[CbCYP716A261, a New ß-Amyrin 28-Hydroxylase Involved in Conyzasaponin Biosynthesis from Conyza blinii].

Conyzasaponins produced by the traditional Chinese herb Conyza blinii are oleanane-type saponins with a wide range of biological activities. Here, we identified a gene, designated CbCYP716A261, encoding a ß-amyrin 28-hydroxylase in conyzasaponins biosynthesis. Ten full putative CYP sequences were isolated from Conyza blinii transcript tags. The CbCYP716A261 gene product was selected as the putative ß-amyrin 28-hydroxylase by phylogenetic analysis and transcriptional activity analysis of methyl jasmonate-treated Conyza blinii. To identify the enzymatic activity, we performed enzymatic activity experiments in vitro and in vivo. The HPLC results revealed that CbCYP716A261 catalyzes the hydroxylation of ß-amyrin at the C-28 position to yield oleanolic acid. Our findings provide new information about the conyzasaponin biosynthesis pathway and widen the list of isolated ß-amyrin 28-hydroxylases.

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris.

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.

[Acceptability of HIV testing using oral quick self-testing kit in men who have sex with men].

Objective: To evaluate the acceptability of oral quick HIV self-testing in men who have sex with men (MSM). Methods: From April 2013 to April 2014, MSM in Beijing and Nanning of China were recruited for an observational study including baseline survey and follow-up, including questionnaire survey, oral HIV self-testing and clinic-based HIV confirmation testing. The sensitivity and specificity of oral quick self-testing were evaluated through comparing the results of oral quick testing with blood testing. The acceptability and associated factors were evaluated by logistic model. Results: A total of 510 MSM were recruited at baseline survey and 279 accepted follow-up. The sensitivity of the oral self-test was 86.00% (43/50) and specificity was 98.23% (445/453) at baseline survey. At baseline survey, 78.63% (401/510) of the MSM showed willingness to use oral quick HIV self-testing. The associated factors included unprotected anal intercourse with a regular male partner in the past 6 months (aOR=0.30, 95%CI: 0.10-1.00) and preference of oral quick HIV self-testing (aOR=7.32, 95%CI: 1.61- 33.31). At baseline survey, 34.51% (176/510) of the MSM reported that oral quick HIV self-testing was the preferred testing method rather than blood testing, which was associated with their birth places-urban area. Conclusion: The acceptability of oral quick HIV self- testing in MSM in the two cities was high.

[Effect of baseline CD(4)(+) T cell count on drop-out of antiretroviral therapy in HIV infected persons in Guangxi Zhuang Autonomous Region, 2008-2015].

Objective: To investigate the effect of baseline CD(4)(+) T cell count (CD(4)) on drop-out of antiretroviral therapy (ART) in HIV infected persons. Methods: Retrospective cohort was conducted in this study. HIV infected persons aged≥18 years and receiving free ART for the first time in Guangxi Zhuang Autonomous Region (Guangxi) from 2008 to 2015 were selected from the antiretroviral treatment database of National Comprehensive HIV/AIDS Information System, with follow-up conducted till May 30, 2016. Cause-specific Cox proportional hazard models were used to evaluate effect of different CD(4) on the drop-out of ART in the HIV infected persons. Results: A total of 58 502 eligible study participants were included in this retrospective cohort study. The average drop-out ratio was 4.8/100 person-years. After controlling the following baseline covariates: age, sex, marital status, route of HIV infection, WHO clinical stage before ART, initial/current ART regiment, ART regiment adjustment, and year of initiating ART for potential confounding, the adjusted HR of drop-out for HIV infected persons with 200- cells/µl, 351-cells/µl and ≥500 cells/µl were 1.110 (95%CI: 1.053-1.171, P<0.001), 1.391 (95%CI: 1.278-1.514, P<0.001) and 1.695 (95%CI: 1.497-1.918, P<0.001), respectively, in risk for drop-out compared with those with baseline CD(4)<200 cells/µl. Among the HIV infected persons, 56.0% (1 601/2 861) of drug withdrawal was due to poor compliance with medication. Conclusions: With the increase of baseline CD(4) when initiating ART, the risk for the drop-out in HIV infected persons increased significantly. To further reduce the drop-out of ART, it is important to take CD(4) into account in initiating ART and to strengthen the health education on treatment compliancy and training for healthcare providers.

