[Association of methylenetetrahydrofolate reductase gene polymorphisms with polycystic ovary syndrome].

OBJECTIVE: To assess the association of C677T and A1298C polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene with the susceptibility to polycystic ovary syndrome (PCOS). METHODS: Blood samples of 115 PCOS patients and 58 fertile women (for whom PCOS has been excluded) were collected for DNA extraction. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for determining the C677T and A1298C polymorphisms. A database has been set up with Epidata and a significance test was performed with a statistical analysis system. RESULTS: A significant difference has been found in the allele frequencies of MTHFR gene 677 C and T polymorphisms between the two groups (P<0.01), for which T allele has increased the risk for PCOS by 2.06 times. Heterozygous and homozygous genotypes at position 677 (CT and TT) were more common among PCOS cases than controls, with an OR of 3.91 (95%CI: 1.70-8.97) and 4.39 (95%CI: 1.77-10.89), respectively. There was no statistical difference in genotypic distribution of MTHFR gene A1298C polymorphism (P>0.05). In the PCOS group, there was a significant difference with an OR of 6.40 (95%CI: 1.71-23.95) for an increased risk of insulin resistance in homozygous C677T mutations (TT) compared with the wild genotype (CC, P<0.01). The PCOS group and the control group also differed significantly in their red blood cell folate levels (P<0.01), but not in serum vitamin B12 and homocysteine levels (P>0.05). CONCLUSION: MTHFR gene C677T polymorphism is associated with PCOS, for which CT and TT genotypes can increase the risk of PCOS. The TT genotype can also increase the risk of insulin resistance in PCOS patients. The A1298C polymorphism of the MTHFR gene is not associated with the occurrence of PCOS. The folate level in red blood cells of PCOS patients is lower, for whom folate should be supplemented.

Impact of chemiluminescent enzyme immunoassay screening for human parvovirus B19 antigen in Japanese blood donors.

BACKGROUND: To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. STUDY DESIGN AND METHODS: Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA-positive donations. B19V DNA-positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA-positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. RESULTS: The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA-positive donor samples. Of 417 CLEIA-B19V-positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA-positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. CONCLUSION: CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (<4 log IU/mL) in pooled plasma, and thus such screening has further reduced the risk of transfusion-transmitted B19V infection. These results show that CLEIA-B19V screening at the JRC Blood Centers can be an alternative approach to comply with recommendations regarding B19V in the United States and Europe.

Preparation of Amphiphilic Chitosan-Loaded Bentonite Adsorbent and Its Performance in Removing Organic Matter from Coking Wastewater.

An amphiphilic chitosan-loaded bentonite adsorbent (C18CTS-BT) was prepared for the efficient removal of organic matter from coking wastewater. The structure and surface morphology of adsorbents were characterized by FT-IR, XRD, and SEM. The removal of those organics by C18CTS-BT was investigated by comparing the adsorption performances of C18CTS-BT with bentonite (BT) and chitosan-loaded bentonite (CTS-BT). The results showed that compared with BT and CTS-BT, C18CTS-BT showed the performance advantages of having a low dosage, wide pH range, and short adsorption equilibrium time. The optimized treatment process was as follows: the adsorbent dosage was 1.5 g·L-1, the adsorption time was 60 min, and the pH of the system was 7.0. The chemical oxygen demand (COD) of the coking wastewater treated with BT, CTS-BT, and C18CTS-BT decreased from 342 mg·L-1 in the raw water to 264 mg·L-1, 218 mg·L-1, and 146 mg·L-1, corresponding to COD removal rates of 22.81%, 36.26%, and 57.31%, respectively. The results of GC-MS analysis also confirmed that C18CTS-BT could remove most of the organic compounds in coking wastewater, especially long-chain alkanes and their derivatives. The hydrophobic modification of the adsorbent material can effectively improve the removal performance of organic compounds from coking wastewater.

VIS Atlas: A Database of Virus Integration Sites in Human Genome from NGS Data to Explore Integration Patterns.

