Use of FISH-FLOW as a Method for the Identification and Quantification of Bacterial Populations.

The gastrointestinal tract (GIT) harbors the largest group of microbiotas among the microbial communities of the human host. The resident organisms typical of a healthy gut are well adapted to the gastrointestinal environment while alteration of these populations can trigger disorders that may affect the health and well-being of the host. Various investigations have applied different tools to study bacterial communities in the gut and their correlation with gastrointestinal disorders such as inflammatory bowel disease (IBD), obesity, and diabetes. This study proposes fluorescent in situ hybridization, combined with flow cytometry (FISH-FLOW), as an alternative approach for phylum level identification of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria and quantification of target bacteria from the GIT based on analysis of fecal samples, where results are validated by quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (16s rRNA) sequencing. The results obtained via FISH-FLOW experimental approach show high specificity for the developed probes for hybridization with the target bacteria. The study, therefore, suggests the FISH-FLOW as a reliable method for studying bacterial communities in the gut with results correlating well with those of metagenomic investigations of the same fecal samples.

Comprehensive Amelioration of Metabolic Dysfunction through Administration of Lactiplantibacillus plantarum APsulloc 331261 (GTB1™) in High-Fat-Diet-Fed Mice.

The beneficial effects of probiotics for the improvement of metabolic disorders have been studied intensively; however, these effects are evident in a probiotic strain-specific and disease-specific manner. Thus, it is still essential to evaluate the efficacy of each strain against a target disease. Here, we present an anti-obese and anti-diabetic probiotic strain, Lactiplantibacillus plantarum APsulloc331261 (GTB1™), which was isolated from green tea and tested for safety previously. In high-fat-diet-induced obese mice, GTB1™ exerted multiple beneficial effects, including significant reductions in adiposity, glucose intolerance, and dyslipidemia, which were further supported by improvements in levels of circulating hormones and adipokines. Lipid metabolism in adipose tissues was restored through the activation of PPAR/PGC1α signaling by GTB1™ treatment, which was facilitated by intestinal microbiota composition changes and short-chain fatty acid production. Our findings provide evidence to suggest that GTB1™ is a potential candidate for probiotic supplementation for comprehensive improvement in metabolic disorders.

ER Stress-Activated HSF1 Governs Cancer Cell Resistance to USP7 Inhibitor-Based Chemotherapy through the PERK Pathway.

Ubiquitin-specific protease 7 inhibitors (USP7i) are considered a novel class of anticancer drugs. Cancer cells occasionally become insensitive to anticancer drugs, known as chemoresistance, by acquiring multidrug resistance, resulting in poor clinical outcomes in patients with cancer. However, the chemoresistance of cancer cells to USP7i (P22077 and P5091) and mechanisms to overcome it have not yet been investigated. In the present study, we generated human cancer cells with acquired resistance to USP7i-induced cell death. Gene expression profiling showed that heat stress response (HSR)- and unfolded protein response (UPR)-related genes were largely upregulated in USP7i-resistant cancer cells. Biochemical studies showed that USP7i induced the phosphorylation and activation of heat shock transcription factor 1 (HSF1), mediated by the endoplasmic reticulum (ER) stress protein kinase R-like ER kinase (PERK) signaling pathway. Inhibition of HSF1 and PERK significantly sensitized cancer cells to USP7i-induced cytotoxicity. Our study demonstrated that the ER stress-PERK axis is responsible for chemoresistance to USP7i, and inhibiting PERK is a potential strategy for improving the anticancer efficacy of USP7i.

Multifunctional effects of Lactobacillus sakei HEM 224 on the gastrointestinal tract and airway inflammation.

Mucosal tissues serve as the first defense line and their commensal microbiota play a role in sustaining of host health. This study aimed to isolate and evaluate a putative probiotic strain on various mucosal regions. Lactobacillus sakei HEM 224 was isolated from traditional Korean kimchi and identified. In the safety assessment L. sakei HEM 224 showed negative results for hemolysis, biogenic amine production and transferable antibiotic resistance. The probiotic potential of strain HEM 224 in diverse mucosal areas was shown in two different models, viz. a murine model with colitis induced by dextran sulfate sodium (DSS) and an allergic airway inflammation model induced by ovalbumin (OVA). In the colitis model, oral administration of L. sakei HEM 224 improved colitis physiology with immunomodulation, enhancing barrier components and gut microbiota alteration. In the allergic airway inflammation model, the intranasal administration of the strain decreased type 2 inflammation and enhanced epithelial barrier integrity from the airways. These results demonstrate that L. sakei HEM 224 can ameliorate inflammatory conditions in both the gastrointestinal and respiratory tracts through the reinforcement of the epithelial barrier and immunomodulation.

Lactobacillus johnsonii BFE6154 Ameliorates Diet-Induced Hypercholesterolemia.

The functional characteristics of Lactobacillus johnsonii BFE6154, first isolated from Maasai traditional fermented milk, were previously identified in vitro, but its cholesterol-lowering properties have not been verified yet. In this study, we investigated the effect of L. johnsonii BFE6154 on cholesterol regulation and the mode of action. Stimulation of Caco-2 intestinal epithelial cells with L. johnsonii BFE6154 downregulated the gene expression of Niemann-Pick C1-like 1 (NPC1L1) through the activation of liver X receptor (LXR). Also, stimulation of HepG2 cells with the metabolites produced by L. johnsonii BFE6154 revealed an increase in the gene expression of low-density lipoprotein receptor (LDLR). Oral administration of L. johnsonii BFE6154 in mice receiving a high-fat and high-cholesterol diet (HFHCD), reduced total cholesterol and low-density lipoprotein-cholesterol (LDL) and increased high-density lipoprotein-cholesterol (HDL) in the blood, compared to the control. Diet-induced hypercholesterolemic mice receiving L. johnsonii BFE6154 showed a suppression of cholesterol absorption under the control of NPC1L1 in the intestine. Furthermore, L. johnsonii BFE6154 consumption ameliorated the hepatic cholesterol level and LDLR expression, which was reduced by HFHCD. These molecular modulations led to the increase of cholesterol excretion and the decrease of cholesterol levels in the feces and liver, respectively. Taken together, these results suggest that L. johnsonii BFE6154 may protect against diet-induced hypercholesterolemia through the regulation of cholesterol metabolism in the intestine and liver.

Lactiplantibacillusplantarum APsulloc331261 (GTB1™) promotes butyrate production to suppress mucin hypersecretion in a murine allergic airway inflammation model.

Introduction: Allergic airway diseases are one of the serious health problems in worldwide and allergic airway inflammation is a prerequisite led to the exacerbated situation such as mucus hypersecretion, epithelial barrier damage and microbiota dysbiosis. Because of side effects and low efficiencies of current therapeutics, the need for novel alternatives has been urged. Probiotics in which have diverse and beneficial modulatory effects have been applied to the airway inflammation model and the underlying mechanism needs to be investigated. Methods: We aimed to evaluate whether our target strain, Lactiplantibacillus plantarum APsulloc331261 (GTB1TM) isolated from green tea, can ameliorate allergic airway inflammation in mice and to figure out the mechanism. We induced allergic airway inflammation to mice by ovalbumin (OVA) and administered GTB1 orally and the immune and epithelial barrier markers were assessed. The gut metabolite and microbiota were also analysed, and the in vitro cell-line experiment was introduced to confirm the hypothesis of the study. Results: GTB1 ameliorated type 2 inflammation and suppressed mucin hypersecretion with the inhibition of MUC5AC in inflamed mice. Moreover, GTB1 increased the butyrate production and the relative abundance of butyrate producer, Clostridium cluster IV. We assumed that butyrate may have a potential role and investigated the effect of butyrate in mucin regulation via human airway epithelial cell line, A549. Butyrate significantly reduced the gene expression of MUC5AC in A549 cells suggesting its regulatory role in mucus production. Conclusion: Therefore, our study demonstrates that the oral administration of GTB1 can ameliorate allergic airway inflammation and mucin hypersecretion by butyrate production.

Effect of Oral Intake of Lactiplantibacillus plantarum APsulloc 331261 (GTB1TM) on Diarrhea-Predominant Irritable Bowel Syndrome: A Randomized, Double-Blind, Placebo-Controlled Study.

Irritable bowel syndrome (IBS) causes intestinal discomfort, gut dysfunction, and poor quality of life. This randomized, double-blind placebo-controlled trial evaluated the efficacy of Lactiplantibacillus (Lp., formerly Lactobacillus) plantarum APsulloc 331261 (GTB1TM) from green tea leaves in participants with diarrhea-predominant irritable bowel syndrome (IBS-D). Twenty-seven participants meeting the Rome IV diagnostic criteria were randomized for GTB1 or placebo ingestion for four weeks and follow-up for two weeks. The efficacy endpoints included adequate global relief of symptoms, assessment of intestinal discomfort symptom severity and frequency, stool frequency, satisfaction, and fecal microbiome abundance. Of all participants, 94.4% and 62.5% reported global relief of symptoms in the GTB1 and placebo groups, respectively, with significant differences (p = 0.037). GTB1 significantly reduced the severity and frequency of abdominal pain, bloating, and feeling of incomplete evacuation. The frequencies of diarrhea were decreased -45.89% and -26.76% in the GTB1 and placebo groups, respectively (p = 0.045). Hence, GTB1 ingestion improved IBS-D patient quality of life. After four weeks treatment, the relative abundance of Lactobacillus was higher in the GTB1 than in the placebo group (p = 0.010). Our results showed that GTB1 enhanced intestinal discomfort symptoms, defecation consistency, quality of life, beneficial microbiota, and overall intestinal health.

Two putative probiotic strains improve diet-induced hypercholesterolemia through modulating intestinal cholesterol uptake and hepatic cholesterol efflux.

AIMS: Two putative probiotic strains, Lacticaseibacillus (Lc.) rhamnosus BFE5264 and Lactiplantibacillus (Lp.) plantarum NR74, have been shown to suppress cholesterol uptake and promote cholesterol efflux in Caco-2 cells. However, an in vivo beneficial effect of these strains on plasma cholesterol levels has not been verified yet; neither have the underlying mechanisms of regulating cholesterol metabolism clarified thus far. This study has focused on these two aspects. METHODS AND RESULTS: A murine model has been used, and the animals receiving a high-fat/high-cholesterol diet showed elevated plasma cholesterol levels. However, supplementation of Lc. rhamnosus BFE5264 and Lp. plantarum NR74 resulted in the down regulation of Niemann-Pick C1-like 1 (NPC1L1) in the intestine in addition to counteracting the diet-induced suppression of low-density lipoprotein receptor expression in the liver. ATP Binding Cassette Subfamily A Member 1 (ABCA1) was only significantly increased upon administration of Lc. rhamnosus BFE5264. CONCLUSIONS: The present findings demonstrate that supplementation with Lc. rhamnosus BFE5264 and Lp. plantarum NR74 may improve diet-induced hypercholesterolemia by suppression of cholesterol absorption in the small intestine and by supporting the regulation of cholesterol metabolism in the liver. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to understanding the beneficial effects of probiotics on host cholesterol metabolism and underlying mechanisms related to hypercholesterolemia.

Structural specificities of cell surface ß-glucan polysaccharides determine commensal yeast mediated immuno-modulatory activities.

Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, ß-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/ß-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.