm-1 Wnt3a concentration gradient and demonstrated that this gradient is sufficient to affect the determination of the pole-to-pole axis of cell division during the later stages of mitosis.

The active form of vitamin D3, 1,25(OH)2D3 has been found to exert multiple effects on the suppression of progression of inflammatory bowel disease (IBD). Vitamin D3 has been gathering attention as a therapy for IBD. However, the clinical trials conducted to date revealed that a relatively high dosage of vitamin D3 was required to see a significant therapeutic effect. Thus, effective formulation and delivery of vitamin D3 to colonic inflammatory lesions will be required. Herein we describe the preparation of a nanostructured lipid carrier (NLC) for the encapsulation of 1,25(OH)2D3 for colonic delivery via oral administration. The optimized fabrication procedure enabled the incorporation of 1,25(OH)2D3 in the NLC by minimizing the destruction of chemically unstable 1,25(OH)2D3. The obtained NLCs orally delivered 1,25(OH)2D3 to the colon in mice and maintained a high concentration of 1,25(OH)2D3 in the colonic tissue for at least 12 h. The NLC showed multiple effects on the suppression of symptoms of colitis induced by dextran sodium sulfate, namely maintaining crypt structure, reducing the tissue concentration of inflammatory cytokines, suppressing the infiltration of polymorphonuclear leukocytes, and augmenting anti-inflammatory CX3CR1high macrophages. Our NLCs containing 1,25(OH)2D3 may be an alternative treatment for IBD therapy.

Inflammatory bowel disease (IBD) is a refractory disease of the gastrointestinal tract that is believed to develop in genetically susceptible individuals. Glycosylation, a type of post-translational modification, is involved in the development of a wide range of diseases, including IBD, by modulating the function of various glycoproteins. To identify novel genes contributing to the development of IBD, we analyzed single nucleotide polymorphisms (SNPs) of glycosylation-related genes in IBD patients and identified MAN2A1, encoding alpha-mannosidase II (α-MII), as a candidate gene. α-MII plays a crucial, but not exclusive, role in the maturation of N-glycans. We also observed that intestinal epithelial cells (IECs), which establish the first-line barrier and regulate gut immunity, selectively expressed α-MII with minimal expression of its isozyme, alpha-mannosidase IIx (α-MIIx). This led us to hypothesize that IEC-intrinsic α-MII is implicated in the pathogenesis of IBD. To test this hypothesis, we generated IEC-specific α-MII-deficient (α-MIIΔIEC) mice. Although α-MII deficiency has been shown to have a minimal effect on N-glycan maturation in most cell types due to the compensation by α-MIIx, ablation of α-MII impaired the maturation of N-glycans in IECs. α-MIIΔIEC mice were less susceptible to dextran sulfate sodium-induced colitis compared with control littermates. In accordance with this, neutrophil infiltration in the colonic mucosa was attenuated in α-MIIΔIEC mice. Furthermore, gene expression levels of neutrophil-attracting chemokines were downregulated in the colonic tissue. These results suggest that IEC-intrinsic α-MII promotes intestinal inflammation by facilitating chemokine expression. We propose SNPs in MAN2A1 as a novel genetic factor for IBD.Key words: inflammatory bowel disease, alpha-mannosidase II, intestinal epithelial cell, N-glycosylation.

Background: Computational simulation using mathematical models is a useful method for understanding the complex behavior of a living system. The majority of studies using mathematical models to reveal biological mechanisms use one of the two main approaches: the bottom-up or the top-down approach. When we aim to analyze a large-scale network, such as a comprehensive knowledge-integrated model of a target phenomenon, for example a whole-cell model, the variation of analyses is limited to particular kind of analysis because of the size and complexity of the model. Results: To analyze a large-scale network of neural differentiation, we developed a hybrid method that combines both approaches. To construct a mathematical model, we extracted network motifs, subgraph structures that recur more often in a metabolic network or gene regulatory network than in a random network, from a large-scale network, detected regulatory motifs among them, and combined these motifs. We confirmed that the model reproduced the known dynamics of HES1 and ASCL1 before and after differentiation, including oscillation and equilibrium of their concentrations. The model also reproduced the effects of overexpression and knockdown of the Id2 gene. Our model suggests that the characteristic change in HES1 and ASCL1 expression in the large-scale network is controlled by a combination of four feedback loops, including a novel large loop discovered in this study. Conclusion: The model extracted by our hybrid method has the potential to reveal the critical mechanisms of neural differentiation. The hybrid method is applicable to other biological events.

