Exposure to dechlorane 602 induces perturbation of gut immunity and microbiota in female mice.

The homeostasis of gut immunity and microbiota are associated with the health of the gut. Dechlorane 602 (Dec 602) with food web magnification potential has been detected in daily food. People who were orally exposed to Dec 602 may encounter increased risk of health problems in the gut. In order to reveal the influence of short-term exposure of Dec 602 on gut immunity and microbiota, adult female C57BL/6 mice were administered orally with Dec 602 (low/high doses: 1.0/10.0 µg/kg body weight per day) for 7 days. Lymphocytes were examined by flow cytometry. Gut microbiota was measured by 16S rRNA gene sequencing. Results showed that fecal IgA was upregulated after exposure to the high dose of Dec 602, suggesting that there might be inflammation in the gut. Then, changes of immune cells in mesenteric lymph nodes and colonic lamina propria were examined. We found that exposure to the high dose of Dec 602 decreased the percentages of the anti-inflammatory T regulatory cells in mesenteric lymph nodes. In colonic lamina propria, the production of gut protective cytokine interleukin-22 by CD4+ T cells was decreased, and a decreased trend of interleukin-22 production was also observed in type 3 innate lymphoid cells in the high dose group. Furthermore, an altered microbiota composition toward inflammation in the gut was observed after exposure to Dec 602. Additionally, the altered microbiota correlated with changes of immune parameters, suggesting that there were interactions between influenced microbiota and immune parameters after exposure to Dec 602. Taken together, short-term exposure to Dec 602 induced gut immunity and microbiota perturbations, and this might be the mechanisms for Dec 602 to elicit inflammation in the gut.

Exposure to endosulfan increases endothelial permeability by transcellular and paracellular pathways in relation to cardiovascular diseases.

Exposure to environmental pollutants results in out-of-balance of vascular homeostasis. Endothelial dysfunction leads to a disruption of the endothelial permeability characteristics, associated with cardiovascular diseases. We previously reported that endosulfan could cause endothelial dysfunction, but the role of endosulfan in permeability of endothelial cells has been unexplored. To elucidate molecular mechanism of endosulfan-induced changes in endothelial permeability, human umbilical vein endothelial cells (HUVECs) were exposed to endosulfan, followed by endothelial permeability analysis. The results showed that permeability of HUVECs was enhanced at 48 h after exposure to endosulfan in a dose-dependent manner. Immunofluorescence analysis demonstrated the disruptions of actin cytoskeleton and focal adhesion in endosulfan-exposed cells. Endosulfan activated MMP3/LAMC1/FAK signaling pathway, and downregulated ROCK and PXN in transcellular pathway. Endosulfan affected adherens junctions via E-cadherin and ß-catenin, and impaired gap junctions through downregulation of Cx43 in paracellular pathway. We predicted four closely related human cardiovascular diseases in Nextbio, including shock, coronary arteriosclerosis, disorder of cardiac function and hypertensive disorder in relation to endosulfan exposure. Some genes such as ROCK2 and PXN were predicted to be key genes in these diseases. These findings suggest that endosulfan increased endothelial permeability by paracellular and transcellular pathways, implicating the potential correlation between endosulfan and cardiovascular diseases.

Gene expression profiling to identify the toxicities and potentially relevant disease outcomes due to endosulfan exposure.

Endosulfan, one of the most toxic organochlorine pesticides, belongs to a group of persistent organic pollutants. Gene expression profiling offers a promising approach in health hazard identification of chemicals. The aim of this study was to use gene expression profiling to identify the toxicities and potentially relevant human diseases due to endosulfan exposure. We performed DNA microarray analysis to analyze gene expression profiles in human endothelial cells exposed to 20, 40 and 60 µM endosulfan in combination with an endothelial phenotype. Microarray results showed that endosulfan increased the number of altered genes in a dose-dependent manner, and changed the expression of 161 genes across all treatment groups. qRT-PCR closely matched the microarray data for the genes tested. Significantly enriched biological processes for overlapping down-regulated genes include the neurological system process, signal transduction, and homeostatic process in all the dose groups. These down-regulated genes were associated with cytoskeleton organization and DNA repair at low doses, and involved in cell cycle, apoptosis, p53 pathway and carcinogenesis at high doses. Those up-regulated genes were linked to the inflammatory response and transcriptional misregulation in cancer at higher doses. These findings are consistent with our established endothelial phenotypes. Endosulfan may be relevant to human diseases including liver cancer, prostate cancer and leukemia using the NextBio Human Disease Atlas. These results provide molecular evidence supporting the toxicities and carcinogenic potential of endosulfan in humans.

