Sirt3 improves monosodium urate crystal-induced inflammation by suppressing Acod1 expression.

BACKGROUND: Previous studies have revealed that Sirt3 deficiency is associated with several inflammatory responses. The purpose of this study is to investigate the role and potential molecular mechanisms of Sirt3 in the inflammation induced by monosodium urate (MSU) crystals. METHODS: The Sirt3 expression level in the peripheral blood mononuclear cells (PBMCs) of patients with gout was measured. Function and molecular mechanism of Sirt3 in MSU crystal-induced inflammation were investigated in bone marrow-derived macrophages (BMDMs), C57BL/6 mouse, and Sirt3-/- mouse. RESULTS: Sirt3 expression was decreased in the PBMCs of patients with gout. Sirt3 agonist (Viniferin) inhibited the acetylation levels of mitochondrial proteins including the SOD2 protein. RNA sequencing, bio-informatics analysis, RT-PCR, and Western blot demonstrated that Sirt3 could suppress the expression of Acod1 (Irg1), which plays an important role in gout. In BMDMs treated with palmitic acid (C16:0) plus MSU crystals, Acod1 knockdown repressed mitochondrial reactive oxygen species (mtROS) over-production, macrophage migration, and mitochondrial fragmentation, and Acod1 improved AMPK activity. The over-expression of Acod1 did not significantly affect the level of itaconic acid, but greatly decreased the levels of some important intermediate metabolites of the tricarboxylic acid (TCA) cycle. These data indicate that Acod1 exerts a pro-inflammatory role in MSU crystal-induced inflammation and is independent of the metabolic level of itaconic acid. Sirt3 deficiency exacerbates inflammatory response induced by MSU crystals in vitro and in vivo. CONCLUSION: The current study has shown that Sirt3 can alleviate the MSU crystal-induced inflammation by inhibiting the expression of Acod1.

[Overexpression of fibroblast growth factor receptor like 1 (FGFRL1) inhibits proliferation and migration of HCT116 human colon cancer cells, and promotes their apoptosis].

Objective To investigate the effect of fibroblast growth factor receptor like 1 (FGFRL1) overexpression on the biological behavior of HCT116 human colon cancer cell line. Methods A recombinant plasmid, named as pcDNA3.1-FGFRL1 which expresses FGFRL1 in mammal cells, was constructed. After a transfection of HCT116 cells with pcDNA3.1-FGFRL1, the stable expression cell line was obtained via continual selection with G418, and FGFRL1 expression was analyzed by real time quantitative PCR and Western blotting. In the following experiment, cells were divided into three groups: the blank group (untreated HCT116 cells), the negative group (empty vector stably transfected cells) and the experience group (pcDNA3.1-FGFRL1 stably transfected cells). Cell proliferation was detected by CCK-8 assay. Cell migration ability was analyzed with TranswellTM assay and their apoptosis was evaluated by flow cytometry. Results FGFRL1 mRNA and protein levels increased significantly in FGFRL1 overexpression group. After the overexpression of FGFRL1, proliferation and migration of HCT116 cells dropped significantly, while their apoptosis increased significantly. Conclusion Overexpression of FGFRL1 inhibits the proliferation and migration of colon cancer HCT116 cells and promotes their apoptosis.

Targeting Tristetraprolin Expression or Functional Activity Regulates Inflammatory Response Induced by MSU Crystals.

The RNA-binding protein tristetraprolin (TTP) is an anti-inflammatory factor that prompts the mRNA decay of target mRNAs and is involved in inflammatory diseases such as rheumatoid arthritis (RA). TTP is regulated by phosphorylation, and protein phosphatase 2A (PP2A) can dephosphorylate TTP to activate its mRNA-degrading function. Some small molecules can enhance PP2A activation. Short interfering RNA (siRNA) targeting TTP expression or PP2A agonist (Arctigenin) was administered to monosodium urate (MSU) crystal-induced J774A.1 cells, and the expression of inflammatory related genes was detected by RT-PCR and Western blot assays. The effects of Arctigenin in mouse models of acute inflammation induced by MSU crystals, including peritonitis and arthritis, were evaluated. The data indicated that TTP expression levels and endogenous PP2A activity were increased in MSU-crystal treated J774A.1 cells. TTP knockdown exacerbated inflammation-related genes expression and NLRP3 inflammasome activation. However, PP2A agonist treatment (Arctigenin) suppressed MSU crystal-induced inflammation in J774A.1 cells. Arctigenin also relieved mitochondrial reactive oxygen species (mtROS) production and improved lysosomal membrane permeability in MSU crystal-treated J774A.1 cells. Moreover, TTP knockdown reversed the anti-inflammatory and antioxidant effects of Arctigenin. Oral administration of Arctigenin significantly alleviated foot pad swelling, the number of inflammatory cells in peritoneal lavage fluids and the production of IL-1ß in the mouse model of inflammation induced by MSU crystals. Collectively, these data imply that targeting TTP expression or functional activity may provide a potential therapeutic strategy for inflammation caused by MSU crystals.