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface., Microfold cells (M cells) sit at the gut epithelial surface to sample antigens and maintain local immune homeostasis. Here the authors show that M cells are feedback-regulated by M cell-originated osteoprotegerin (OPG) to suppress RNAKL-induced M cell differentiation, and that OPG deficiency alters both gut colitis and infection phenotypes.

The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases (IBDs); however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin not only establishes a first-line barrier against pathogens, but also serves as a niche for microbial growth. We hypothesized that dysbiosis may cause abnormal mucin utilization and microbial metabolic dysfunction. To test this hypothesis, we analyzed short-chain fatty acids (SCFAs) and mucin components in the stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn’s disease (CD) patients from Japan. The levels of n-butyrate were significantly lower in the stools of both the CD and UC patients than in those of the healthy subjects. Correlation analysis identified 7 bacterial species positively correlated with n-butyrate levels, among which the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans act as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in the colonic lamina propria. Importantly, the availability of mucin-associated O-glycans to the microbiota was significantly reduced in n-butyrate-deficient UC patients. Taken together, our findings highlight the biological significance of the symbiosynthesis pathway in the production of n-butyrate, which maintains gut immune homeostasis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

Summary: The gut microbiota metabolizes the nutrients to produce various metabolites that play crucial roles in host metabolism. However, the links between the microbiota established by different nutrients and the microbiota-influenced changes in the plasma lipids remain unclear. Diets rich in cornstarch, fructose, branched chain amino acids, soybean oil (SO), or lard established a unique microbiota and had influence on glucose metabolism, which was partially reproduced by transferring the microbiota. Comparison of plasma lipidomic analysis between germ-free and colonized mice revealed significant impacts of the microbiota on various lipid classes, and of note, the microbiota established by the SO diet, which was associated with the greatest degree of glucose intolerance, caused the maximum alteration of the plasma lipid profile. Thus, the gut microbiota composed of dietary nutrients was associated with dynamic changes in the lipids potentially having differential effects on glucose metabolism.

The intestinal microbiota has been implicated in a wide range of diseases, including inflammatory bowel disease, obesity and cancer. Food ingredients are considered a major determinant of gut microbial composition, as exemplified by high-fat diet-induced dysbiosis that can affect host physiology. Accumulating studies show that microbial metabolites are key regulators of the intestinal epithelial barrier and gut immunity. In particular, short-chain fatty acids produced by bacterial fermentation of indigestible polysaccharides have profound impacts on host physiology beyond the gut. In this review, we describe the influences of the diet-microbiota-metabolite axis on host physiology, and especially on the immune and metabolic systems.

Polyamines produced by both prokaryotes and eukaryotes are bioactive substances with pleiotropic effects. Accumulating evidence has demonstrated that polyamines contribute to anti-inflammatory responses by suppressing the expression of proinflammatory cytokines in mononuclear cells and macrophages. However, the effects of polyamines on CD4+ T cell responses remain to be elucidated. Here, we investigated the effect of polyamines on cell fate decisions of naive CD4+ T cells in vitro. We found that endogenously generated polyamines are essential for the development of T helper 2 (Th2) cells. Treatment with DL-2-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, diminished GATA3 expression in CD4+ T cells under Th2-skewed conditions. Supplementation of exogenous polyamines rescued GATA3 downregulation caused by DFMO treatment in CD4+ T cells. Transcriptome analysis revealed that deprivation of endogenous polyamines resulted in upregulated Th9-related genes, such as Il9, Irf4, and Batf3, even under the Th2-skewing conditions. Depletion of intracellular polyamines reduced GATA3 expression but increased IL-9-producing CD4+ T cells under both Th2 and Th9-skewing conditions. Furthermore, oral administration of DFMO increased IL-9-producing CD4+ T cells in small intestine in mice. Thus, our data indicate that polyamines play a critical role in the regulation of the Th2/Th9 balance.

