Bacteroides and related species: The keystone taxa of the human gut microbiota.

Microbial communities play a significant role in maintaining ecosystems in a healthy homeostasis. Presently, in the human gastrointestinal tract, there are certain taxonomic groups of importance, though there is no single species that plays a keystone role. Bacteroides spp. are known to be major players in the maintenance of eubiosis in the human gastrointestinal tract. Here we review the critical role that Bacteroides play in the human gut, their potential pathogenic role outside of the gut, and their various methods of adapting to the environment, with a focus on data for B. fragilis and B. thetaiotaomicron. Bacteroides are anaerobic non-sporing Gram negative organisms that are also resistant to bile acids, generally thriving in the gut and having a beneficial relationship with the host. While they are generally commensal organisms, some Bacteroides spp. can be opportunistic pathogens in scenarios of GI disease, trauma, cancer, or GI surgery, and cause infection, most commonly intra-abdominal infection. B. fragilis can develop antimicrobial resistance through multiple mechanisms in large part due to its plasticity and fluid genome. Bacteroidota (formerly, Bacteroidetes) have a very broad metabolic potential in the GI microbiota and can rapidly adapt their carbohydrate metabolism to the available nutrients. Gastrointestinal Bacteroidota species produce short-chain fatty acids such as succinate, acetate, butyrate, and occasionally propionate, as the major end-products, which have wide-ranging and many beneficial influences on the host. Bacteroidota, via bile acid metabolism, also play a role in in colonization-resistance of other organisms, including Clostridioides difficile, and maintenance of gut integrity.

Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well-Characterized Serum Samples.

Standard two-tiered testing (STTT) is the recommended algorithm for laboratory diagnosis of Lyme disease (LD). Several limitations are associated with STTT that include low sensitivity in the early stages of disease, as well as technical complexity and subjectivity associated with second-tier immunoblotting; therefore, modified two-tiered testing (MTTT) algorithms that utilize two sequential first-tier tests and eliminate immunoblotting have been evaluated. Recently, a novel MTTT that uses a VlsE chemiluminescence immunoassay followed by a C6 enzyme immunoassay has been proposed. The purpose of this study was to evaluate the performance of the VlsE/C6 MTTT using well-characterized serum samples. Serum samples from the CDC Lyme Serum Repository were tested using three MTTTs, VlsE/C6, whole-cell sonicate (WCS)/C6, and WCS/VlsE, and three STTTs (immunoblotting preceded by three different first-tier assays: VlsE, C6, and WCS). Significant differences were not observed between the results of the MTTTs assessed; however, the VlsE/C6 MTTT resulted in the highest specificity (100%) when other diseases were tested and the lowest sensitivity (75%) for LD samples. Significant differences were present between the results for various MTTTs and STTTs evaluated. Specifically, all MTTTs resulted in higher sensitivities than the STTTs for all LD groups combined and were significantly more accurate (i.e., higher proportion of correct classifications) for this group, with the exception of the WCS/ViraStripe STTT. Additionally, when other diseases were tested, only the results of the VlsE/C6 MTTT differed significantly from those of the WCS/ViraStripe STTT, with the VlsE/C6 MTTT resulting in a 6.2% higher accuracy. Overall, the VlsE/C6 MTTT offers an additional laboratory testing algorithm for LD with equivalent or enhanced performance compared to that of the other MTTTs and STTTs evaluated in this study.

miR-204 mediated loss of Myeloid cell leukemia-1 results in pancreatic cancer cell death.

BACKGROUND: Pancreatic cancer is one of the most lethal human malignancies, with an all-stage 5-year survival of <5%, mainly due to lack of effective available therapies. Cancer cell survival is dependent upon up-regulation of the pro-survival response, mediated by anti-apoptotic proteins such as Mcl-1. RESULTS: Here we show that over-expression of Mcl-1 in pancreatic patient tumor samples is linked to advancement of the disease. We have previously shown that triptolide, a diterpene triepoxide, is effective both in vitro and in vivo, in killing pancreatic cancer cells. Decrease of Mcl-1 levels, either by siRNA or by treatment with triptolide results in cell death. Using pancreatic cancer cell lines, we have shown that miR-204, a putative regulator of Mcl-1, is repressed in cancer cell lines compared to normal cells. Over-expression of miR-204, either by a miR-204 mimic, or by triptolide treatment results in a decrease in Mcl-1 levels, and a subsequent decrease in cell viability. Using luciferase reporter assays, we confirmed the ability of miR-204 to down-regulate Mcl-1 by directly binding to the Mcl-1 3' UTR. Using human xenograft samples treated with Minnelide, a water soluble variant of triptolide, we have shown that miR-204 is up-regulated and Mcl-1 is down-regulated in treated vs. control tumors. CONCLUSION: Triptolide mediated miR-204 increase causes pancreatic cancer cell death via loss of Mcl-1.

