Proteomic and microbial assessments on the effect of Antrodia cinnamomea in C57BL/6 mice.

Antrodia cinnamomea (AC) is a nutraceutical fungus and studies have suggested that AC has the potential to prevent or alleviate diseases. However, little is known about the AC-induced phenotypes on the intestine-liver axis and gut microbial alterations. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-Biotyper to elaborate the AC-induced phenotypes on the intestine-liver axis and gut microbial distribution of C57BL/6 mice. The experimental outcomes showed that the hepatic density may increase by elevating hepatic redox regulation, lipid degradation and glycolysis-related proteins and alleviating cholesterol biosynthesis and transport-related proteins in C57BL/6 mice with AC treatment. Moreover, AC facilitates intestinal glycolysis, TCA cycle, redox and cytoskeleton regulation-related proteins, but also reduces intestinal vesicle transport-related proteins in C57BL/6 mice. However, the body weight, GTT, daily food/water intake, and fecal/urine weight were unaffected by AC supplementation in C57BL/6 mice. Notably, the C57BL/6-AC mice had a higher gut microbial abundance of Alistipes shahii (AS) than C57BL/6-Ctrl mice. In summary, the AC treatment affects intestinal permeability by regulating redox and cytoskeleton-related proteins and elevates the gut microbial abundance of AS in C57BL/6 mice that might be associated with increasing hepatic density and metabolism-related proteins of the liver in C57BL/6 mice. Our study provides an insight into the mechanisms of AC-induced phenotypes and a comprehensive assessment of AC's nutraceutical effect in C57BL/6 mice.

Anti-Cancer Effects of Radix Angelica Sinensis (Danggui) and N-Butylidenephthalide on Gastric Cancer: Implications for REDD1 Activation and mTOR Inhibition.

BACKGROUND/AIMS: Radix Angelica Sinensis (danggui in Chinese) is widely used in traditional chinese medicine (TCM). N-butylidenephthalide (BP), a bioactive compound in danggui, is a potential antitumor agent for various cancer types. However, its clinical effect and mechanism in the treatment of gastric cancer remain undetermined. METHODS: The in vivo protective effect of danggui in patients with gastric cancer were validated using data from Taiwan's National Health Insurance Research Database (NHIRD). The genes induced by BP-treatment were analyzed by whole transcriptome RNA sequencing (RNA-seq) and validated by real-time PCR, western blot and siRNA transfection. The effect of BP on AGS cell migration and invasion was evaluated in transwell assays. The antitumor effects of BP were evaluated in vivo in an AGS xenograft animal model. RESULTS: Danggui users were found to have an increased survival rate when compared with danggui nonusers (log-rank test p = 0.002) . The use of danggui highly associated with decreased mortality (the adjusted hazard ratio (HR) of danggui user was 0.72 [95 % CI, 0.57-0.92] (p = 0.009). The in vitro results showed that BP inhibited gastric cancer cell proliferation, and triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Using RNA-seq analysis we found that REDD1 was the highest transcript induced by BP in gastric cancer cells. BP induce an increase of REDD1 expression that inhibits mTOR signaling, thus inhibiting gastric cancer growth. We used RNA interference to demonstrate that the knock-down of REDD1 attenuated the BP-induced mTORC1 activation and growth inhibition. BP suppressed the growth of AGS xenografts tumor in vivo. CONCLUSION: Danggui can prolong the survival rate of gastric cancer patients in Taiwan. BP caused gastric cancer cell death through the activation of mitochondria-intrinsic pathway and induced the REDD1 expression leading to mTOR signal pathway inhibition in gastric cancer cells. BP inhibited the in vivo growth of AGS xenograft tumors. These results may provide the basis for a new therapeutic approach toward the treatment of gastric cancer progression.

Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

Intestinal dual-specificity phosphatase 6 regulates the cold-induced gut microbiota remodeling to promote white adipose browning.