OBJECTIVES: Intestinal mucins escape digestion and enter the large bowel where they are degraded by microbiome. To what extent and how mucins impact large-bowel physiology remain unclear. This study examined the large-bowel fermentation characteristics of mucins and mucin-derived O-glycan sugars and whether mucins and N-acetylglucosamine (GlcNAc) affect gut immunity. METHODS: Mucin secretion from the terminal ileum was determined from feces of ileorectostomized male Wistar rats (age 6 weeks) fed AIN76-based control diet (CD) for 15 d (Expt. 1). Normal male Wistar rats (age 6 weeks; 4 weeks for Expt. 4) were fed CD ± porcine stomach mucin (PM) at 6 or 12g/kg diet, equivalent to 1.5 and 3 times daily mucin secretion, for 14 d (Expt. 2); CD ± GlcNAc, fucose, or N-acetylneuraminic acid at 10g/kg diet for 14 d (Expt. 3); or CD ± PM (15 g/kg diet) or GlcNAc (10 g/kg diet) for 29 d (Expt. 4). Short-chain fatty acids (SCFAs), microbial composition, and cecal O-glycan content were assessed. IgA(+) plasma and regulatory T cells and inflammatory cytokine expression in the cecum were evaluated (Expt. 4). RESULTS: Daily mucin secretion corresponded to 43.2 mmol of O-glycans. PM was efficiently fermented in the cecum, as evidenced by comparable amounts of cecal O-glycans between groups. PM-fed rats harbored more mucin-degrading bacteria. Cecal SCFA concentrations, particularly n-butyrate, were higher in 12g/kg PM diet-fed rats versus CD (P

Mouse mothers transfer metabolic mode Obesity and metabolic diseases tend to go together, and humans who become obese are also prone to type 2 diabetes and cardiovascular problems. Starting with the observation that offspring of germ-free mice tended to become obese on high-fat diets, Kimura et al. investigated how the presence of the microbiota might be protective in mice (see the Perspective by Ferguson). Short-chain fatty acids (SCFAs) from the microbiota are known to suppress insulin signaling and reduce fat deposition in adipocytes. Further experiments showed that SCFAs in the bloodstream were able to pass from a non–germ-free mother's gut microbiota across the placenta and into the developing embryos. The authors found that in the embryos, the SCFA propionate mediates not only insulin levels through GPR43 signaling but also sympathetic nervous system development through GPR41 signaling. A high-fiber diet promoted propionate production from the maternal microbiota, and maternal antibiotic treatment resulted in obese-prone offspring. Science , this issue p. eaaw8429 ; see also p. 978