Triptolide induces the expression of miR-142-3p: a negative regulator of heat shock protein 70 and pancreatic cancer cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates HSP70, a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. Because microRNAs (miRNA) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here, we show that triptolide as well as quercetin, but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expression of miR-142-3p inhibited cell proliferation, measured by electric cell-substrate impedance sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We showed that miR-142-3p directly binds to the 3'UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR-142-3p-induced cell death. We found that miR-142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water-soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo. This is the first description of an miRNA-mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention.

Minnelide reduces tumor burden in preclinical models of osteosarcoma.

Osteosarcoma is the most common bone cancer in children and adolescents with a 5-year survival rate of about 70%. In this study, we have evaluated the preclinical therapeutic efficacy of the novel synthetic drug, Minnelide, a prodrug of triptolide on osteosarcoma. Triptolide was effective in significantly inducing apoptosis in all osteosarcoma cell lines tested but had no significant effect on the human osteoblast cells. Notably, Minnelide treatment significantly reduced tumor burden and lung metastasis in the orthotopic and lung colonization models. Triptolide/Minnelide effectively downregulated the levels of pro-survival proteins such as heat shock proteins, cMYC, survivin and targets the NF-κB pathway.

MUC1c regulates cell survival in pancreatic cancer by preventing lysosomal permeabilization.

BACKGROUND: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. The cytosolic end of MUC1 (MUC1-c) is extensively involved in a number of signaling pathways. MUC1-c is reported to inhibit apoptosis in a number of cancer cells, but the mechanism of inhibition is unclear. METHOD: Expression of MUC1-c was studied in the pancreatic cancer cell line MIAPaCa-2 at the RNA level by using qRTPCR and at the protein level by Western blotting. MUC1-c expression was inhibited either by siRNA or by a specific peptide inhibitor, GO-201. Effect of MUC1-c inhibition on viability and proliferation and lysosomal permeabilization were studied. Association of MUC1-c with HSP70 was detected by co-immunoprecipitation of MUC1-c and HSP70. Localization of MUC1-c in cellular organelles was monitored by immunofluorescence and with immuno- blotting by MUC1-c antibody after subcellular fractionation. RESULTS: Inhibition of MUC1-c by an inhibitor (GO-201) or siRNA resulted in reduced viability and reduced proliferation of pancreatic cancer cells. Furthermore, GO-201, the peptide inhibitor of MUC1-c, was effective in reducing tumor burden in pancreatic cancer mouse model. MUC1-c was also found to be associated with HSP70 in the cytosol, although a significant amount of MUC1 was also seen to be present in the lysosomes. Inhibition of MUC1 expression or activity showed an enhanced Cathepsin B activity in the cytosol, indicating lysosomal permeabilization. Therefore this study indicates that MUC1-c interacted with HSP70 in the cytosol of pancreatic cancer cells and localized to the lysosomes in these cells. Further, our results showed that MUC1-c protects pancreatic cancer cells from cell death by stabilizing lysosomes and preventing release of Cathepsin B in the cytosol.

Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways.

BACKGROUND & AIMS: Pancreatic adenocarcinoma, among the most lethal human malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. This study investigates the mechanism by which triptolide kills pancreatic cancer cells. METHODS: Cells were treated with triptolide and viability and caspase-3 activity were measured using colorimetric assays. Annexin V, propidium iodide, and acridine orange staining were measured by flow cytometry. Immunofluorescence was used to monitor the localization of cytochrome c and Light Chain 3 (LC3) proteins. Caspase-3, Atg5, and Beclin1 levels were down-regulated by exposing cells to their respective short interfering RNA. RESULTS: We show that triptolide induces apoptosis in MiaPaCa-2, Capan-1, and BxPC-3 cells and induces autophagy in S2-013, S2-VP10, and Hs766T cells. Triptolide-induced autophagy has a pro-death effect, requires autophagy-specific genes, atg5 or beclin1, and is associated with the inactivation of the Protein kinase B (Akt)/mammalian target of Rapamycin/p70S6K pathway and the up-regulation of the Extracellular Signal-Related Kinase (ERK)1/2 pathway. Inhibition of autophagy in S2-013 and S2-VP10 cells results in cell death via the apoptotic pathway whereas inhibition of both autophagy and apoptosis rescues cell death. CONCLUSIONS: This study shows that triptolide kills pancreatic cancer cells by 2 different pathways. It induces caspase-dependent apoptotic death in MiaPaCa-2, Capan-1, and BxPC-3, and induces caspase-independent autophagic death in metastatic cell lines S2-013, S2-VP10, and Hs766T, thereby making it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.