Gut microbiota rearrangement induced by cold temperature is crucial for browning in murine white adipose tissue. This study provides evidence that DUSP6, a host factor, plays a critical role in regulating cold-induced gut microbiota rearrangement. When exposed to cold, the downregulation of intestinal DUSP6 increased the capacity of gut microbiota to produce ursodeoxycholic acid (UDCA). The DUSP6-UDCA axis is essential for driving Lachnospiraceae expansion in the cold microbiota. In mice experiencing cold-room temperature (CR) transitions, prolonged DUSP6 inhibition via the DUSP6 inhibitor (E/Z)-BCI maintained increased cecal UDCA levels and cold-like microbiota networks. By analyzing DUSP6-regulated microbiota dynamics in cold-exposed mice, we identified Marvinbryantia as a genus whose abundance increased in response to cold exposure. When inoculated with human-origin Marvinbryantia formatexigens, germ-free recipient mice exhibited significantly enhanced browning phenotypes in white adipose tissue. Moreover, M. formatexigens secreted the methylated amino acid Nε-methyl-L-lysine, an enriched cecal metabolite in Dusp6 knockout mice that reduces adiposity and ameliorates nonalcoholic steatohepatitis in mice. Our work revealed that host-microbiota coadaptation to cold environments is essential for regulating the browning-promoting gut microbiome.

The Alteration of Akkermansiaceae/Lachnospiraceae Ratio Is a Microbial Feature of Antibiotic-Induced Microbiota Remodeling.

Antibiotic treatment has been shown to cause gut microbiota dysbiosis. However, lacking critical features defining gut microbiota dysbiosis makes it challenging to prevent. By co-occurrence network analysis, we found that despite short antibiotic courses eliminating certain microbial taxa, the Akkermansia genus played the role of a high-centrality hub to maintain microbiota homeostasis. When the antibiotic courses continued, the elimination of Akkermansia induced a significant microbiota remodeling of the gut microbiota networks. Based on this finding, we found that under long-term antibiotic stress, the gut microbiota was rearranged into a stable network with a significantly lower Akkermansiaceae/Lachnospiraceae (A/L) ratio and no microbial hub. By functional prediction analysis, we confirmed that the gut microbiota with a low A/L ratio also had enhanced mobile elements and biofilm-formation functions that may be associated with antibiotic resistance. This study identified A/L ratio as an indicator of antibiotic-induced dysbiosis. This work reveals that besides the abundance of specific probiotics, the hierarchical structure also critically impacts the microbiome function. Co-occurrence analysis may help better monitor the microbiome dynamics than only comparing the differentially abundant bacteria between samples.

The composition of the maternal breastmilk microbiota influences the microbiota network structure during early infancy.

BACKGROUND/PURPOSE(S): Human breastmilk (BM) is important for microbiome maturation in infants across different body sites. Streptococcus and Staphylococcus are considered universally predominant genera in the BM microbiota. However, whether the differential abundance of Streptococcus and Staphylococcus in BM can differentially affect microbiome maturation in infants remains unclear. METHODS: We recruited exclusively breastfeeding mothers from among the donors of the human milk bank established at National Cheng-Kung University Hospital. The donor mothers provided 35 BM samples at three months (3 M; before introducing children to complementary feeding) and 23 BM samples at six months (6 M; after introducing children to complementary feeding) postpartum. At both time points, samples from different body sites, including nasal swabs, oral swabs and stool, were collected from the mothers and their infants. RESULTS: Maternal BMI was inversely associated with coagulase-negative Staphylococcus (CoNS) abundance in breastmilk. Staphylococcus caprae representation in BM CoNS showed a negative correlation with Streptococcus abundance. Network analysis revealed that infants fed Staphylococcus-dominated BM had better gut and nasal microbiota networks than infants fed Streptococcus-abundant BM during early infancy. CONCLUSION: Our work suggests that maternal metabolic status plays a crucial role in Staphylococcus/Streptococcus competition in BM, which in turn can impact the development of the infant microbiota. Our microbiota co-occurrence network analysis might serve as a helpful bioinformatic tool to monitor microbiota maturation during early infancy.