Design and evaluation of a multi-epitope assembly peptide vaccine against Acinetobacter baumannii infection in mice.

AIM: To design a multi-epitope assembly peptide (MEP) of Acinetobacter baumannii and evaluate its immunogenicity and protective immunity in Balb/c mice. METHODS: The T- and B-cell epitopes of outer membrane proteins FilF and NucAb from A. baumannii were predicted and identified by using bioinformatics software and immunological tests. Peptides with predicted high adhesin probability from A. baumannii Ata protein was used as the backbone, two B-cell epitopes and one CD4+ T-cell epitope from FilF were linked to the N-terminal of the backbone, and two B-cell epitopes and one CD4+ T-cell epitope from NucAb were linked to the C-terminal of the backbone to construct the MEP. The gene of the MEP was expressed in E. coli BL21, and its immunogenicity and protective efficacy were evaluated in Balb/c mice. RESULTS: A recombinant protein with a molecular weight of about 37 kDa was successfully purified, and was identified as the recombinant multi-epitope assembly peptide (rMEP) by Western blot analysis. The animal tests showed that the rMEP was highly immunogenic and could induce high levels of IgG antibody and provide potent protection (88.9%) against lethal doses of A. baumannii. CONCLUSIONS: This is the first report of the design and study of a rMEP vaccine against A. baumannii. The results indicate that the rMEP is a promising vaccine candidate for the control of infections caused by A. baumannii.

MF59 adjuvant enhances the immunogenicity and protective immunity of the OmpK/Omp22 fusion protein from Acineterbacter baumannii through intratracheal inoculation in mice.

Acinetobacter baumannii (A baumannii) is an emerging nosocomial pathogenic bacterium which leads to hospital infections. The increase in drug-resistant A baumannii strains makes it difficult to control by using common antibiotics. The development of effective vaccines is an alternative means to avoid A baumannii infections. In the present study, Balb/c mice were inoculated intratracheally with 30 µg of OmpK/Omp22 fusion protein alone or OmpK/Omp22 formulated with MF59 adjuvant. After two times of boosting at day 14 and 21, the antigen-specific antibody levels and the protective immunity against A baumannii challenge were evaluated. The results showed that the OmpK/Omp22 formulated with MF59 immunized mice produced much higher level of antigen-specific antibodies compared to mice immunized with OmpK/Omp22 alone (P < 0.01). Mice immunized with 30 µg of OmpK/Omp22 formulated with MF59 also provided more potent protection post-challenge, which showed lower bacterial loads in the blood and lung tissue, lower level of blood inflammatory cytokines and higher survival rate (83.3%) than mice immunized with OmpK/Omp22 alone (P < 0.001). In conclusion, this study demonstrated that OmpK/Omp22 fusion protein adjuvanted with MF59 induced superior immune response and better protection than OmpK/Omp22 alone through intratracheal inoculation in mice.

Effect of S1P5 on proliferation and migration of human esophageal cancer cells.

AIM: To investigate the sphingosine 1-phosphate (S1P) receptor expression profile in human esophageal cancer cells and the effects of S1P5 on proliferation and migration of human esophageal cancer cells. METHODS: S1P receptor expression profile in human esophageal squamous cell carcinoma cell line Eca109 was detected by semi-quantitative reverse transcription polymerase chain reaction. Eca109 cells were stably transfected with S1P5-EGFP or control-EGFP constructs. The relation between the responses of cell proliferation and migration to S1P and S1P5 expression was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and migration assay, respectively. RESULTS: Both normal human esophageal mucosal epithelium and Eca109 cells expressed S1P1, S1P2, S1P3 and S1P5, respectively. Esophageal mucosal epithelium expressed S1P5 at a higher level than Eca109 cell line. S1P5 over-expressing Eca109 cells displayed spindle cell morphology with elongated and extended filopodia-like projections. The proliferation response of S1P5-transfected Eca109 cells was lower than that of control vector-transfected cells with or without S1P stimulation (P < 0.05 or 0.01). S1P significantly inhibited the migration of S1P5-transfected Eca109 cells (P < 0.001). However, without S1P in transwell lower chamber, the number of migrated S1P5-transfected Eca109 cells was greater than that of control vector-transfected Eca109 cells (P < 0.001). CONCLUSION: S1P binding to S1P5 inhibits the proliferation and migration of S1P5-transfected Eca109 cells. Esophageal cancer cells may down-regulate the expression of S1P5 to escape the inhibitory effect.