Dye-functional mesoporous silica material for fluorimetric detection of Cr(III) in aqueous solution and biological imaging in living systems.

A dye-functionalized silica nanomaterial, SBA-RT was prepared by the immobilization of the Rhodamine-based chemosensor R6G-TETA within the channels of SBA-15. SBA-RT exhibits several different properties compared to the free R6G-TETA, such as higher selectivity, blue-shift of the UV-vis spectra due to special spatial environment in the channels of the mesoporous material. It presents Cr(III)-selective fluorimetric and colorimetric responses in aqueous solution. The fluorescence responses are reversible by treating with EDTA and do not vary over a broad pH range suitable for Cr(III) bioimaging application. Through isolating of the metal ions within the mesopores of the silica, SBA-RT can extract Cr(III) from the solution with only trace amounts remaining. The fluorescence images experiment demonstrated the possibility of further application in monitoring Cr(III) in living cells and organisms.

PTHrP regulation and calcium balance in sea bream (Sparus auratus L.) under calcium constraint.

Juvenile gilthead sea bream were exposed to diluted seawater (2.5 per thousand salinity; DSW) for 3 h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30 days. Branchial Ca2+ influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca2+ and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca2+ (R2 = 0.30, N = 39, P < 0.05), and plasma sPTHrP and body mass of the fish (R2 = 0.37, N = 148, P < 0.001) were found. Immunoreactive sPTHrP was demonstrated in pituitary gland pars intermedia cells that border the pars nervosa and co-localises with somatolactin. In the pituitary gland, pthrp, pth1r and casr mRNAs were downregulated after both short- and long-term exposure to DSW. A correlation between pituitary gland pthrp mRNA expression and plasma Ca2+ (R2 = 0.71, N = 7, P < 0.01) was observed. In gill tissue, pthrp and pth1r mRNAs were significantly upregulated after 30 days exposure to DSW, whereas no effect was found for casr mRNA expression. We conclude that in water of low salinity, declining pituitary gland pthrp mRNA expression accompanied by constant plasma sPTHrP levels points to a reduced sPTHrP turnover and that sPTHrP, through paracrine interaction, is involved in the regulation of branchial calcium handling, independently of endocrine pituitary gland sPTHrP.

Influences of enteral nutrition combined with probiotics on gut microflora and barrier function of rats with abdominal infection.

