DNA methylation and mRNA expression of IGF-1 and MMP-2 after form-deprivation myopia in guinea pigs.

PURPOSE: The molecular mechanism of form-deprivation myopia is unclear. This study was aimed to investigate the roles of scleral DNA methylation and mRNA expression of IGF-1 and MMP-2 in a guinea pig model of form-deprivation myopia. METHODS: Seventy 2-week-old male guinea pigs were assigned to three groups: (1) zero week group that was used to collect baseline data; (2) monocular deprivation treatment (MDT) group, in which a thin slice of opaque latex glove was placed over the right eyes of the animals for four weeks, and the left eyes were untreated and served as the monocular contralateral control (MCC) group; (3) control group (CG), in which the animals grew four weeks, but received no manipulation. Animals in each group were evenly divided for DNA methylation assay and quantitative PCR (qPCR). After eye enucleation, the sclerae were harvested for DNA methylation assay and qPCR. The DNA methylation pattern in the promoter and exon regions of IGF-1 and MMP-2, along with the mRNA expression level of them, were determined by base-specific cleavage and mass spectrometry and qPCR, respectively. RESULTS: After four weeks of form-deprivation, DNA methylation at 4/8 cytosine-guanine sites in the IGF-1 promoter was significantly lower in the MDT eyes than in the MCC or CG eyes. In addition, the level of IGF-1 mRNA was moderately higher in MDT eyes compared to the MCC eyes and CG eyes. DNA methylation at 4/14 cytosine-guanine sites in the MMP-2 gene was very low, and no significant change was observed between the MDT eyes and the MCC or CG ones. However, the level of MMP-2 mRNA in MDT eyes was significant higher compared with MCC eyes and CG eyes, with an increase of 217% and 222%, respectively. CONCLUSIONS: In our guinea pig model of form-deprivation myopia, the methylation of four cytosine-guanine sites in the IGF-1 gene promoter was significantly lower in the sclera after four weeks of MDT, and the transcription level of scleral IGF-1 was moderately higher. Hence, the IGF-1 gene methylation might play a role in the pathogenesis of form-deprivation myopia in guinea pigs. The level of MMP-2 mRNA in the sclera of MDT eyes was significantly higher, but not regulated by the methylation pathway, as the methylation status of MMP-2 was unchanged.

Development of high-throughput genotyping method of all 18 HR HPV based on the MALDI-TOF MS platform and compared with the Roche Cobas 4800 HPV assay using clinical specimens.

BACKGROUND: To develop a new 18 high-risk human papillomavirus (HR HPV) detection and genotyping assay, which is important to evaluate the risk degree of HR HPV for causing cancers. METHODS: All 18 HR HPV and ß-globin relative DNA fragments were synthesized and cloned to a plasmid pUC57 to obtain their recombinant plasmids. Based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, each of the 18 HR HPV genotypes were investigated using their constructed recombinant plasmids. The new 18 HR HPV genotyping assay was tested using 356 clinical specimens and the results were compared to ones detected by the Roche Cobas 4800 HPV assay (Cobas). The discrepant results between two assays were resolved by sequencing and genotyping methods. RESULTS: The new 18 HR HPV MALDI-TOF MS genotyping assay was developed using HPV recombination plasmids. The sensitivity was 103 to 102 copies/reaction for the all 18 HR HPV. This new developed HR HPV genotyping test was used to detect the clinical specimens. When the results on clinical samples detected by the new MALDI-TOF MS HPV test were compared with ones detected by the Roche Cobas 4800 HPV assay in terms of 14 HR HPV, the concordance was 80.1% (kappa coefficient, 0.60; 95% confidence interval [CI], 0.52-0.69). The discrepant results were resolved by sequencing and genotyping and suggests that the developed HR HPV assay is more sensitive and specific. CONCLUSIONS: The new developed 18 HR HPV detection method based on MALDI-TOF MS platform is a high-throughput assay for the all 18 HR HPV genotypes and a powerful complement to current detection methods.

Polymorphisms in three genes are associated with hemorrhagic stroke.