[Study of epidemiological characteristics and viral sources of dengue fever outbreak in Guangxi Zhuang Autonomous Region, 2014].

Objective: To understand the epidemiological characteristics and viral sources of dengue fever outbreak in Guangxi Zhuang Autonomous Region (Guangxi) in 2014. Methods: A combined analysis of epidemiological characteristics and genetic characteristics were performed in this study. The time, population and area distributions of the cases were analyzed. Serum samples were collected from dengue fever cases to detect NS1 antigen by using commercial ELISA kits according to the guideline of the manufacture. RT-PCR assay was conducted to detect dengue virus in NS1 positive samples. Phylogenetic tree based on E gene sequence of dengue virus were further analyzed. Results: During September-December 2014, an outbreak of dengue fever caused by dengue virus type 1 and 2 occurred in Guangxi, a total of 854 cases were reported without death, including 712 laboratory confirmed cases and 142 clinical diagnosed cases, in which 79.63% (680/854) occurred during 22 September-21 October 2014. All the cases had typical dengue fever symptoms. Most cases occurred in Nanning and Wuzhou, in which 83.61% (714/854) were in age group 15-59 years; 46.60% (398/854) were staff or people engaged in commercial service. A total 526 serum samples were tested for dengue virus serotype by RT-PCR assay. Among 414 positive samples, 345 were positive for dengue virus type 1 (DENV-1) and 69 were positive for dengue virus type 2 (DENV-2), no DENV-3 and DENV-4 were detected. The results of phylogenetic analysis of E gene sequence indicated that the sequences of 99.12%(113/114) of DENV-1 strains in Nanning in China shared 100.00% homology with the isolate (SG EHI D1/529Y13) from Singapore in 2013, which belonged to the genotype Ⅰ; All the DENV-2 isolates from Wuzhou shared 99.80% homology with the isolate (D14005) from Guangdong province, which belonged to genotype Cosmopolitan. Conclusions: The outbreak was caused by DENV-1 from Singapore and DENV-2 from Guangdong province in China. It is necessary to strengthen the surveillance and early warning for imported dengue fever, conduct vector control and improve the diagnosis of suspected dengue fever cases for the effective control of dengue fever outbreak.

Allelic loss in esophageal squamous cell carcinoma patients with and without family history of upper gastrointestinal tract cancer.

Chromosomal regions with frequent allelic loss may point to major susceptibility genes that will assist in understanding molecular events involved in esophageal carcinogenesis. Esophageal squamous cell carcinoma samples and blood from 46 patients, including 23 patients with and 23 patients without a family history of upper gastrointestinal cancer, were screened using laser microdissected DNA and tested for loss of heterozygosity (LOH) at 18 marker loci representing 14 chromosomal regions (on 2q, 3p, 4p, 4p, 5q, 6q, 8p, 9p, 9q, 11p, 13q, 14q, 15q, and 17p) identified in an earlier genome-wide scan to have frequent LOH. Clinical/pathological and lifestyle risk factor data were also collected. For all 46 tumors combined, the lowest frequency LOH for any of the 18 markers was 37%, and 8 markers showed LOH in > or =75% of informative tumors. One marker (D13S894 on 13q) showed greater LOH in patients with a positive family history (93% versus 50%; P = 0.04), whereas two markers (D6S1027 on 6q and D9S910 on 9q) had significantly more LOH in patients with metastasis, and one marker (D4S2361 on 4p) showed significantly higher LOH in patients with a lower pathological tumor grade. No relation was seen between LOH and lifestyle risk factors. This study confirms the previously observed high frequency LOH for these 14 chromosomal regions, including a locus on 13q where LOH is more common in patients with a family history of upper gastrointestinal cancer than in those without such history, suggesting that a gene in this area may be involved in genetic susceptibility to esophageal cancer.