Human pituitary tumor-transforming gene 1 overexpression reinforces oncogene-induced senescence through CXCR2/p21 signaling in breast cancer cells.

INTRODUCTION: hPTTG1 (human pituitary tumor-transforming gene 1) is an oncogene overexpressed in breast cancer and several other types of cancer. Increased hPTTG1 expression has been shown to be associated with poor patient outcomes in breast cancer. Although hPTTG1 overexpression plays important roles in promoting the proliferation, invasion, and metastasis of cancer cells, it also has been suggested to induce cellular senescence. Deciphering the mechanism by which hPTTG1 overexpression induces these contradictory actions in breast cancer cells is critical to our understanding of the role of hPTTG1 in breast cancer development. METHODS: MCF-10A and MCF-7 cells were used to identify the mechanism of hPTTG1-induced senescence. The interplay between hPTTG1 overexpression and chemokine C-X-C motif receptor 2 (CXCR2)/p21-dependent senescence in tumor growth and metastasis of MCF-7 cells was investigated by orthotopic transplantation of severe combined immunodeficiency (SCID) mice. Additionally, human invasive ductal carcinoma (IDC) tissue arrays were used to confirm the hPTTG1/CXCR2/p21 axis established in vitro. RESULTS: In this study, we investigated the mechanism of hPTTG1-induced senescence as well as its role in breast cancer progression and metastasis. Herein, we showed that hPTTG1 overexpression reinforced senescence through the CXCR2/p21 signaling. Furthermore, hPTTG1 overexpression activated NF-κB signaling to transactivate the expression of interleukin (IL)-8 and growth-regulated oncogene alpha (GROα) to execute CXCR2 signaling in MCF-7 cells. When CXCR2 expression was knocked down in hPTTG1-overexpressing MCF-7 cells, hPTTG1-induced senescence was abrogated by alleviating CXCR2-induced p21 expression. In a mouse model, CXCR2-mediated senescence limited hPTTG1-induced tumor growth and metastasis. Moreover, CXCR2 knockdown in hPTTG1-overexpressing MCF-7 tumors dramatically accelerated tumor growth and metastasis. Our in vitro and in vivo results demonstrated that hPTTG1 overexpression reinforces senescence through CXCR2 signaling, and the evasion of CXCR2/p21-dependent senescence was critical to hPTTG1 exerting its oncogenic potential. Interestingly, although CXCR2-dependent senescence restrained hPTTG1-induced tumor progression, when MCF-7 cells and hPTTG1-overexpressing MCF-7 cells were co-transplanted into the mammary fat pads of SCID mice, hPTTG1-overexpressing senescent cells created a metastasis-promoting microenvironment that promoted lung metastasis of the MCF-7 cells. Immunohistochemical analysis of human breast tumor samples also confirmed the importance of the hPTTG1/CXCR2 axis in promoting breast cancer metastasis. CONCLUSIONS: Our findings provide novel molecular insights into hPTTG1-induced senescence and identify a novel mechanism by which hPTTG1 promotes metastasis by regulating the senescence-associated microenvironment.

Single-cell RNA sequencing uncovers the individual alteration of intestinal mucosal immunocytes in Dusp6 knockout mice.