BACKGROUND: Multiligand receptor for advanced glycation end products (RAGE), osteoprotegerin, and Golgb1 genes may be implicated in atherosclerosis and vascular diseases. Single nucleotide polymorphisms (SNPs) rs1035798 in RAGE gene, rs2073617 and rs2073618 in TNFRSF11B, and rs3732410 in Golgb1 will be investigated on whether there is an association with hemorrhagic stroke (HS) in Chinese population. METHODS: A total of 600 subjects including 199 HS patients and 401 controls were assayed. These samples were divided into two groups: the ≤50 year and >50 year groups. Genotyping of SNPs was determined using the SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. The association between genotype and HS risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) with multivariate unconditional logistic regression analyses. RESULTS: Our data showed that in the ≤50 year group, the rs1035798 major allele homozygote C/C in RAGE gene was associated with an increased risk of HS, while Golgb1 rs3732410 minor allele homozygote G/G was associated with a decreased risk of HS. In the >50 year group, the major allele homozygote G/G of rs2073618 was found to be associated with an increased risk of HS. CONCLUSIONS: The polymorphisms rs1035798 of RAGE gene, rs2073618 of TNFRSF11B, and rs3732410 of Golgb1 might be involved in the risk of HS at different stage of ages.

Bioremediation of hexavalent chromate using permeabilized Brevibacterium sp. and Stenotrophomonas sp. cells.

Bioremediation has been found to be a useful method for removing hexavalent chromium (Cr(VI)), which is very toxic, from wastewater. Two strains of bacteria that were able to reduce Cr(VI) effectively were isolated from Cr(VI) contaminated soil samples and identified as Brevibacterium sp. K1 and Stenotrophomonas sp. D6, respectively, based on 16S rRNA gene sequence analyses. Brevibacterium sp. K1 and Stenotrophomonas sp. D6 could grow in Luria-Broth medium containing K2Cr2O7 at 1000 and 1600 mg/L, respectively, and they completely reduced the Cr(VI) in LB medium containing K2Cr2O7 at 200 mg/L within 72 h. Further analyses revealed that permeabilized K1 and D6 cells reduced Cr(VI) more effectively than did the resting cells. Triton X-100 was the best permeabilizing agent that was tested. The permeabilized cells of both strains could completely reduce Cr(VI) in industrial wastewater twice before needing to be replenished. The results suggested that these chromate-reducing bacteria are potential candidates for practical use biotreating industrial effluents containing Cr(VI) with Stenotrophomonas sp. D6 being the more effective bacterium.

A novel low temperature PCR assured high-fidelity DNA amplification.

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94-96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'-5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

Comparative evaluations on bio-treatment of hexavalent chromate by resting cells of Pseudochrobactrum sp. and Proteus sp. in wastewater.

Two marine bacterial strains, B5 and H24, were isolated from long-term Cr(VI) contaminated seawater and identified as Pseudochrobactrum and Proteus, respectively, based on 16S rRNA gene sequence analyses. Both strains were examined for their tolerance to Cr(VI) and other metal salts and their abilities to reduce Cr(VI) to trivalent chromium [Cr(III)]. Growing cells of Pseudochrobactrum sp. B5 and Proteus sp. H24 could tolerate Cr(VI) at a concentration of 2000 and 1500 mg/l and completely reduce 1000 mg/l Cr(VI) in LB medium within 96 and 144 h, respectively. Resting cells of the two strains were able to reduce 200mg/l Cr(VI) in Tris-HCl buffer within 16 and 24h, respectively. Furthermore, resting cells of both strains were able to reduce Cr(VI) in industrial wastewaters three times consecutively. Overall, this study provides evidence of the potential for application of chromate-reducing bacteria to direct Cr(VI) decontamination of industrial effluents.

Distinct and effective biotransformation of hexavalent chromium by a novel isolate under aerobic growth followed by facultative anaerobic incubation.

A bacterial isolate (G161) with high Cr(VI)-reducing capacity was isolated from Cr(VI)-contaminated soil and identified as Leucobacter sp. on the basis of 16S rRNA gene sequence analysis. The isolate was a Gram-positive, aerobic rod. The hexavalent chromate-reducing capability of the isolate was investigated under three conditions of oxygen stress. The isolate was found to reduce Cr(VI) under all conditions but performed most effectively during aerobic growth followed by facultative anaerobic incubation. Under these conditions, the isolate tolerated K(2)Cr(2)O(7) concentrations up to 1,000 mg/l and completely reduced 400 mg/l K(2)Cr(2)O(7) within 96 h. The strain reduced Cr(VI) over a wide range of pH (6.0-11.0) and temperatures (15-45 °C) with optimum performance at pH 8.0 and 35 °C. The presence of other metals, such as Ca(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+), induced no effect or else played a stimulatory role on Cr(VI)-reduction activity of the strain. The strain was tested for Cr(VI) removal in wastewaters and proved capable of completely reducing the contained Cr(VI). This is the novel report of a bacterial growth and Cr(VI)-reduction process under sequential aerobic growth and facultative anaerobic conditions. The study suggested that the isolate possesses a distinct capability for Cr(VI) reduction which could be harnessed for the detoxification of chromate-contaminated wastewaters.