Frequent inactivation of the TP53 gene in esophageal squamous cell carcinoma from a high-risk population in China.

Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal cancers worldwide, and north central China has some of the highest rates in the world. Previous studies from tumors in this area of China have shown high frequencies of allelic loss on chromosome 17p13-11, which includes the region where the TP53 gene is found. We examined 56 ESCC patients using single-strand conformation polymorphism and DNA sequencing to assess the frequency and spectrum of TP53 mutation and the association between allelic loss at microsatellite marker TP53 and TP53 mutations. Ninety-six % of cases were found to have at least one genetic alteration, including TP53 mutation (77%), allelic loss within the TP53 gene (73%), and/or loss of heterozygosity at the TP53 microsatellite marker (80%); 75% had two or more such alterations, including 59% with both a point mutation and an intragenic allelic loss ("two hits"). The majority of mutations observed were in exon 5, where the most common type of nucleotide substitution was a G:C-->A:T or C:G-->T:A transition, including half that occurred at CpG sites. Allelic loss was most commonly found in exon 4 but was very common in exon 5 as well. Taken together, the multiple genetic alterations of TP53 in this population at high risk for ESCC indicate that there is a very high degree of genetic instability in these tumors, that TP53 is a primary target for inactivation, and that this tumor suppressor gene plays a critical role in the carcinogenesis process for ESCC.

Observations of fragile sites in patients with lymphoma and leukemia.

Chromosomal fragile sites analyses were performed in peripheral lymphocytes of 37 patients with lymphoma and 16 patients with leukemia, and also of 50 healthy individuals as controls. The results were: 1) The rates of chromosomal aberration and frequency of expression of fragile sites in patients with lymphoma and leukemia were significantly higher than those of normal controls. 2) There was a statistical association between 21 of 44 fragile sites and specific cancer breakpoints in patients with lymphoma and this was also the case with 19 of 30 fragile sites and specific cancer breakpoints in patients with leukemia. 3) Concordance between fragile sites and location of oncogenes in the diseases was established. The possible important role of fragile sites in the pathogenesis of lymphoma and leukemia is discussed.

[G-banded chromosome analysis of bone marrow and peripheral blood cells from small cell lung cancer (SCLC) patients].

Detailed G-banded chromosome analysis was carried out on the bone marrow and/or PHA-stimulated peripheral blood cells from 17 SCLC patients (14 males and 3 females), who were diagnosed cytologically or pathologically or both. Twelve of them had no prior treatment and 16 had a heavy smoking history. High chromosome aberration rates were found in the bone marrow (31% for average structural aberration rate and 63% for numerical aberration rate) and peripheral blood cells (37% for average structural aberration rate and 49% for numerical aberration rate). The smoking index, as a whole, was positively correlated to the structural chromosome aberration rate, indicating that smoking is one of the most important environmental factors in causing chromosome aberrations. But some patients gave a high aberration rate dis-proportional to their smoking index, suggesting that genetically determined susceptibility to smoking or even other factors also play an important role. The structural chromosome aberrations in the bone marrow and peripheral blood cells were mainly clustered on chromosome 3 and chromosomes 1, 9 and 11, respectively. The aberration types were manifold and complicated. No consistent or specific aberration as del 3p14-23 for SCLC was found in this study.

High frequency allelic loss on chromosome 17p13.3-p11.1 in esophageal squamous cell carcinomas from a high incidence area in northern China.