AIM: To investigate the influences of enteral, parenteral nutrition and probiotics delivered by gut on intestinal microecology, epithelial tight junctions, immune and barrier function of rats with abdominal infection. METHODS: Rat abdominal infection models established with cecal ligation and perforation method, were divided into three groups: parenteral nutrition (PN group, n = 7), PN+enteral nutrition (EN group, n = 7) and PN + EN + probiotics (probiotics group, n = 7) via the needle jejunostomy and neck vein for five days. The total nutritional supplement of the three groups was isonitrogenic and isocaloric. Probiotics was delivered by jejunostomy 10 mL/d (1 x 10(8) cfu/mL). The rats were killed on the sixth day. The feces in the cecum were cultured for anaerobic bacterial growth and analyzed with bacterial group DNA fingerprint profile with random amplified polymorphic DNA. The transmembrane binding proteins (occludin) and IgA level in plasma cells of intestine epithelium in colon and terminal ileum were measured by an immunohistochemistry method. The ultrastructure of intestinal epithelial tight junctions in colon and small intestine was observed by electron-microscopy. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocations, and endotoxin in the blood from portal vein was detected. RESULTS: (1) The amount of bacteria of gut species in EN group and probiotic group was higher than that in PN group. The DNA-profiles in EN group and probiotic group were similar to that of normal rats. The number of DNA-profiles in probiotics group was much more than that in PN group and EN group. Moreover, there were strange stripes in PN group. (2) The expression of occludin and IgA in the small and large intestine in EN group (2.309 +/- 0.336, 15.440 +/- 2.383) and probiotic group (2.938 +/- 0.515, 16.230 +/- 3.183) was improved as compared with PN group (1.207 +/- 0.587, P < 0.05, 11.189 +/- 2.108, P < 0.01). The expression of occludin in probiotic group (intestine: 2.93 +/- 0.515; cecum: 3.40 +/- 0.617) was higher than that in EN group (intestine: 2.309 +/- 0.336; cecum: 2.076 +/- 0.670; P < 0.05). The expression of IgA, especially in EN group (intestine: 15.440 +/- 2.383) and probiotic EN group (large intestine: 12.516 +/- 1.542) significantly increased as compared with PN group (intestine: 11.189 +/- 2.108; cecum: 10.160 +/- 1.643; P<0.01). The intestinal epithelial tight junctions and microvilli of the probiotic group were more intact than those in the PN group. (3) The bacterial translocations in blood, liver, lung and mesenteric lymph nodes, and the levels of endotoxin were significantly reduced in probiotic (0.082 +/- 0.029) and EN (0.125 +/- 0.040) groups as compared with PN group (0.403 +/- 0.181, P < 0.05). CONCLUSION: Application of EN combined with probiotics could improve the expression of transmembrane binding proteins (occludin) and IgA, correct the intestinal flora disturbance, maintain gut barrier functions and tight junctions, and reduce the occurrence of gut bacterial translocation.

PTHrP potentiating estradiol-induced vitellogenesis in sea bream (Sparus auratus, L.).

In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E2-naïve and E2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E2 in combination with sbPTHrP a higher vitellogenin production was seen than with E2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E2-naïve liver cells, both in vivo and in vitro.

Stanniocalcin in the euryhaline flounder (Platichthys flesus): primary structure, tissue distribution, and response to altered salinity.

Stanniocalcin (STC) is a homodimeric glycoprotein hormone that was first discovered in fish, where it is largely produced by a unique endocrine gland, the corpuscles of Stannius (CS). In bony fish, it is thought to be an important regulator of calcium and phosphate uptake from the aquatic environment. This report describes the molecular cloning of STC from euryhaline flounder (Platichthys flesus) CS cDNA and genomic DNA. The flounder STC encodes a prehormone of 251 amino acids (aa) with a signal peptide of 17 aa, followed by another 15 aa sequence before the mature protein of 219 aa. The deduced aa sequence of flounder STC shows 62.9-89.0% similarity and 50.4-83.1% identity with other known fish STC sequences, but only 42.3% identity with mouse STC1, 24.4% identity with fugu and zebrafish STC2, and 22.3% identity with mouse STC2. Primary structural analysis demonstrated that flounder STC gene contains five exons in contrast to the four exons present in mammalian STC gene. RT-PCR revealed the expression of flounder STC mRNA to be widely spread in many tissues and organs, similar to the situation in mammals and other fish. Quantitative PCR (Q-PCR) was conducted to measure relative STC expression levels in the CS, which showed STC mRNA expression levels in seawater-adapted fish CS were about 3-fold higher than in freshwater-adapted fish CS.

Polyhydroxyalkanoate biosynthesis in Pseudomonas pseudoalcaligenes YS1.

Pseudomonas pseudoalcaligenes strain YS1 isolated from oil contaminated soil was able to produce polyhydroxybutyrate blended with medium-chain-length polyhydroxyalkanoates (mcl PHA). PHA synthesis genes were cloned from this strain. A fadB (gene for fatty acid degradation) deleted mutant Escherichia coli KM32B (FADB::Tet) was constructed to express the cloned PHA synthesis gene phaC1(Pp) or phaC2(Pp). The fadB deleted mutant KM32B harboring phaC1(Pp) or phaC2(Pp) showed mcl PHA accumulation while the intact E. coli KM32 did not. The results demonstrated that P. pseudoalcaligenes YS1 possessed at least two PHA synthesis pathways; one of them was responsible for production of mcl PHA.

PCR cloning of polyhydroxyalkanoate biosynthesis genes from Burkholderia caryophylli and their functional expression in recombinant Escherichia coli.

The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.