Systematic Insights into the Relationship between the Microbiota-Gut-Brain Axis and Stroke with the Focus on Tryptophan Metabolism.

Stroke, as a serious cerebral vascular disease with high incidence and high rates of disability and mortality, has limited therapeutic options due to the narrow time window. Compelling evidence has highlighted the significance of the gut microbiota and gut-brain axis as critical regulatory factors affecting stroke. Along the microbiota-gut-brain axis, tryptophan metabolism further acquires increasing attention for its intimate association with central nervous system diseases. For the purpose of exploring the potential role of tryptophan metabolism in stroke and providing systematic insights into the intricate connection of the microbiota-gut-brain axis with the pathological procedure of stroke, this review first summarized the practical relationship between microbiota and stroke by compiling the latest case-control research. Then, the microbiota-gut-brain axis, as well as its interaction with stroke, were comprehensively elucidated on the basis of the basic anatomical structure and physiological function. Based on the crosstalk of microbiota-gut-brain, we further focused on the tryptophan metabolism from the three major metabolic pathways, namely, the kynurenine pathway, serotonin pathway, and microbial pathway, within the axis. Moreover, the effects of tryptophan metabolism on stroke were appreciated and elaborated here, which is scarcely found in other reviews. Hopefully, the systematic illustration of the mechanisms and pathways along the microbiota-gut-brain axis will inspire more translational research from metabolic perspectives, along with more attention paid to tryptophan metabolism as a promising pharmaceutical target in order to reduce the risk of stroke, mitigate the stroke progression, and ameliorate the stroke prognosis.

Fusobacterium nucleatum upregulates MMP7 to promote metastasis-related characteristics of colorectal cancer cell via activating MAPK(JNK)-AP1 axis.

BACKGROUND: Colorectal cancer (CRC) is the third most common malignant tumor. Fusobacterium nucleatum (F. nucleatum) is overabundant in CRC and associated with metastasis, but the role of F. nucleatum in CRC cell migration and metastasis has not been fully elucidated. METHODS: Differential gene analysis, protein-protein interaction, robust rank aggregation analysis, functional enrichment analysis, and gene set variation analysis were used to figure out the potential vital genes and biological functions affected by F. nucleatum infection. The 16S rDNA sequencing and q-PCR were used to detect the abundance of F. nucleatum in tissues and stools. Then, we assessed the effect of F. nucleatum on CRC cell migration by wound healing and transwell assays, and confirmed the role of Matrix metalloproteinase 7 (MMP7) induced by F. nucleatum in cell migration. Furthermore, we dissected the mechanisms involved in F. nucleatum induced MMP7 expression. We also investigated the MMP7 expression in clinical samples and its correlation with prognosis in CRC patients. Finally, we screened out potential small molecular drugs that targeted MMP7 using the HERB database and molecular docking. RESULTS: F. nucleatum infection altered the gene expression profile and affected immune response, inflammation, biosynthesis, metabolism, adhesion and motility related biological functions in CRC. F. nucleatum was enriched in CRC and promoted the migration of CRC cell by upregulating MMP7 in vitro. MMP7 expression induced by F. nucleatum infection was mediated by the MAPK(JNK)-AP1 axis. MMP7 was highly expressed in CRC and correlated with CMS4 and poor clinical prognosis. Small molecular drugs such as δ-tocotrienol, 3,4-benzopyrene, tea polyphenols, and gallic catechin served as potential targeted therapeutic drugs for F. nucleatum induced MMP7 in CRC. CONCLUSIONS: Our study showed that F. nucleatum promoted metastasis-related characteristics of CRC cell by upregulating MMP7 via MAPK(JNK)-AP1 axis. F. nucleatum and MMP7 may serve as potential therapeutic targets for repressing CRC advance and metastasis.

Fecal microbiota transplantation inhibits colorectal cancer progression: Reversing intestinal microbial dysbiosis to enhance anti-cancer immune responses.

Many lines of evidence demonstrate the associations of colorectal cancer (CRC) with intestinal microbial dysbiosis. Recent reports have suggested that maintaining the homeostasis of microbiota and host might be beneficial to CRC patients, but the underlying mechanisms remain unclear. In this study, we established a CRC mouse model of microbial dysbiosis and evaluated the effects of fecal microbiota transplantation (FMT) on CRC progression. Azomethane and dextran sodium sulfate were used to induce CRC and microbial dysbiosis in mice. Intestinal microbes from healthy mice were transferred to CRC mice by enema. The vastly disordered gut microbiota of CRC mice was largely reversed by FMT. Intestinal microbiota from normal mice effectively suppressed cancer progression as assessed by measuring the diameter and number of cancerous foci and significantly prolonged survival of the CRC mice. In the intestine of mice that had received FMT, there were massive infiltration of immune cells, including CD8+ T and CD49b+ NK, which is able to directly kill cancer cells. Moreover, the accumulation of immunosuppressive cells, Foxp3+ Treg cells, seen in the CRC mice was much reduced after FMT. Additionally, FMT regulated the expressions of inflammatory cytokines in CRC mice, including down-regulation of IL1a, IL6, IL12a, IL12b, IL17a, and elevation of IL10. These cytokines were positively correlated with Azospirillum_sp._47_25, Clostridium_sensu_stricto_1, the E. coli complex, Akkermansia, Turicibacter, and negatively correlated with Muribaculum, Anaeroplasma, Candidatus_Arthromitus, and Candidatus Saccharimonas. Furthermore, the repressed expressions of TGFb, STAT3 and elevated expressions of TNFa, IFNg, CXCR4 together promoted the anti-cancer efficacy. Their expressions were positively correlated with Odoribacter, Lachnospiraceae-UCG-006, Desulfovibrio, and negatively correlated with Alloprevotella, Ruminococcaceae UCG-014, Ruminiclostridium, Prevotellaceae UCG-001 and Oscillibacter. Our studies indicate that FMT inhibits the development of CRC by reversing gut microbial disorder, ameliorating excessive intestinal inflammation and cooperating with anti-cancer immune responses.

