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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.

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.

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.

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.

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.

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.

Water is essential for living organisms. Terrestrial organisms are incessantly exposed to the stress of losing water, desiccation stress. Avoiding the mortality caused by desiccation stress, many organisms acquired molecular mechanisms to tolerate desiccation. Larvae of the African midge, Polypedilum vanderplanki, and its embryonic cell line Pv11 tolerate desiccation stress by entering an ametabolic state, anhydrobiosis, and return to active life after rehydration. The genes related to desiccation tolerance have been comprehensively analyzed, but transcriptional regulatory mechanisms to induce these genes after desiccation or rehydration remain unclear. Here, we comprehensively analyzed the gene regulatory network in Pv11 cells and compared it with that of Drosophila melanogaster, a desiccation sensitive species. We demonstrated that nuclear transcription factor Y subunit gamma-like, which is important for drought stress tolerance in plants, and its transcriptional regulation of downstream positive feedback loops have a pivotal role in regulating various anhydrobiosis-related genes. This study provides an initial insight into the systemic mechanism of desiccation tolerance.

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.

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.