Dietary Lactobacillus plantarum ST-III alleviates the toxic effects of triclosan on zebrafish (Danio rerio) via gut microbiota modulation.

The probiotics, Lactobacillus plantarum ST-III, plays an important role in modulating microbiota and alleviating intestinal metabolic disorders. Herein, we reported that Lactobacillus increases biodiversity of zebrafish gut flora, and attenuates toxic effects from chronic triclosan (TCS) exposure. Lactobacillus-feeding recovered the species and amount of microorganisms in the intestines of zebrafish, and inhibited toxin production by saprophytic bacterial growth. Abnormal physiological indexes and malonaldeyhde content resulting from TCS exposure were effectively alleviated. Additionally, lipid-metabolism disorders, such as increased triglyceride and total cholesterol levels, were attenuated by a probiotics diet. The number of CD4+ T cell lymphocytes in the lamina propria of the duodenal mucosa was decreased in zebrafish receiving a Lactobacillus diet compared to the TCS-exposed group, showing a consistent expression trend for six immune genes (NF-κB, IL-1ß, TNF-α, lysozyme, TLR4α, IL-10) in the intestinal mucosa. Histopathological observations of intestines, spleen and kidney showed that TCS exposure produced severe damage to the morphology and structure of immune and metabolism-related organs. Lactobacillus was capable of mitigating this damage, but bile salt hydrolase, an active extract of Lactobacillus, was not an effective mitigation strategy. The Lactobacillus-induced decrease in the number of inflammatory cells confirmed its role in preventing inflammatory injury. Three behavioral tests (T-maze, bottom dwelling and social interaction) indicated that a probiotics diet improved zebrafish movement and learning/memory capacity, effectively alleviating anxiety behavior due to TCS exposure. These findings inform development of beneficial strategies to alleviate intestinal metabolic syndromes and neurodegenerative diseases resulting from exposure to environmental contaminants through modifying gut flora with a probiotics diet.

Toxicity evaluation of ß-diketone antibiotics on the development of embryo-larval zebrafish (Danio rerio).

This study evaluated the effects of ß-diketone antibiotics (DKAs) on the development of embryo-larval zebrafish (Danio rerio). When exposure to DKAs, developmental malformations, such as hatching delay, curved body axis, pericardial edema, uninflated swim bladder and yolk sac edema, were observed at 120 h postfertilization (hpf). The estimated 120 hpf nominal concentrations of no observed effect concentration and lowest observed effect concentration for DKAs were 18.75 and 37.50 mg/L, respectively, suggesting that DKAs have much lower toxicity than other persistent pollutants. Following DKA exposure, embryonic heart rates were significantly reduced as compared to the controls at 48 and 60 hpf. The peak bending motion frequency appeared 1 h earlier than in control embryos. The 2.34 and 9.38-mg/L treatment groups had a higher basal swim rate than control groups at 120 hpf in both light and light-to-dark photoperiod experiments. The occurrence of high speed swim rates was enhanced approximately threefold to sevenfold in the 2.34 and 9.38 mg/L treatments compared to the control. Glutathione (GSH) concentrations in the 2.34 and 9.38-mg/L treatments were significantly higher than the control at 72 hpf, suggesting that GSH production was induced at the end of the hatching period. When exposed to DKAs, zebrafish superoxide dismutase enzyme (SOD) activities were significantly inhibited in the early embryonic period, demonstrating that the clearing ability in zebrafish was lower than the generation rate of free radicals. In summary, the combined DKAs were developmentally toxic to zebrafish in their early life stages and had the ability to impair individual behaviors that are of great importance in the assessment of their ecological fitness.

An extremophile Microbacterium strain and its protease production under alkaline conditions.

Extremophiles are potential resources for alkaline protease production. In order to search for alkaline protease producers, we isolated and screened alkaliphilic microorganisms from alkaline saline environments. The microorganism HSL10 was identified as a member of the genus Microbacterium by morphological observation, Gram staining and sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region. By colony-forming unit counting under alkali or salt stress, it was further identified as an alkaliphilic microbe with mild halotolerance. In addition, it was capable of secreting alkaline proteases, evidenced by larger hydrolyzation zones in the skim milk-containing medium at pH 9.0 than at pH 7.0. Subsequently, we demonstrated that both NaCl and yeast extract significantly promoted protease production by HSL10. Finally, we established a sensitive colorimetric method for the detection of protease production by HSL10 under neutral and alkaline conditions, by using the Bradford reagent for substrate staining to improve the contrast between the hydrolyzation zone and the substrate background on agar plates. HSL10 was the first example of an alkaliphilic protease-producing member in Microbacterium, and its isolation and characterization have both academic and commercial importance.