Background: The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases (IBDs); however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin not only establishes a first-line barrier against pathogens, but also serves as a niche for microbial growth. Methods: To elucidate relationships between dysbiosis, abnormal mucin utilization, and microbial metabolic dysfunction, we analyzed short-chain fatty acids (SCFAs) and mucin components in the stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan. Findings: The levels of n-butyrate were significantly lower in the stools of both the CD and UC patients than in those of the healthy subjects. Correlation analysis identified 7 bacterial species positively correlated withn-butyrate levels, among which the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans act as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in the colonic lamina propria. Importantly, the availability of mucin-associated O-glycans to the microbiota was significantly reduced inn-butyrate-deficient UC patients. Interpretation: Our findings demonstrated that mucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormality in the mucin utilization may lead to decreased n-butyrate production in UC patients. Funding Statement: This study was supported by grants from the Japan Society for the Promotion of Science (#16H01369, 17KT0055, and 18H04680 to KH), Health Labour Sciences Research Grant (KH and JK), AMED-Crest (#16gm0000000h0101, 17gm1010004h0102, and 18gm1010004h0103 to KH and JK), AMED (#18ek0109303h0001 to KH and JK), Yakult Foundation (KH), Keio Gijuku Academic Development Funds (KH), The Aashi Grass Foundation, and The Canon Foundation (JK). Declaration of Interests:The authors have declared that no competing interests exist. Ethics Approval Statement: The protocol was approved by the ethics committees of Osaka University (#13165-2), The University of Tokyo (#25-42-1122), Keio University (#150421-1), Shizuoka University (#14-11), and NIBIOHN (#72) prior to subject inclusion. All animal experiments were performed according to protocols approved by the Animal Use Committees of Keio University and Shizuoka University. The rats and mice were maintained in accordance with the guidelines for the care of laboratory animals of Shizuoka University and Keio University, respectively.

Summary Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by ∼50% in Peyer’s patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.

SummaryImage-based deep learning systems, such as convolutional neural networks (CNNs), have recently been applied to cell classification, producing impressive results; however, application of CNNs has been confined to classification of the current cell state from the image. Here, we focused on cell movement where current and/or past cell shape can influence the future cell fate. We demonstrate that CNNs prospectively predicted the future direction of cell movement with high accuracy from a single image patch of a cell at a certain time. Furthermore, by visualizing the image features that were learned by the CNNs, we could identify morphological features, e.g., the protrusions and trailing edge that have been experimentally reported to determine the direction of cell movement. Our results indicate that CNNs have the potential to predict the future cell fate from current cell shape, and can be used to automatically identify those morphological features that influence future cell fate.

The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to conquer fatal desiccation stress. The Pv11 cell line, derived from embryos of the midge, shows desiccation tolerance by pretreatment with trehalose before desiccation; they can resume proliferation after rehydration. To address the underlying molecular mechanisms, we desiccated Pv11 cells after pretreatment with the medium containing trehalose and induced proliferation by rehydration. We collected the cells at each before and after desiccation and rehydration step and performed CAGE-seq of mRNA of those cells. By analysing differentially expressed genes (DEGs) among the results of CAGE-seq, we detected 384 DEGs after trehalose treatment and 14 DEGs after rehydration. Hierarchical clustering of the identified DEGs indicated that rehydration returns their expression pattern to that in the control culture state. DEGs involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction were upregulated by trehalose treatment. DEGs for rehydration supported that DNA repair is one of the potential mechanisms involves recovery. This study provided initial insight into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells with a potential for proliferation following rehydration.

Multicellular organisms develop from the fertilized eggs through numbers of cell divisions. While the pattern of cell division has specific rules for healthy development, some fluctuations for this pattern are allowable. In order to uncover this robust mechanism of development, the position of cells in each embryo must be analyzed quantitatively. In the embryonic developmental biology, various studies are attempted to acquire the quantitative criteria from time-series three-dimensional microscopic images by image analysis such as segmentation, to understand the mechanism of development. Unfortunately, due to the fact of inaccuracy of nuclei detection and segmentation, it is a hard task to evaluate an embryo quantitatively from bioimages automatically. Based on these demands of quantitative analysis, we developed QCA Net, which accurately performs nuclear segmentation of three-dimensional fluorescence microscopic images for early-stage mouse embryos. QCA Net is based on Convolutional Neural Network. We trained QCA Net using a part of one early-stage mouse embryo. As a test, QCA Net performed segmentation of different 11 mouse embryos images. We succeeded in accurately acquiring the shape of the nucleus without fusion of nuclear regions. Besides, we achieved accurate extraction of the time-series data of nuclear number, volume, surface area, and center of gravity coordinates as the quantitative criteria of mouse development, from the segmentation images acquired by QCA Net. To our surprise, these results suggested that QCA Net recognized and distinguished the nucleus and a polar body formed in meiosis process. We consider that QCA Net can drastically contribute to performing segmentation of various bioimages in the embryonic developmental biology. The various quantitative criteria obtained from segmented images can uncover various unknown mechanisms of embryonic development, such as the robust mechanism of development.