HLA-B27 modulates intracellular growth of Salmonella pathogenicity island 2 mutants and production of cytokines in infected monocytic U937 cells.

BACKGROUND: Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells. METHODS/PRINCIPAL FINDINGS: To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis. CONCLUSIONS: The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.

Prevent cervical cancer by screening with reliable human papillomavirus detection and genotyping.

The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for "cervical cancer risk" screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 "true-negative" samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy.

Iron deprivation suppresses hepatocellular carcinoma growth in experimental studies.

PURPOSE: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death, and iron overload is a significant risk factor in the development of HCC. In this study, we investigated the potential application of depriving iron by a novel iron chelator, thiosemicarbazone-24 (TSC24), in HCC treatment. EXPERIMENTAL DESIGN: Two HCC cell lines and HFE knockout (HFE(-/-)) mice were used to determine iron chelation efficiency of TSC24. The anticancer effects of TSC24 on HCC were analyzed in vitro and in athymic xenograft mouse models. RESULTS: Treatment with TSC24 significantly decreased the cellular iron concentration in hepatoma cells and the serum iron concentration in HFE(-/-) mice by blocking iron uptake and interfering with normal regulation of iron levels. Moreover, the viability of HCC cell lines was reduced by TSC24. Confirming the mechanism of the agent, this decrease in viability could be partially rescued by addition of exogenous iron. TSC24 also suppressed tumor growth in athymic mice bearing human HCC xenografts in a concentration-dependent manner, without apparent toxicity in parallel with a decrease in the serum iron level. Further studies revealed that TSC24 efficiently triggered cell-cycle arrest and apoptosis in Hep3B and HepG2 cell lines. CONCLUSIONS: TSC24 is a potent iron chelator that suppresses human HCC tumor growth by disrupting iron homeostasis, reducing available iron, and triggering cell-cycle arrest and apoptosis, without apparent host toxicity at effective doses. Thus, TSC24 shows great potential for the treatment of HCC.

Experimental therapy of ovarian cancer with synthetic makaluvamine analog: in vitro and in vivo anticancer activity and molecular mechanisms of action.

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer.

Microarray analysis of response of Salmonella during infection of HLA-B27- transfected human macrophage-like U937 cells.

BACKGROUND: Human leukocyte antigen (HLA)-B27 is strongly associated with the development of reactive arthritis (ReA) in humans after salmonellosis. Human monocytic U937 cells transfected with HLA-B27 are less able to eliminate intracellular Salmonella enterica serovar Enteritidis than those transfected with control HLA antigens (e.g. HLA-A2). To investigate further the mechanisms by which HLA-B27-transfected cells allow increased replication of these bacteria, a DNA-based microarray was used for comparative genomic analysis of S. Enteritidis grown in HLA-B27- or HLA-A2-transfected cells. The microarray consisted of 5080 oligonucleotides from different serovars of Salmonella including S. Enteritidis PT4-specific genes. Bacterial RNA was isolated from the infected HLA-B27- or HLA-A2-transfected cells, reverse-transcribed to cDNA, and hybridized with the oligonucleotides on the microarrays. Some microarray results were confirmed by RT-PCR. RESULTS: When gene expression was compared between Salmonella grown in HLA-B27 cells and in HLA-A2 cells, 118 of the 4610 S. Enteritidis-related genes differed in expression at 8 h after infection, but no significant difference was detectable at 2 h after infection. These differentially expressed genes are mainly involved in Salmonella virulence, DNA replication, energy conversion and metabolism, and uptake and metabolism of nutrient substances, etc. The difference suggests HLA-B27-dependent modulation of Salmonella gene expression, resulting in increased Salmonella replication in HLA-B27-positive cells. Among the up-regulated genes were those located in Salmonella pathogenicity island (SPI)-2, which play a central role in intracellular survival and replication of Salmonella. CONCLUSIONS: This is the first report to show the regulation of Salmonella gene expression by HLA-B27 during infection of host cells. This regulation probably leads to increased Salmonella survival and replication in HLA-B27-positive cells. SPI-2 genes seem to contribute significantly to the increased replication.

[The expression of gene related to salt tolerance from Sinorhizobium meliloti 042BM in Escherichia coli and purification of its fusion protein].

A 1.9kb DNA fragment related to salt tolerance of S. meliloti strain 042BM containing two open reading frames were obtained by PCR amplification and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named as rstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed, and transformed into E. coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond, and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.