Increased levels of circulating oxidized mitochondrial DNA contribute to chronic inflammation in metabolic syndrome, and MitoQ-based antioxidant therapy alleviates this DNA-induced inflammation.

Here, the aim was to investigate the role of circulating oxidized mitochondrial DNA (ox-mtDNA) in metabolic syndrome (MetS)-associated chronic inflammation and evaluate the effect of Mito-Quinone (MitoQ)-based antioxidant therapy on inflammation. A total of 112 MetS patients and 111 healthy control individuals (HCs) were recruited. Peripheral blood was collected, and mononuclear cells (PBMCs) were separated. In a preclinical study, MitoQ, a mitochondrial-targeted antioxidant, was administered to Sprague-Dawley (SD) rats fed a high-fat diet (HFD). In vitro, H2O2- or MitoQ-treated HUVECs served as the oxidative or antioxidative cell models to detect the cell-free ox-mtDNA level. Plasma or cell-free ox-mtDNA levels were measured by qPCR. Additionally, THP-1 cells were incubated with plasma cell-free DNA (cfDNA) from MetS patients and HCs or cell-free ox-mtDNA to detect TLR9-NF-κB pathway activation. Plasma ox-mtDNA levels and TLR9 expression levels in PBMCs were increased in MetS patients. In vivo, HFD-fed rats showed elevated plasma ox-mtDNA and TLR9 expression levels in cardiac-residing immune cells, but MitoQ administration attenuated these increases. In vitro, a significant lower level of cell-free ox-mtDNA was detected in MitoQ-treated cells, compared with H2O2-treated cells. Coincubation of plasma cfDNA from MetS patients or cell-free ox-mtDNA and THP-1 cells increased TLR9-NF-κB p65 expression, and promoted IL-1ß, IL-6 and IL-8 secretion in THP-1 cells. In conclusion, increased circulating ox-mtDNA contributes to chronic inflammation in MetS by activating the TLR9-NF-κB pathway. MitoQ-based antioxidant therapy effectively alleviates inflammation by reducing ox-mtDNA release.

Lactobacillus plantarum-Derived Postbiotics Ameliorate Acute Alcohol-Induced Liver Injury by Protecting Cells from Oxidative Damage, Improving Lipid Metabolism, and Regulating Intestinal Microbiota.

Here, the aim was to evaluate the protective effect of Lactobacillus plantarum-derived postbiotics, i.e., LP-cs, on acute alcoholic liver injury (ALI). After preincubation with LP-cs, HL7702 human hepatocytes were treated with alcohol, and then the cell survival rate was measured. C57BL/6 male mice were presupplemented with or without LP-cs and LP-cs-loaded calcium alginate hydrogel (LP-cs-Gel) for 3 weeks and given 50% alcohol gavage to establish the mouse model of ALI, LP-cs presupplementation, and LP-cs-Gel presupplementation. The histomorphology of the liver and intestines; the levels of serum AST, ALT, lipid, and SOD activity; liver transcriptomics; and the metagenome of intestinal microbiota were detected in all mouse models. In vitro, LP-cs significantly increased the survival rate of alcohol-treated cells. In vivo, presupplementation with LP-cs and LP-cs-Gel restored the levels of serum AST, ALT, and SOD activity, as well as TC and TG, after acute alcohol intake. In the LP-cs-presupplemented mice, the genes involved in fatty acid metabolic processes were upregulated and the genes involved in steroid biosynthesis were downregulated significantly as compared with the ALI mice. LP-cs significantly increased the abundance of intestinal microbiota, especially Akkermansia muciniphila. In conclusion, LP-cs ameliorates ALI by protecting hepatocytes against oxidative damage, thereby, improving lipid metabolism and regulating the intestinal microbiota. The effect of LP-cs-Gel is similar to that of LP-cs.

Lactobacillus plantarum ST-III culture supernatant protects against acute alcohol-induced liver and intestinal injury.