Integration of oncogenic DNA viruses into the human genome is a key step in most virus-induced carcinogenesis. Here, we constructed a virus integration site (VIS) Atlas database, an extensive collection of integration breakpoints for three most prevalent oncoviruses, human papillomavirus, hepatitis B virus, and Epstein-Barr virus based on the next-generation sequencing (NGS) data, literature, and experimental data. There are 63,179 breakpoints and 47,411 junctional sequences with full annotations deposited in the VIS Atlas database, comprising 47 virus genotypes and 17 disease types. The VIS Atlas database provides (1) a genome browser for NGS breakpoint quality check, visualization of VISs, and the local genomic context; (2) a novel platform to discover integration patterns; and (3) a statistics interface for a comprehensive investigation of genotype-specific integration features. Data collected in the VIS Atlas aid to provide insights into virus pathogenic mechanisms and the development of novel antitumor drugs. The VIS Atlas database is available at https://www.vis-atlas.tech/.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

A linked donor-recipient study to evaluate parvovirus B19 transmission by blood component transfusion.

Parvovirus B19V infection can be a serious infection for hematology patients with underlying hemolysis or compromised erythropoiesis syndromes. Although case reports of B19V transmission by blood component transfusion (as contrasted to manufactured plasma derivatives) are rare, no studies have systematically determined a rate of transmission to recipients transfused with B19V DNA-positive components. We used a linked donor and recipient repository and a sensitive, quantitative B19V DNA polymerase chain reaction (PCR) assay to assess such transmission in B19V-susceptible (ie, anti-B19V immunoglobulin G [IgG] negative) recipients. We assessed 112 B19V DNA-positive components from 105 donors (of 12 529 tested donations) transfused into a population of surgical patients with a pretransfusion B19V IgG seroprevalence of 78%. We found no transmission to 24 susceptible recipients from transfusion of components with B19V DNA at concentrations less than 10(6) IU/mL (upper 95% confidence interval, 11.7%). We found an anamnestic IgG response in one pretransfusion seropositive recipient transfused with a component containing greater than 10(10) IU/mL B19V DNA. These findings show either that transmission from components with less than 10(6) IU/mL does not occur, or, if it does, it is an uncommon event. These data do not support the need to routinely screen blood donations with a sensitive B19V DNA nucleic acid assay.

Boosting oxygen reduction and permeability properties of doped iron-porphyrin membrane cathode in microbial fuel cells.

To achieve a membrane cathode with excellent performance, iron-porphyrin (Fe(por)) was doped to boost the catalytic and permeability properties in microbial fuel cell (MFC). The membrane cathode with the optimal 0.05 g of Fe(por) (denoted as Fe(por)-0.05) had the highest current density of 10.3 A m-2 and the lowest charge transfer resistance of 12.6 ± 0.3 Ω. The ring-disk electrode (RDE) results further proved that the oxygen reduction reaction (ORR) occurred on the Fe(por)-0.05 through a direct four-electron transfer pathway. Moreover, the membrane cathode performed better permeability properties under electric filed and the Fe(por)-0.05 + E (E was electric field) obtained the lowest flux attenuation ratio of 14.1 ± 0.2%, which was related to its superior hydrophilicity and strong electrostatic repulsion force. Iron-porphyrin can simultaneously enhance the ORR activity and permeability of membrane cathode, providing a new direction for the practical application in MFCs.

Enhanced oxygen reduction activity and high-quality effluent of membrane filtration electrodes with Prussian blue in microbial fuel cells.

Membrane filtration electrode based microbial fuel cell provides a promising route to simultaneously recover energy and produce high-quality effluent during water treatment. Enhancing effluent quality and oxygen reduction reaction (ORR) activity of the membrane electrode still remains a major challenge. In this study, filtration types of membrane electrodes with Prussian blue (PB) doping and PVDF-PVC-PEG triblock copolymers were prepared by a simple phase inversion fabrication process. The PB-0.2 membrane electrode with optimal 0.2 wt% of PB obtained the highest current density (12.0 A m-2) and the lowest charge transfer resistance (5.0 ± 0.1 Ω). Rotating disk electrode (RDE) results also demonstrated that the PB-0.2 catalyst exhibited the superior ORR activity with the highest number of transferred electrons (n = 3.90). Furthermore, the MFC with PB-0.2 produced the maximum power density of 1401 ± 17 mW m-2, which was 186.5% higher than that of the control. Moreover, the filtrated effluent tCODeff was 20.6 ± 1.2 mg L-1 for the PB-0.2, which was significantly reduced by 63% compared with the control. These results showed that the addition of PB was an effective strategy to enhance the overall oxygen reduction performance and improve effluent quality of microbial fuel cells.