Regenerative medicine using neural stem cells (NSCs), which self-renew and have pluripotency, has recently attracted a lot of interest. Much research has focused on the transplantation of differentiated NSCs to damaged tissues for the treatment of various neurodegenerative diseases and spinal cord injuries. However, current approaches for distinguishing differentiated from non-differentiated NSCs at the single-cell level have low reproducibility or are invasive to the cells. Here, we developed a fully automated, non-invasive convolutional neural network-based model to determine the differentiation status of human NSCs at the single-cell level from phase-contrast photomicrographs, after training, our model showed an accuracy of identification greater than 94%. To understand how our model distinguished between differentiated and non-differentiated NSCs, we evaluated the informative features it learned for the two cell types and found that it had learned several biologically relevant features related to NSC shape during differentiation. We also used our model to examine the differentiation of NSCs over time, the findings confirmed our model&rsquo, s ability to distinguish between non-differentiated and differentiated NSCs. Thus, our model was able to non-invasively and quantitatively identify differentiated NSCs with high accuracy and reproducibility, and, therefore, could be an ideal means of identifying differentiated NSCs in the clinic.

Neuroblastoma is the most common solid tumour of childhood, and it metastasizes to distant organs. However, the mechanism of metastasis, which generally depends on the cell motility of the neuroblastoma, remains unclear. In many solid tumours, it has been reported that shear stress promotes metastasis. Here, we investigated the relationship between shear stress and cell motility in the MYCN-amplified human neuroblastoma cell line IMR32, using a microfluidic device. We confirmed that most of the cells migrated downstream, and cell motility increased dramatically when the cells were exposed to a shear stress of 0.4 Pa, equivalent to that expected in vivo . We observed that the morphological features of focal adhesion were changed under a shear stress of 0.4 Pa. We also investigated the relationship between malignancy and the motility of IMR32 cells under shear stress. Decreasing the expression of MYCN in IMR32 cells via siRNA transfection inhibited cell motility by a shear stress of 0.4 Pa. These results suggest that MYCN-amplified neuroblastoma cells under high shear stress migrate to distant organs due to high cell motility, allowing cell migration to lymphatic vessels and venules.

Attempts to grow mono-crystalline cubic silicon carbide (3C-SiC) have been made using vapor phase hetero-epitaxial growth with Si [1], TiC [2], and sapphire [3] as substrates, and with bulk growth using the sublimation method. In 1983, Nishino et al. reported hetero-epitaxial growth of 3C-SiC on a carbonized Si(001) surface [5]. Since then, 3C-SiC heteroepitaxy on Si substrate using the CVD or MBE methods has been studied intensively, because it has been proved that using Si as a substrate facilitates the upsizing of SiC, while reducing manufacturing costs, and that carbonization of the Si surface significantly improves the reproducibility of the 3C-SiC grown on it.

The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to overcome fatal desiccation stress. The Pv11 cell line, derived from P. vanderplanki embryo, shows desiccation tolerance when treated with trehalose before desiccation and resumes proliferation after rehydration. However, the molecular mechanisms of this desiccation tolerance remain unknown. Here, we performed high-throughput CAGE-seq of mRNA and a differentially expressed gene analysis in trehalose-treated, desiccated, and rehydrated Pv11 cells, followed by gene ontology analysis of the identified differentially expressed genes. We detected differentially expressed genes after trehalose treatment involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction that were upregulated. In the desiccation phase, L-isoaspartyl methyltransferase and heat shock proteins were upregulated and ribosomal proteins were downregulated. Analysis of differentially expressed genes during rehydration supported the notion that homologous recombination, nucleotide excision repair, and non-homologous recombination were involved in the recovery process. This study provides initial insights into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells.