Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02–2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

Comparison between ziehl-neelsen staining and fluorescent staining of sputum samples to detect acid fast bacilli in suspected case of pulmonary tuberculosis at tertiary care hospital, Amreli, Gujarat

In India, Tuberculosis (TB) is one of the major community health problems.Pulmonary tuberculosis (PTB) is a respiratory disease. Causative organism for this is acid fast bacilli known as . It is the most ordinary disease affecting the lower socio-economic class in developing countries. Microbiological diagnosis is the heart for the effective treatment of pulmonary TB (PTB). The look forrapid and efficient method has resulted in several staining techniques. Objective of the study was to compare the results of ZN stain (RNTCP) with fluorescent stain by use of microscopy. The study was carried out in Microbiology Department, SMCGH, Amreli. 350 sputum samples (Spot and early morning sample) collected from 175 suspected case of the pulmonary tuberculosis. All 350 samples were processed by ZN stain and Fluorescent stain to detect acid fast bacilli. By use of microscope, the results of the stained smears were given according to RNTCP guideline.Out of 350 sputum smears, 52 (14.85%) and 61 (17.4%) were positive by ZN and FM staining respectively. Males are predominantly affected than females. Majority of the patients were in age above 50 years. Early morning samples were more reliable than spot samples for detection of acid fast bacilli for ZN stain, but not for fluorescent stain.Fluorescent staining with LED microscopy was more efficient than ZN staining for detection of acid fast bacilli from sputum smear.

A Novel In Vitro Simulator to Investigate Promotion of Reconstruction of Damaged Neuronal Cell Colony Differentiated from iPS Cells with the Aid of Micro Dynamic Stimulation

Neuronal cells are equipped with the function of a sensor that senses stimulation and elongates neurites to connect nearby neuronal cells in forming a neuronal network, as they are generally said to be hard to recover from physical damage, such as in the case of a spinal cord injury. Therefore, in this study, a novel in vitro simulator in which micro dynamic stimulations are applied to a damaged neuronal cell colony artificially is proposed to investigate the possibility of promoting the reconstruction of damaged neuronal cells on a colony basis. A neuronal cell colony differentiated from iPS cells is physically damaged by cutting off treatment, and micro dynamic stimulations are applied to the colony by utilizing a developed mini-vibration table system. NeuroFluor NeuO is used to establish a method for fluorescent staining of the living neuronal cells, and morphologies of the reconstructing neurons are analysed, revealing a relationship between the stimulation and the reconstructing process of the damaged neurons. It is found that significant differences are observed in the reconstructing efficiency between the statically cultured damaged neuronal cell colony and the dynamically stimulated one. The results suggest that applying appropriate micro dynamic stimulations is a promising approach to promote the reconstruction of a damaged neuronal cell colony.

Bone Marrow Mesenchymal Stem Cell (BMSC) Exosomes Inhibit Angiogenesis and Enhance Autophagy Through Decreasing miR-21 in Cervical Cancer

We aimed to explore the mechanism underlying the role of miR-21 derived from bone marrow mesenchymal stem cell exosomes (BMSC-exos) in cervical cancer (CC) and the relation between angiogenesis and autophagy. In this study, BMSC-exos were co-cultured with CC stem cells followed by analysis of miR-21 expression by RT-qPCR, autophagy after hunger-induced feeding by Acridine Orange fluorescent staining, angiogenesis by tube formation assay. Co-culture of BMSC-exos effectively reduced miR-21 expression in CC stem cells and enhanced autophagy as demonstrated by upregulated Beclin1 and LC3B with assembly of autophagosome (p < 0.05), but the autophagy restored later. Moreover, in the presence of BMSC-exos, CC stem cell angiogenesis was suppressed by 79%. In conclusion, BMSC-exos enhance autophagy and inhibit angiogenesis in CC through decreasing miR-21, which provides a novel insight into etiology of CC.

A Novel Small Molecule, LCG-N25, Inhibits Oral Streptococcal Biofilm

Dental caries is a chronic oral infectious disease caused by cariogenic biofilm adhered on the tooth surface. Our previous study demonstrated that a repurposed natural compound napabucasin (NAP) showed good antimicrobial activity against oral streptococcal biofilms. The current study designed a novel small molecule, namely LCG-N25, using NAP as a lead compound, and aimed to investigate its potential as an antimicrobial agent in the control of dental caries. LCG-N25 was designed and synthesized with reference to the structure of NAP. The minimal inhibitory concentrations and the minimal bactericidal concentrations of LCG-N25 against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii were evaluated by microdilution method. The antimicrobial activity of LCG-N25 was further evaluated by crystal violet staining, colony forming units counting, biofilm metabolism assay, dead/live fluorescent staining, and scanning electron microscopy. The effect of LCG-N25 on the extracellular polysaccharides of biofilms was determined by both anthrone-sulfuric acid method and fluorescent staining. The microbial composition of streptococcal biofilms after LCG-N25 treatment was further visualized and quantified by fluorescence in situ hybridization. Besides, the cytotoxicity of LCG-N25 was evaluated by Cell Counting Kit-8 assay, and repeated exposure of S. mutans to LCG-N25 treatment was performed to assess if this novel compound could induce drug resistance of this cariogenic bacterium. We found that LCG-N25 exhibited a good antibacterial activity, low-cytotoxicity, and did not induce drug resistance of cariogenic S. mutans. These findings suggest that LCG-N25 may represent a promising antimicrobial agent that can be used as an adjuvant to the management of dental caries.

Ursolic acid induces apoptosis and anoikis in colorectal carcinoma RKO cells

Background Ursolic acid (UA) is an anti-cancer herbal compound. In the present study, we observed the effects of UA on anchorage-dependent and -independent growth of human colorectal cancer (CRC) RKO cells. Methods RKO cells were cultured in conventional and detached condition and treated with UA. Cell viability was evaluated by CCK-8 assay. Cell cycle was analyzed by flow cytometry. Apoptosis was identified by Hoechst 33258 staining and flow cytometry analysis. Activities of caspases were measured by commercial kits. Reactive oxygen species (ROS) was recognized by DCFH-DA fluorescent staining. Anoikis was identified by EthD-1 fluorescent staining and flow cytometry analysis. Expression and phosphorylation of proteins were analyzed by western blot. Results UA inhibited RKO cell viability in both a dose- and time-dependent manner. UA arrested the cell cycle at the G0/G1 phase, and induced caspase-dependent apoptosis. UA inhibited Bcl-2 expression and increased Bax expression. In addition, UA up-regulated the level of ROS that contributed to UA activated caspase-3, − 8 and − 9, and induced apoptosis. Furthermore, UA inhibited cell growth in a detached condition and induced anoikis in RKO cells that was accompanied by dampened phosphorylation of FAK, PI3K and AKT. UA also inhibited epithelial-mesenchymal transition (EMT) as indicated by the down-regulation of N-Cad expression and up-regulation of E-Cad expression. Conclusions UA induced caspase-dependent apoptosis, and FAK/PI3K/AKT singling and EMT related anoikis in RKO cells. UA was an effective anti-cancer compound against both anchorage-dependent and -independent growth of RKO cells.