E. coli diversity: low in colorectal cancer.

BACKGROUND: Escherichia coli are mostly commensals but also contain pathogenic lineages. It is largely unclear whether the commensal E. coli as the potential origins of pathogenic lineages may consist of monophyletic or polyphyletic populations, elucidation of which is expected to lead to novel insights into the associations of E. coli diversity with human health and diseases. METHODS: Using genomic sequencing and pulsed field gel electrophoresis (PFGE) techniques, we analyzed E. coli from the intestinal microbiota of three groups of healthy individuals, including preschool children, university students, and seniors of a longevity village, as well as colorectal cancer (CRC) patients, to probe the commensal E. coli populations for their diversity. RESULTS: We delineated the 2280 fresh E. coli isolates from 185 subjects into distinct genome types (genotypes) by PFGE. The genomic diversity of the sampled E. coli populations was so high that a given subject may have multiple genotypes of E. coli, with the general diversity within a host going up from preschool children through university students to seniors. Compared to the healthy subjects, the CRC patients had the lowest diversity level among their E. coli isolates. Notably, E. coli isolates from CRC patients could suppress the growth of E. coli bacteria isolated from healthy controls under nutrient-limited culture conditions. CONCLUSIONS: The coexistence of multiple E. coli lineages in a host may help create and maintain a microbial environment that is beneficial to the host. As such, the low diversity of E. coli bacteria may be associated with unhealthy microenvironment in the intestine and hence facilitate the pathogenesis of diseases such as CRC.

SREBP1 promotes the invasion of colorectal cancer accompanied upregulation of MMP7 expression and NF-κB pathway activation.

BACKGROUND: Sterol-regulatory element binding protein 1 (SREBP1), an intracellular cholesterol sensor located in the endoplasmic reticulum, regulates the intracellular cholesterol by the Insig-Srebp-Scap pathway. Over-expression of SREBP1 can cause dyslipidemia. SREBP1 can regulate the metabolic pathway, and then promote the proliferation of tumor cells. However, there is no relevant research of metastasis and invasion in the field of colorectal cancer (CRC). METHODS: Expression of SREBP1 was manipulated in CRC cell lines with low and high level SREBP1 expression by transfectiong with plasmids containing the SREBP1 gene, or by shRNA. The effect of SREBP1 on cell migration was assayed. The expression of SREBP1, p65 and MMP7 were detected by western blot. Human umbilical vein endothelial cell was used for detection of angiogenesis by adding the culture supernatant from HT29 and SW620. The level of reactive oxygen species (ROS) was detected by Dihydroethidium (DHE) staining. NF-κB inhibitor SN50 was used to test the relationship of SREBP1, NF-κB pathway and MMP7. RESULTS: We found that the expression of SREBP1 in colon adenocarcinoma was significantly higher than that in noncancerous tissues, especially in the invasive tumor front including tumor budding. In vitro, SREBP1 over-expressed in colon cancer cell lines HT29 promoted angiogenesis in endothelial cells, increased ROS levels, phosphorylation of NF-κB-p65 and increases MMP7 expression. The effect of SREBP1 on expression of MMP7 was lost following treatment with the NF-κB inhibitor SN50. CONCLUSION: Our results suggest that SREBP1 can promote the invasion and metastasis of CRC cells by means of promoting the expression of MMP7 related to phosphorylation of p65.

Rapid and sensitive detection of the vanA resistance gene from clinical Enterococcus faecium and Enterococcus faecalis isolates by loop-mediated isothermal amplification.

OBJECTIVES: Vancomycin resistance in Enterococcus spp., mediated mainly by the vanA resistance gene, has become a major health concern as it has spread worldwide. Therefore, a rapid method is urgently required to detect the vanA gene for timely and appropriate antimicrobial control of resistant Enterococcus infections. METHODS: The loop-mediated isothermal amplification (LAMP) assay was optimised for vanA detection in Enterococcus spp. isolates. RESULTS: The LAMP primer set designed in this study could reliably recognise seven distinct regions of the vanA gene and amplify the gene within 25min at an isothermal temperature of 65°C with high specificity. The sensitivity of the optimised assay was high, with a detection limit for vanA as low as 100pg/µL, which is 100-fold more sensitive than the PCR assay. A special advantage of this optimised LAMP method is that the vanA gene could be detected directly from clinical specimens. CONCLUSION: This optimised LAMP assay has great application potential for efficient detection of vanA in clinical diagnosis and epidemiological studies.