Overexpression of S-adenosylhomocysteine hydrolase and down-regulation of prohibitin and c-Myc binding protein in the human esophageal squamous cell line HEEC exposed to N-nitrosomethylbenzylamine.

Numerous studies have shown that N-nitrosamines and their precursors are probable etiological factors for esophageal cancer. Certain N-nitrosamines have been shown to induce esophageal cancer in animal models. However, the molecular mechanisms by which N-nitrosamines promote esophageal carcinogenesis remain poorly understood. In this study, we compared the protein expression profiles of the human esophageal squamous cell line HEEC before and after treatment with various concentrations of N-nitrosomethylbenzylamine (NMBA). There were no marked changes in protein expression in HEEC cells exposed to 2 or 10 µg/ml NMBA. Twenty-eight differentially expressed protein spots were identified in HEEC cells exposed to 50 µg/ml NMBA. Two tumor suppressor proteins, prohibitin and c-Myc binding protein, were found to be down-regulated in NMBA-treated HEEC cells. S-adenosylhomocysteine hydrolase, a regulator of biological methylation, was found to be up-regulated in NMBA-treated HEEC cells. These findings may contribute to the further study of the molecular mechanism by which N-nitrosamines promote esophageal carcinogenesis.

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Enterobacter cloacae from a Teaching Hospital in China.

Of 59 clinical isolates of Enterobacter cloacae from a teaching hospital in Sichuan, China, 18 isolates were shown to be resistant to oxyimino cephalosporins and aztreonam. Enterobacterial repetitive consensus PCR revealed that these isolates comprised 7 distinct genotypes. The presence of plasmids in the 18 clinical isolates was revealed by conjugational transfer of plasmids from E. cloacae to Escherichia coli with the further isolation of the plasmids in the transconjugants. Subsequent nucleotide sequencing and beta-lactamase isoelectric focusing indicated that the plasmids encoded blaSHV, blaCTX-M and/or blaTEM genes, including genes for CTX-M-22 (13 strains), TEM-1 (12 strains), TEM-29 (1 strain), TEM-141 (1 strain), TEM-157 (1 strain), SHV-5 (1 strain), SHV-12 (1 strain), and SHV-70 (1 strain). The widespread presence of extended-spectrum beta-lactamases in E. cloacae isolated from the southwest of China was likely due to the dissemination of resistance plasmids with the predominant genotype of blaCTX-M-22.

Characterization of CTX-M-22 and TEM-141 Encoded by a single plasmid from a clinical isolate of Enterobacter cloacae in China.

We analyzed the resistance to expanded-spectrum cephalosporins of an Enterobacter cloacae clinical isolate, EC002, by transconjugation, isoelectric-focusing analysis, and cloning experiments. It produced two beta-lactamases with isoelectric point values of 5.4 and 8.7, corresponding to TEM-141, a novel variant of TEM-1, and CTX-M-22, encoded by a transferable plasmid.

Down-regulation of Bcl-XL by RNA interference suppresses cell growth and induces apoptosis in human esophageal cancer cells.

AIM: To determine the inhibitory effect of the vector-generated small interfering RNAs (siRNAs) on the expression of the Bcl-X(L) gene in established human esophageal cancer cells, and to investigate the effect of the Bcl-X(L) siRNAs on cell growth and apoptosis in esophageal cancer cells. METHODS: Three siRNA-expressing vectors targeting different sites of the Bcl-X(L) gene were constructed from pTZ-U6+1 vector. Cultured esophageal cancer cells were transfected with the siRNA-expressing vector (or the control vector) using lipofectamine 2000. Bcl-X(L) gene expression was determined with semiquantitative RT-PCR assay and Western blotting. Among the three siRNA-expressing vectors, the most highly functional vector and its effect on cell growth and apoptosis in esophageal cancer cells was further analyzed. RESULTS: Of the three siRNA-expressing vectors, siRNA-expressing vector No.1 was the most potent one which suppressed Bcl-X(L) mRNA production to 32.5% of that in the untreated esophageal cancer cells. Western blotting analysis showed that siRNA-expressing vector No.1 markedly down-regulated the expression of Bcl-X(L) in human esophageal cancer cells. Treatment of esophageal cancer cells with siRNA-expressing vector No.1 resulted in inhibition of cell growth and induction of apoptosis. CONCLUSION: Down-regulation of Bcl-X(L) by vector-generated small interfering RNAs can suppress cell growth and induce apoptosis in human esophageal cancer cells.