Parvovirus B19 infection transmitted by transfusion of red blood cells confirmed by molecular analysis of linked donor and recipient samples.

BACKGROUND: Extremely high viremic levels of parvovirus B19 (B19V) can be found in acutely infected, but asymptomatic donors. However, reports of transmission by single-donor blood components are rare. In this prospective study, paired donor-recipient samples were used to investigate the transfusion risk. STUDY DESIGN AND METHODS: Posttransfusion plasma or blood samples from recipients were tested for B19V DNA by polymerase chain reaction, generally at 4 and 8 weeks, and for anti-B19V immunoglobulin (Ig)G by enzyme immunoassay, at 12 and 24 weeks. To rule out infection unrelated to transfusion, pretransfusion samples and linked donor's samples for each B19V DNA-positive recipient were assayed for B19V DNA and anti-B19V IgG and IgM. To confirm transmission, sequencing and phylogenetic analysis were performed. RESULTS: A total of 14 of 869 (1.6%) recipients were B19V DNA positive, but only 1 of 869 (0.12%; 95% confidence interval, 0.0029%-0.6409%) was negative for B19V DNA and anti-B19V IgG before transfusion and seroconverted posttransfusion. This newly infected patient received 5 × 10(10) IU B19V DNA in one red blood cell (RBC) unit from an acutely infected anti-B19V-negative donor in addition to RBCs from three other donors that cumulatively contained 1320 IU of anti-B19V IgG. DNA sequencing and phylogenetic analysis showed that sequences from the linked donor and recipient were identical (Genotype 1), thus establishing transfusion transmission. CONCLUSIONS: The 0.12% transmission rate documented here, although low, could nonetheless result in hundreds or thousands of infections annually in the United States based on calculated confidence limits. Although most would be asymptomatic, some could have severe clinical outcomes, especially in neonates and those with immunocompromised or hemolytic states.

West Nile virus infection in plasma of blood and plasma donors, United States.

This study investigated the association of ongoing West Nile virus (WNV) infections with neutralizing antibody titers in US plasma-derived intravenous immune globulin released during 2003-2008. Titers correlated closely with the prevalence of past WNV infection in blood donors, with 2008 lots indicating a prevalence of 1%.

A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations.

BACKGROUND: Hepatitis A virus (HAV), the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. RESULTS: We developed an antibiotic resistance titration assay (ARTA) based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd) resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 microg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG) preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. CONCLUSION: The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the endpoint dilution ELISA. The ARTA reduced the labour, time, and cost of HAV titrations making it suitable for high throughput screening of sera and antivirals, determination of anti-HAV antibodies in human immune globulin preparations, and research applications that involve the routine evaluation of HAV titers.

[Identification of the gene encoding transglutaminase zymogen from Streptomyces hygroscopicus and its expression in Escherichia coli].

OBJECTIVE: We identified a microbial transglutaminase (MTGase) gene from Streptomyces hygroscopicus; cloned and expressed it in Escherichia coli. We also analyzed the active sites sequence of S. hygroscopicus MTGase through homologous sequence comparison. METHODS: Wild-type microbial transglutaminase zymogen (pro-MTGase) was purified from liquid culture of S. hygroscopicus (CCTCC M203062). N-terminal amino acid sequence of this pro-MTGase was determined. According to the N-terminal sequence and the corresponding nucleotide sequence of MTGase from other three Streptomyces species, PCR primers of S. hygroscopicus pro-MTGase were designed and the completed gene of pro-MTGase was amplified and sequenced. The gene was sub-cloned into pET-20b(+) vector downstream pelB signal peptide to construct the expression vector pET/pro-MTG. RESULTS: The nucleotide sequence showed 92% homologue with that of S. platensis and S. caniferus. Rosetta (DE3) pLysS carrying the expression vector was induced with IPTG at 24 and expressed pro-MTGase as extracellular soluble protein. SDS-PAGE showed the expressed recombinant pro-MTGase was about 44 kDa, similar to the wild-type pro-MTGase purified from S. hgroscopicus. Recombinant pro-MTGase was activated with trypsin and the enzyme activity reached to 0.24U/mL. CONCLUSION: This is the first report of the gene encoding microbial pro-transglutaminase from S. hygroscopicus, and also this is the first report of expression extracellular soluble pro-MTGase in E. coli in our country.