The beneficial effects of probiotics have been studied in inflammatory bowel disease, nonalcoholic steatohepatitis, and alcoholic liver disease (ALD). Probiotic supplements are safer and more effective; however, their potential mechanisms are unclear. An objective of the current study was to examine the effects of extracellular products of Lactobacillus plantarum on acute alcoholic liver injury. Mice on a standard chow diet were supplemented with Lactobacillus plantarum ST-III culture supernatant (LP-cs) for two weeks and administered alcohol at 6 g/kg body weight by gavage. Alcohol-induced liver injury was assessed by measuring plasma alanine aminotransferase activity levels and triglyceride content determined liver steatosis. Intestinal damage and tight junctions were assessed using histochemical staining. LP-cs significantly inhibited alcohol-induced fat accumulation, inflammation, and apoptosis by inhibiting oxidative stress and endoplasmic reticulum stress. LP-cs significantly inhibited alcohol-induced intestinal injury and endotoxemia. These findings suggest that LP-cs alleviates acute alcohol-induced liver damage by inhibiting oxidative stress and endoplasmic reticulum stress via one mechanism and suppressing alcohol-induced increased intestinal permeability and endotoxemia via another mechanism. LP-cs supplements are a novel strategy for ALD prevention and treatment.

[Expression detection of med-ORF12 encoding a stereochemical ketoreductase possibly involved in medermycin biosynthesis].

UNLABELLED: Medermycin, an aromatic polyketide antibiotic produced by streptomyces, possesses a stereochemical-pyran-ring lactone critical for its strong anticancer activity. The med-ORF12 located in the biosynthetic gene cluster of medermycin encodes a stereochemical ketoreductase. Based on many indirect data, we proposed it to be involved in the enantioselective reduction at C-3 in the formation of the pyran ring of medermycin. The direct genetic evidence about the function of med-ORF12 in the medermycin-producing strain has yet to be obtained. Enzymatic features, expression and regulation pattern of Med-ORF12 in the medermycin-producer still remain obscure. OBJECTIVE AND METHODS: The present study aimed to investigate the expression profiles of med-ORF12 and relationship between Med-ORF12 and medermycin accumulation in medermycin-producers using prokaryotic expression, protein purification, polyclonal antiserum preparation, western blot. RESULTS: First, we established a prokaryotic expression system of med-ORF12 using a pET vector and optimized the induction conditions to accumulate the soluble Med-ORF12. Subsequently, we acquired the polyclonal antiserum against Med-ORF12 by immunizing the New Zealand rabbit with the purified protein. Finally, we detected the expression pattern of med-ORF12 in the medermycin producers with the obtained polyclonal antiserum, and found that med-ORF12 could express with a fairly high amount during the late stationary phase of the medermycin-producers, consistent with the accumulation of medermycin as a secondary metabolite. CONCLUSION: These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.

Up-stream mechanisms for up-regulation of miR-125b from triclosan exposure to zebrafish (Danio rerio).

Triclosan (TCS) exposure has widely adverse biological effects such as influencing biological reproduction and endocrine disorders. While some studies have addressed TCS-induced expression changes of miRNAs and their related down-stream target genes, no data are available concerning how TCS impairs miRNA expression leading us to study up-stream regulating mechanisms. Four miRNAs (miR-125b, miR-205, miR-142a and miR-203a) showed differential expression between TCS-exposure treatments and the control group; their functions mainly involved fatty acid synthesis and metabolism. TCS exposure led to the up-regulation of mature miR-125b that was concomitant with consistent changes in pri-mir-125b-1 and pri-mir-125b-3 among its 3 pri-mir-125bs. Up-regulation of miR-125b originated from direct shear processes involving the two up-regulated precursors, but not pri-mir-125b2. Increased expression of pri-mir-125b-1 and pri-mir-125b-3 resulted from nfe2l2- and c/ebpα-integration with positive control elements of promoters for the two precursors. The overexpression of transcriptional factors, nfe2l2 and c/ebpα, initiated the promoter activity for the miR-125b precursor. CpG islands and Nfe2l2 were involved in constitutive expression of mir-125b-1 and mir-125b-3. The activities of two promoter regions, -487 to -1bp for pri-mir-125b1 and -1327 to +14bp for pri-mir-125b-3 having binding sites for NFE2 and Nfe2l2/MAF:NFE2, were higher than other regions, further demonstrating that the transcriptional factor Nfe2l2 was involved in the regulation of pri-mir-125b1 and pri-mir-125b-3. TCS's estrogen activity resulted from its effects on GPER, a novel membrane receptor, rather than the classical ERα and ERß. These results explain, to some extent, the up-stream mechanism for miR-125b up-regulation, and also provide a guidance to future mechanistic study on TCS-exposure.

Immunotoxicity of ß-Diketone Antibiotic Mixtures to Zebrafish (Danio rerio) by Transcriptome Analysis.