Allelic loss on chromosome 17p has been reported frequently in esophageal squamous cell carcinoma (ESCC) and generally encompasses the p53 locus at 17p13.1. However, a good correlation between allelic loss on 17p and mutation of p53 has not been found. This suggests the possibility that unknown tumor suppressor genes near p53 may be involved in the development of ESCC. To evaluate this possibility, we analyzed 30 microsatellite markers covering the entire short arm of chromosome 17 in 56 ESCC patients from a high risk population in northern China, including 34 with a family history of upper gastrointestinal (UGI) cancer and 22 without a family history of any cancer. Cancer lifestyle risk factors and clinical/pathological characteristics were also collected. We found frequent allelic loss (>/=65%) at 28 of the 30 markers evaluated in these ESCC patients. The highest frequencies of allelic loss (> or =80%) were found in three smaller regions: deletion region I located at 17p13.3-p13.2 (between D17S849 and D17S1828); deletion region II located at 17p13.2-p13.1 (between D13S938 and TP53); deletion region III located at 17p13.1-p12 (between D17S804 and D17S799). A number of genes have already been identified in these deleted regions, including: OVCA1, OVCA2 and HIC-1 in deletion region I; p53 in deletion region II; ZNF18, ZNF29, ALDH3 and ALDH10 in deletion region III. These results will help us direct future testing of candidate genes and narrow the search region for major new tumor suppressor genes that may play a role in the pathogenesis of ESCC.

Allelic loss on chromosome bands 13q11-q13 in esophageal squamous cell carcinoma.

Allelic loss on chromosome 13 occurs frequently in esophageal squamous cell carcinoma. However, studies of the two known tumor suppressor genes located on 13q, RB1 and BRCA2, have shown few mutations, suggesting that other genes are likely to be involved in the development of this tumor type. To identify a minimal deletion interval, we first analyzed 42 microsatellite markers spanning chromosome bands 13q11-q13 in 56 esophageal squamous cell carcinoma patients, including 34 with a family history of upper gastrointestinal cancer and 22 without a family history of cancer. Lifestyle risk factors and clinical/pathologic characteristics were also collected. Two commonly deleted regions were identified: one was located on band 13q12.11, between markers D13S787 and D13S221; the other was located on bands 13q12.3-q13.1 from markers D13S267 to D13S219. We observed higher allelic loss frequencies for eight of the microsatellite markers in those patients with a family history of upper gastrointestinal cancer compared to patients without such a history. This study suggests that one or more unidentified tumor suppressor genes are located on chromosome arm 13q that play a role in the development of esophageal squamous cell carcinoma.

Identification of novel regions of allelic loss from a genomewide scan of esophageal squamous-cell carcinoma in a high-risk Chinese population.

Esophageal cancer is one of the most common fatal cancers worldwide. Deletions of genomic regions are thought to be important in esophageal carcinogenesis. We conducted a genomewide scan for regions of allelic loss using microdissected DNA from 11 esophageal squamous-cell carcinoma patients with a family history of upper gastrointestinal tract cancer from a high-risk region in north central China. Allelic patterns of 366 fluorescently labeled microsatellite markers distributed at 10-cM intervals over the 22 autosomal chromosomes were examined. We identified 14 regions with very high frequency (>/= 75%) loss of heterozygosity (LOH), including broad regions encompassing whole chromosome arms (on 3p, 5q, 9p, 9q, and 13q), regions of intermediate size (on 2q, 4p, 11p, and 15q), and more discrete regions identified by very high frequency LOH for a single marker (on 4q, 6q, 8p, 14q, and 17p). Among these 14 regions were 7 not previously described in esophageal squamous-cell carcinoma as having very high frequency LOH (on 2q, 4p, 4q, 6q, 8p, 14q, and 15q). The very high frequency LOH regions identified here may point to major susceptibility genes, including potential tumor suppressor genes and inherited gene loci, which will assist in understanding the molecular events involved in esophageal carcinogenesis and may help in the development of markers for genetic susceptibility testing and screening for the early detection of this cancer. Genes Chromosomes Cancer 27:217-228, 2000. Published 2000 Wiley-Liss, Inc.