[Molecular cloning and expression of the immunodominant protein Ag85A from Mycobacterium tuberculosis H37Rv strain].

OBJECTIVE: To provide the target gene and target antigen for the development of new vaccine against tuberculosis. METHODS: According to the gene sequence encoding protein Ag85A from Mycobacterium tuberculosis H37Rv strain, we designed a pair of oligonucleotide primers, obtained the gene by using polymerase chain reaction, and inserted the gene into the BamH I and EcoR I site of plasmid pBK-CMV to construct recombinant plasmid, and after that, the recombinant plasmid was transferred into E. coli XL1-Blue MRF' and induced with IPTG. The expression product of the gene was analyzed by using SDS-PAGE and western-blotting. RESULTS: The gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain was successfully amplified by using PCR. A recombinant shuttle plasmid was constructed. The recombinant plasmid stably expressed recombinant Ag85A protein relative molecular mass 32 x 10(3) in E. coli XL1-Blue MRF'. CONCLUSION: A recombinant plasmid which contains the gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain has been successfully constructed. The recombinant plasmid can stably express recombinant protein relative molecular mass 32 x 10(3) in E. coli XL1-Blue MRF'. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of new vaccine against tuberculosis.

[Research on preparation of mycobacterium tuberculosis DNA microarray].

OBJECTIVE: To optimize and develop the technique for mycobacterium tuberculosis DNA microarray. METHODS: The process included preparation of DNA samples, spotting and past-spotting treatment of arrays. DNA microarrays were prepared by spotting fluorescence labeled PCR products of target genes onto specially treated glass slides with robotics. The fluorescent signals before and after treatment were scanned with a scanner, and the DNA attachment rate was calculated from the obtained data by software. RESULTS: A foundation for optimizing the conditions of Mycobacterium tuberculosis DNA microarrays has been laid. The support aldehyde-modified glass slide is useful for anchoring DNA at Some distance. DMSO as spotting solution is of benefit to preparation of Mycobacterium tuberculosis DNA microarray. Drying the chip at 37 degrees C temperature after spotting can enhance the DNA combination rate. CONCLUSION: Several key steps of this technique have been optimized. This study has provided a foundation for optimizing the DNA attachment conditions in creating mycobacterium tuberculosis DNA microarray.

[Construction and identification of recombinant shuttle-plasmid with ESAT-6 from Mycobacterium tuberculosis].

OBJECTIVE: To construct a recombinant BCG secretively expressing ESAT-6 of Mycobacterium tuberculosis. METHODS: alpha-antigen(alpha-Ag) signal sequence and esat-6 gene were amplified from the genome of Bacille Calmette-Guerin (BCG) and Mycobacterium tuberculosis by PCR respectively. esat-6 gene was cloned in E. coli-BCG shuttle-plasmid pMV261 to get pME. Then a new recombinant plasmid pSME was constructed by inserting BCG alpha-Ag signal sequence into pME. RESULTS: The cloned genes alpha-Ag signal sequence and esat-6 were correctly inserted into the vector pMV261, which was confirmed by restriction endonuclease digestion and PCR amplification of pSME. CONCLUSION: pSME was expected to secretively express ESAT-6 of Mycobacterium tuberculosis in BCG. This study provides the possibility of further researches on the development of new anti-tuberculosis vaccine.

The construction and expression of recombinant shuttle plasmid with OmpL1 gene from leptospira interrogans serovar Lai strain 017 in Bacille Calmette-Guerin.

OBJECTIVE: To construct recombinant BCG against leptospirosis. METHODS: We amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017. Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E. coli-BCG shuttle plasmids pMV261 and pMV361 respectively. The recombinant plasmids were transformed into BCG by electroporation. The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45 degrees C. RESULTS: The expressed product, a 35kD protein was detected by SDS-PAGE. The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG. CONCLUSION: The technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe, highly effective rBCG bearing leptospiral antigen with long-lasting protection.