Fluoroquinolones and tetracyclines are known as ß-diketone antibiotics (DKAs) because of bearing a diketone group in their molecular structure. DKAs are the most widely used antibiotics to prevent generation of disease in humans and animals and to suppress bacterial growth in aquaculture. In recent years, overuse of DKAs has caused serious environmental risk due to their pseudo-persistence in the environment, even though their half-lives are not long. So far, no reports were concerned with the joint immunotoxicity of DKAs. Herein, we reported on the immunotoxicity of DKAs on zebrafish after a 3-month DKAs exposure using transcriptomic techniques. According to transcriptome sequencing, 10 differentially expressed genes were screened out among the genes related to KEGG pathways with high enrichment. The identified 7 genes showed to be consistent between RNA-seq and qRT-PCR. Due to DKAs exposure, the content or activity for a series of immune-related biomarkers (Complement 3, lysozyme, IgM and AKP) showed the inconsistent changing trends as compared with the control group. Histopathological observations showed that the number of goblet cells increased sharply, the columnar epithelial cells swelled, the nucleus became slender in intestinal villi, and numerous brown metachromatic granules occurred in spleens of DKAs-exposed groups. Overall, both detection of biomarkers and histopathological observation corroborated that chronic DKAs exposure could result in abnormal expression of immune genes and enzymes, and variable levels of damage to immune-related organs. These complex effects of DKAs may lead to zebrafish dysfunction and occurrence of diseases related to the immune system.

The joint effects of room temperature ionic liquids and ordered media on fluorescence characteristics of estrogens in water and methanol.

This study investigated the steady-state and time-resolved fluorescence properties of 17α-ethinylestradiol (EE2) and 17ß-estradiol (E2) in the presence of ordered media (ß-cyclodextrins (ß-CD) and cetyltrimethylammonium bromide (CTAB)). In addition, we analyzed the effects of four room temperature ionic liquids (RTILs) on the fluorescence intensities (FIs) of EE2/ß-CD and E2/ß-CD inclusion complexes in methanol. Both ß-CD and CTAB enhanced the fluorescence of EE2 and E2. The FIs of EE2 and E2 with ß-CD or CTAB in methanol were greater than those in water, possibly resulting from decreased oxygen-quenching in H2O molecules. ß-CD and CTAB may form inclusion complexes with estrogen in both water and methanol. The inclusion ratio of the complex was 1:1 and the inclusion constant (K) values in water were greater than those in methanol. The fluorescence lifetimes were 2.50 and 4.13 ns for EE2 and 2.58 and 4.03 ns for E2 in aqueous solution and methanol, respectively. The changing trend of fluorescence lifetimes for EE2 and E2 in ß-CD or CTAB was similar to the steady-state FIs. The four RTILs had a significant quenching effect on the FIs of EE2/ß-CD and E2/ß-CD, and the quenching process for EE2/ß-CD and E2/ß-CD by RTILs was demonstrated to be a dynamic quenching mechanism. Fluorescent data obtained from these complex systems provide a theoretical foundation for understanding the interaction mechanisms between ordered media and RTILs in the analysis of estrogens.

The clinical diagnostic significance of cerebrospinal fluid D-lactate for bacterial meningitis.

BACKGROUND: To study the clinical and laboratory significance of D­lactate in the diagnosis of bacterial meningitis (BM). METHODS: The levels of D­lactate, L­lactate, IL-6, IL-8, and other biochemical markers were determined in 83 CSF samples from different types of meningitis and the controls. RESULTS: The CSF values of D­lactate, L­lactate, IL-6, IL-8, erythrocytes, leukocytes, and protein were higher in patients with BM than those in the controls and patients with viral meningitis. The levels of D­lactate, L­lactate, IL-6, and erythrocytes in the BM group were higher than those in the tuberculous meningitis group. At the cutoff 12.8 µmol/l, D­lactate showed the diagnostic sensitivity of 94.7%. D­lactate gave the area under the curve (AUC) 0.905, which was higher than those of other markers. Using multiple marker detection, the AUC reached 0.956, which was the highest among all the parameters. Pearson correlation analysis revealed that D­lactate was positively correlated to IL-6 and L­lactate (r=0.727, 0.789 and P=0.000, 0.000, respectively). CONCLUSIONS: THE CSF concentrations of D­lactate are significantly increased in the presence of BM. Measurement of D­lactate provides a rapid diagnosis and differential diagnosis for BM. Combination of D­lactate with other biochemical markers improves the specificity.