Single-cell RNA sequencing (scRNA-seq) approach can broadly and specifically evaluate the individual cells with minimum detection bias. To explore the individual compositional and transcriptional alteration of intestinal leukocytes in the Dual Specificity Phosphatase six knockout (D6KO) mice, we performed a scRNA-seq followed by the cell type annotation based on ImmGen database. Composition assessments found that D6KO-derived intestinal leukocytes tend to stay inactivate or immature status. The enrichment analysis showed that D6KO-derived intestinal leukocytes are less sensitive to microbes. The mod PhEA phenotypic analysis showed that the D6KO leukocytes may link to not only immune-associated but also diverse previously non-immune-related diseases. Integrating our dataset with the published dataset GSE124880 generated a comprehensive dataset for exploring intestinal immunity. Down-regulation of Ccl17 gene was found in the D6KO-derived dendritic cells. Our results demonstrated the advantage of applying scRNA-seq for dissecting the individual alteration of intestinal leukocytes, particularly in the D6KO mice at a naive state.

Identification of a gut microbiota member that ameliorates DSS-induced colitis in intestinal barrier enhanced Dusp6-deficient mice.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.

Epigenetic Silencing of Ubiquitin Specific Protease 4 by Snail1 Contributes to Macrophage-Dependent Inflammation and Therapeutic Resistance in Lung Cancer.

There is a positive feedback loop driving tumorigenesis and tumor growth through coordinated regulation of epigenetics, inflammation, and stemness. Nevertheless, the molecular mechanism linking these processes is not well understood. In this study, we analyzed the correlation of de-ubiquitinases (DUBs) expression with survival data from the OncoLnc database. Among the DUBs analyzed, ubiquitin specific protease 4 (USP4) had the lowest negative Cox coefficient. Low expression of USP4 was associated with poor survival among lung cancer patients and was inversely correlated with expression of stemness and inflammation markers. Expression of USP4 were reduced at more advanced stages of lung cancer. Mechanistically, expression of USP4 was downregulated in snail1-overexpressing and stemness-enriched lung cancer cells. Snail1 was induced in lung cancer cells by interaction with macrophages, and epigenetically suppressed USP4 expression by promoter methylation. Stable knockdown of USP4 in lung cancer cells enhanced inflammatory responses, stemness properties, chemotherapy resistance, and the expression of molecules allowing escape from immunosurveillance. Further, mice injected with USP4 knockdown lung cancer cells demonstrated enhanced tumorigenesis and tumor growth. These results reveal that the Snail1-mediated suppression of USP4 is a potential mechanism to orchestrate epigenetic regulation, inflammation and stemness for macrophage-promoted tumor progression.

Antrodia cinnamomea Confers Obesity Resistance and Restores Intestinal Barrier Integrity in Leptin-deficient Obese Mice.

Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. Antrodia cinnamomea (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient (ob/ob) mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, ob+/+) and ob/ob mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in ob/ob mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in ob/ob mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.

An overview of microbiome based strategies on anti-obesity.

With the significant global obesity epidemic and emerging strong scientific evidence that connected gut microbiota to obesity, intervening obesity by targeting gut microbiota has become a trendy strategy. Particularly the application of probiotics has become remarkably popular because of their expected association with gut microbiota modulation. Although there are many literatures on the effects of probiotics in obese animal models, most of them reported the effects of probiotic bacteria on metabolic indications with limited information on anti-obesity itself. Besides, some probiotics have been shown to reduce certain metabolic symptoms but they failed to achieve weight loss. This report reviewed the current literatures on the anti-obesity effects of next-generation probiotics in various animal obesity models and discussed the beneficial potential of fecal microbiota transplantation in treating obesity in humans. The purpose of this article is to help guide further research improve the probiotic bacteria experiments in more precise animal obesity models by standardizing the anti-obesogenesis, obesity control, and treatment assays and hopefully the evidence-based investigations on harnessing gut microbiota through next-generation probiotics or fecal microbiota transplantation will develop new interventions to promote and achieve anti-obesity.

Fecal microbiota transplantation for Clostridium difficile infection in Taiwan: Establishment and implementation.

