水葫芦根际细菌群落结构多样性分析

【目的】了解水葫芦根际细菌群落结构。【方法】运用末端限制性片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术分析富营养化水体中水葫芦根际和水葫芦近、远水样的细菌群落特征及多样性,结合克隆文库技术和培养法分析根际的细菌种群类型。【结果】同一时期水葫芦根际细菌多样性(Shannon-Weiner指数H′或Simpson指数D)更高,水葫芦近水样次之,远水样最小。10月份的细菌多样性高于5月份的。通过水葫芦根际细菌的克隆文库可知变形杆菌门(Proteobacteria)是水葫芦根际细菌的主要类群,占总群体的65.1%,包括噬菌弧菌(Bacteriovorax sp.)、Dechloromonas sp.、Leptothrix sp.、红螺菌科(Rhodospirillaceae)、Rhodoferax sp.和红环菌科(Rhodocyclaceae)等。T-RFLP图谱显示159 bp为最大优势菌,247 bp为第二大优势菌,对照克隆文库及培养结果分析247 bp属于γ-Proteobacteria,159 bp为不动杆菌(Acinetobacter sp.)。【结论】水葫芦根际细菌的群落结构丰富,不同时段水葫芦根际细菌的丰度略有变化,主要类群为变形杆菌门。     

Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis

IntroductionSystemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.MethodsAdult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30 % post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.ResultsTen subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud’s symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71 %) randomized to abatacept and one out of three patients (33 %) randomized to placebo experienced ≥30 % improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (−8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (−2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate −9.8, 95 % confidence interval −16.7 to −3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (−13.5 ± 3.1 vs. −4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.ConclusionsClinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00442611","term_id":"NCT00442611"}}NCT00442611. Registered 1 March 2007.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0669-3) contains supplementary material, which is available to authorized users.     

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.     

Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and uterus and/or renal cancer. Although the identification of germline mutations in the fumarate hydratase (FH) gene in European families supports it as the susceptibility gene for HLRCC, its role in families in North America has not been studied. We screened for germline mutations in FH in 35 families with cutaneous leiomyomas. Sequence analysis revealed mutations in FH in 31 families (89%). Twenty different mutations in FH were identified, of which 18 were novel. Of these 20 mutations, 2 were insertions, 5 were small deletions that caused frameshifts leading to premature truncation of the protein, and 13 were missense mutations. Eleven unrelated families shared a common mutation: R190H. Eighty-one individuals (47 women and 34 men) had cutaneous leiomyomas. Ninety-eight percent (46/47) of women with cutaneous leiomyomas also had uterine leiomyomas. Eighty-nine percent (41/46) of women with cutaneous and uterine leiomyomas had a total hysterectomy, 44% at age < or =30 years. We identified 13 individuals in 5 families with unilateral and solitary renal tumors. Seven individuals from four families had papillary type II renal cell carcinoma, and another individual from one of these families had collecting duct carcinoma of the kidney. The present study shows that mutations in FH are associated with HLRCC in North America. HLRCC is associated with clinically significant uterine fibroids and aggressive renal tumors. The present study also expands the histologic spectrum of renal tumors and FH mutations associated with HLRCC.     

Effects of macrophage-specific adiponectin expression on lipid metabolism in vivo

Epidemiological studies have associated low circulating levels of the adipokine adiponectin with multiple metabolic disorders, including metabolic syndrome, obesity, insulin resistance, type II diabetes, and cardiovascular disease. Recently, we reported that adiponectin selectively overexpressed in mouse macrophages can improve insulin sensitivity and protect against inflammation and atherosclerosis. To further investigate the role of adiponectin and macrophages on lipid and lipometabolism in vivo, we engineered the expression of adiponectin in mouse macrophages (Ad-TG mice) and examined effects on plasma lipoproteins and on the expression levels of genes involved in lipoprotein metabolism in tissues. Compared with the wild-type (WT) mice, Ad-TG mice exhibited significantly lower levels of plasma total cholesterol (-21%, P < 0.05) due to significantly decreased LDL (-34%, P < 0.05) and VLDL (-32%, P < 0.05) cholesterol concentrations together with a significant increase in HDL cholesterol (+41%, P < 0.05). Further studies investigating potential mechanisms responsible for the change in lipoprotein cholesterol profile revealed that adiponectin-producing macrophages altered expression of key genes in liver tissue, including apoA1, apoB, apoE, the LDL receptor, (P < 0.05), and ATP-binding cassette G1 (P < 0.01). In addition, Ad-TG mice also exhibited higher total and high-molecular-weight adipnection levels in plasma and increased expression of the anti-inflammatory cytokine IL-10 as well as a decrease in the proinflammatory cytokine IL-6 in adipose tissue. These results indicate that macrophages engineered to produce adiponectin can influence in vivo gene expression in adipose tissue in a manner that reduces inflammation and macrophage infiltration and in liver tissue in a manner that alters the circulating lipoprotein profile, resulting in a decrease in VLDL and LDL and an increase in HDL cholesterol. The data support further study addressing the use of genetically manipulated macrophages as a novel therapeutic approach for treatment of cardiometabolic disease.     

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.     

Mutagenicity of azo dyes: structure-activity relationships.

Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.     

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.