Long noncoding AFAP1-antisense RNA 1 is upregulated and promotes tumorigenesis in gastric cancer.

Long noncoding RNA serves important roles in gastric cancer (GC). However, the prognostic significance and tumorigenesis effect of AFAP1-antisense RNA 1 (AS1) in GC remain to be clarified. The present study was conducted in order to determine the expression level of AFAP1-AS1 by reverse transcription-quantitative polymerase chain reaction. It was demonstrated that AFAP1-AS1 expression level was higher in GC tissues in comparison with adjacent tissues. By analyzing 66 GC tissue specimens, AFAP1-AS1 expression level was found to be markedly associated with tumor size, clinical stage and differentiation. By performing multivariate Cox regression test, AFAP1-AS1 expression level was confirmed to be an independent factor for poor prognosis in patients with GC. Furthermore, SGC-7901 and BGC-823 cells were used for further investigation following transfection of an AFAP1-AS1 short hairpin RNA lentiviral vector. Knockdown of AFAP1-AS1 significantly inhibited GC cell proliferation, migration and invasion abilities in vitro. Finally, nude mice experiments confirmed that downregulation of AFAP1-AS1 in GC cells suppressed tumor growth in vivo. In conclusion, the results of the present study suggested that AFAP1-AS1 may serve as a valuable prognostic indicator and therapeutic target for GC.

Inhibition effect of Peg-IFNα-2b and Imatinib alone or combination on imatinib-resistant gastrointestinal stromal tumors cell lines.

To investigate the inhibition effect of polyethylene glycol interferon α-2b and imatinib alone or combination on imatinib-resistant GIST cell lines, and to explore the possible mechanism. Imatinib resistant GIST cell lines (GIST-R) were exposured to either Peg-IFNα-2b or imatinib alone or combination. The proliferative inhibition rates and the combination index (CI) values of GIST-R cells were detected by MTT assay. The apoptotic rates of GIST-R cells were detected by flow cytometry. The expression levels of phospho-mammalian target of rapamycin (p-mTOR), and Bcl-2 of GIST-R cells were analyzed by Western blot. GIST-R cells presents remarkable resistance to imatinib, and the resistance index (RI) were (P<0.05). And The proliferative inhibition rate and the apoptotic rate of GIST-R cells in combination of Peg-IFNα2b and Imatinib group were higher than those in Peg-IFNα-2b or imatinib alone group (P<0.05). The CI value of Peg-IFNα-2b and imatinib was less than each alone, which had a synergistic effect (CI=0.63). As compared with the control (GIST-R cells without any treatment), the expression levels of p-mTOR and Bcl-2 proteins of GIST-R cells in combination of Peg-IFNα-2b and imatinib group were decreased (P<0.01). The combination of Peg-IFNα-2b and imatinib generats a synergistic effect in GIST-R cells, and reversal of drug resistance. This effect may be related with apoptosis and down-regulation of the expression of p-mTOR.

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay.

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

[Difference in expression of VEGF, bFGF and their receptors between the young and postmenopausal women with breast cancer].

OBJECTIVE: To study the difference in the expression of VEGF, bFGF and their receptors between young and postmenopausal women with breast cancer. METHODS: The expression of VEGF, FLK-1, bFGF and FLG in 40 young and 30 postmenopausal women with breast cancer was studied by immunohistochemical method (SABC), with its relation with axillary lymph node metastasis and the clinical and pathologic characteristics. The expression index between these two groups was compared. RESULTS: The positive axillary lymph node rate and the mean expression of VEGF, bFGF in the young group were higher than postmenopausal group (P < 0.01 and P < 0.05), respectively. The mean expression of VEGF, bFGF, FLK-1 and FLG of axillary lymph node positive patients was higher than the negative ones both in young and postmenopausal women groups (P < 0.05 and P < 0.01). There was also a significant difference in VEGF, bFGF, FLK-1, FLG and MVC between the stage 0 - II and stage III - IV (P < 0.05 and P < 0.01) in both groups. CONCLUSION: Breast cancer angiogenesis, characterized by the high expression of VEGF and bFGF, is directly correlated with the high tumor aggressiveness in the young women.

An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.