SREBP1 promotes 5-FU resistance in colorectal cancer cells by inhibiting the expression of caspase7.

BACKGROUND: The role of lipid metabolism played in cancer cell growth attracts more attention. SREBP1 is a common lipid regulatory factor. It has been reported that SREBP1 can promote tumor cell resistance. The aim of this study was to investigate its role in chemoresistance of colorectal cancer (CRC). METHODS: The expression of SREBP1 in CRC tissues was analyzed by immunohistochemistry. Using a viability assay, the sensitivity to 5-fluorouracil in two colon cancer cell lines (HT-29 and SW620) was measured and its correlation with different expression levels of SREBP1 protein by western blot was investigated. RESULTS: The protein expression of SREBP1 in CRC tissues was higher than that in normal colon tissues. We found that over-expression of SREBP1 through SREBP1 gene transfection enhances the resistant of CRC cell lines to 5-FU, and SREBP1 silencing through SREBP1 shRNA transfection can promote apoptosis in 5-FU treated SW620 cells. Further study indicated that SREBP1 could inhibit the expression of caspase7 and reduce PARP1 cleavage fragments. CONCLUSION: Our results suggest that SREBP1 protect the 5-FU treated CRC cells through caspase7 dependent PARP1 cleavage in apoptosis pathway and potentially provide a new target in the treatment of CRC.

Cancer killers in the human gut microbiota: diverse phylogeny and broad spectra.

Cancer as a large group of complex diseases is believed to result from the interactions of numerous genetic and environmental factors but may develop in people without any known genetic or environmental risks, suggesting the existence of other powerful factors to influence the carcinogenesis process. Much attention has been focused recently on particular members of the intestinal microbiota for their potential roles in promoting carcinogenesis. Here we report the identification and characterization of intestinal bacteria that exhibited potent anti-malignancy activities on a broad range of solid cancers and leukemia. We collected fecal specimens from healthy individuals of different age groups (preschool children and university students), inspected their effects on cancer cells, and obtained bacteria with potent anti-malignancy activities. The bacteria mostly belonged to Actinobacteria but also included lineages of other phyla such as Proteobacteria and Firmicutes. In animal cancer models, sterile culture supernatant from the bacteria highly effectively inhibited tumor growth. Remarkably, intra-tumor administration of the bacterial products prevented metastasis and even cleared cancer cells at remote locations from the tumor site. This work demonstrates the prevalent existence of potent malignancy-killers in the human intestinal microbiota, which may routinely clear malignant cells from the body before they form cancers.

A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus.

Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62°C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.

Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type ß-lactamases, are among the leading causes of nosocomial infections worldwide. Among the ß-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of ß-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of ß-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

Effects of erythromycin and rifampicin on immunomodulatory gene expression and cellular function in human polymorphonuclear leukocytes.

BACKGROUND: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). METHODS: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. RESULTS: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. CONCLUSIONS: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs.

Hydrogen peroxide induces overexpression of angiotensin-converting enzyme in human umbilical vein endothelial cells.

Oxidative stress has been linked to endothelial dysfunction in atherosclerosis and hypertension. The present study was designed to investigate the effect of hydrogen peroxide (H2O2) on angiotensin-converting enzyme (ACE), a key regulator of the renin-angiotensin system, and the mechanisms underlying ACE regulation in human umbilical vein endothelial cells (HUVECs). We used Tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, enzyme-linked immunosorbent assay (ELISA) for cAMP measurement, real-time PCR for mRNA detection, and Western blot for protein analysis in the study. Our results demonstrated that H2O2 (50-1000 µM) decreased HUVECs viability by inducing apoptosis. Notably, H2O2 upregulated ACE expression in a concentration-dependent manner. H2O2 100 µM significantly enhanced cyclic adenosine monophosphate (cAMP) expression by 1.48-fold (P<0.05). Additionally, forskolin 10 µM, a cAMP agonist, was also found to enhance ACE expression by 1.78-fold (P<0.05); in contrast, H-89 10 µM, a protein kinase A (PKA) inhibitor, abolished H2O2-induced ACE expression and prevented the enhancing effect of forskolin-induced ACE expression. Similar effects on ACE mRNA were also observed. cAMP-response element-specific decoy oligodeoxynucleotides (CRE-dODN) containing binding sites for cAMP-response element-binding protein (CREB) inhibited ACE expression at both the mRNA and protein levels. Negative control CRE-dODN had no effect on ACE expression. We conclude that H2O2 upregulates the expression of ACE through the activation of cAMP/PKA/CREB signal pathway in HUVECs, indicating a role of oxidative stress in the pathophysiology of hypertension.

Difference of sodium currents between pediatric and adult human atrial myocytes: evidence for developmental changes of sodium channels.

Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.