Clostridium difficile infection (CDI) remains a major public health issue, and fecal microbiota transplantation (FMT) has become one of the standard therapies for recurrent or refractory CDI. When compared to medical therapies, such as metronidazole or vancomycin, FMT has a high rate of treatment response with acceptable safety and efficiency. Following promulgation of the amendments in September 2018 in Taiwan, FMT has been indicated for recurrent or refractory CDI. The Taiwan Microbiota Consortium contributed to the Taiwan FMT Expert Consensus, which established basic norms and stipulated essential principles, including the indications for transplantation, eligible locations and personnel, donor screening policies, fecal sample handling, and post-FMT follow-up. However, establishing an eligible FMT team in a qualified hospital remains a clinical challenge, and the requirement for facilities and well-screened donors impedes the implementation of FMT. In this review, we aim to provide domestic FMT teams with explicit instructions to facilitate realization and increase the practice of FMT. Based on the Taiwan FMT Expert Consensus and current regulations, we performed a literature review and integrated the experiences of Taiwanese multidisciplinary experts into this article. The content intends to offer clinicians up-to-date evidence and highlight the essential points of FMT.

HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.

Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.

Dual-specificity phosphatase 6 deficiency regulates gut microbiome and transcriptome response against diet-induced obesity in mice.

The gut microbiota plays profound roles in host metabolism and the inflammatory response associated with the development of obesity. Dusp6-deficient mice have been shown to be resistant to diet-induced obesity, but the mechanism behind this remains unclear. 16S ribosomal RNA gene analysis demonstrated that dusp6-deficient mice harbour unique gut microbiota with resistance to diet-induced-obesity-mediated alteration of the gut microbiome. Using a germ-free mouse model, we found that faecal/gut microbiota derived from dusp6-deficient mice significantly increased energy expenditure and reduced weight gain in recipient wild-type mice fed on a high-fat diet. On analysis of the intestinal transcriptome of dusp6-deficient mice, we found that dusp6 deficiency mainly induced biological processes involved in metabolism and the extracellular matrix, particularly the peroxisome proliferator-activated receptor gamma (Pparγ) pathway and tight-junction genes. Furthermore, dusp6-deficient mice have a high-fat-diet-specific transcriptomic response to reverse the expression of genes associated with intestinal barrier functions and mucosal immunity involved in microbiome homeostasis. This study demonstrates that dusp6 deficiency is a strong genetic factor shaping gut microbiota, and that it confers obesity protection by ameliorating the gut microbiota response to diet-mediated stress.

Lipidome and transcriptome profiling of pneumolysin intoxication identifies networks involved in statin-conferred protection of airway epithelial cells.

Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin.

Genetic and functional analyses of the gene encoding synaptophysin in schizophrenia.

OBJECTIVES: Synaptophysin (SYP) has been shown to be critical for regulating neurotransmitter release and synaptic plasticity, a process thought to be disrupted in schizophrenia. In addition, abnormal SYP expression in different brain regions has been linked to this disorder in postmortem brain studies. We investigated the involvement of the SYP gene in the susceptibility to schizophrenia. METHODS: We searched for genetic variants in the promoter region, all exons, and both UTR ends of the SYP gene using direct sequencing in a sample of patients with schizophrenia (n=586) and non-psychotic controls (n=576), both being Han Chinese from Taiwan, and conducted an association and functional study. RESULTS: We identified 2 common SNPs (c.*4+271A>G and c.*4+565T>C) in the SYP gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. In addition, we identified 6 rare variants in 7 out of 586 patients, including 1 variant (g.-511T>C) located at the promoter region, 1 synonymous (A104A) and 2 missense variants (G293A and A324T) located at the exonic regions, and 2 variants (c.*31G>A and c.*1001G>T) located at the 3'UTR. No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated the influence of g.-511T>C and c.*1001G>T on the regulatory function of the SYP gene, while that the influence of c.*31G>A may be tolerated. In silico analysis demonstrated the functional relevance of other rare variants. CONCLUSION: Our study lends support to the hypothesis of multiple rare mutations in schizophrenia, and provides genetic clues that indicate the involvement of SYP in this disorder.