FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries.

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Intercellular adhesion molecule-1 gene K469E polymorphism and ischemic stroke: a case-control study in a Chinese population.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) was involved in the pathogenetic mechanisms responsible for ischemic stroke (IS). Population-based sample have revealed gene-gender interaction in blood pressure which is major risk for IS. We sought to evaluate whether ICAM-1 K469E polymorphism was involved in the causation of IS and whether it was different between female and male. METHODS: A 1:1 case-control study was conducted. The K469E polymorphism of ICAM-1 gene were analyzed by polymerase chain reaction (PCR) and restriction enzyme analysis in Chinese patients with IS (n = 309) and elderly subjects without IS (n = 309). RESULTS: ICAM-1 K469E polymorphism was significantly associated with IS. Interestingly, a further analysis stratified by sex found that there was significance between 469E genotypes and IS in female, but not in male. Multiple regression analysis revealed that ICAM-1 K469E polymorphism was still significantly associated with IS, compared with ICAM-1 KK genotype in all population (OR = 1.60, P = 0.030). Stratified by sex, EE combined EK was contributory factor to IS in female (OR = 3.03, P = 0.004), but not in male. After adjustment for confounding factors, the interaction between female and ICAM-1 EK/EE genotypes was found (OR = 3.54, P = 0.001). CONCLUSIONS: It is suggested that the ICAM-1 469E allele may be important in the pathogenesis of ischemic stroke, especially in female but not in male.

Differentially expressed genes between female and male adult Anopheles anthropophagus.

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

Association of platelet-derived growth factor-D gene polymorphism with ischemic stroke in a Chinese case-control study.

Stroke is a multiple genetic disease. Platelet-derived growth factor-D has been found to be involved in the pathogenesis of atherosclerosis, suggesting possible association between platelet-derived growth factor-D and the development of ischemic stroke. However, little information on the relationship between platelet-derived growth factor-D and stroke is currently available. The aim of this study was to investigate the association between platelet-derived growth factor-D genetic variation and the risk of ischemic stroke in a Chinese population. We conducted a case-control study with 309 ischemic stroke patients and 309 sex and age (<5 years)-matched controls. DNA was extracted from the whole blood of each participant. Platelet-derived growth factor-D C/G polymorphism at position +3166 (rs7950273) was detected by TaqMan SNP genotyping assay. Overall, the combined rates of platelet-derived growth factor- D CG and GG are 51% in patients in contrast with 46% in controls. There were no significant differences in the genotype frequencies of platelet-derived growth factor-D +3166 polymorphisms between the patients and controls with history or family history of hypertension or diabetes (P = 0.770). However, among people without history or family history of hypertension or diabetes, platelet-derived growth factor-D CG/GG is significantly more frequently expressed in patients (60%) than in controls (43%) (odds ratio 1.97; 95% confidence interval 1.19-3.26). This significant association holds after adjustment for age, sex, smoking and alcohol intaking (odds ratio 1.86; 95% confidence interval 1.11-3.10) (P = 0.018). Our study found that the G allele of rs7950273 of the platelet-derived growth factor-D gene is associated with higher risk of ischemic stroke in a Chinese population without history or family history of hypertension or diabetes. Future studies with larger and ethnically diverse populations are needed to further evaluate the platelet-derived growth factor-D polymorphism and stroke association, as well as its pathophysiological mechanisms.

[Study on immuno-effect with GRA4 or SAG2 gene recombinant BCG vaccine of Toxoplasma gondii].

OBJECTIVE: To compare the immuno-protection induced by the recombinant BCG vaccine of Toxoplasma gondii GRA4 gene (rBCG-GRA4) and SAG2 gene (rBCG-SAG2) in BALB/c mice. METHODS: 108 SPF BALB/c mice were divided into 6 groups: PBS, BCG, rBCG, rBCG-GRA4, rBCG-SAG2 and rBCG-GRA4+SAG2, each with 18 mice. Each mouse was injected by 100 microl corresponding materials for 2 times. Blood was taken from tail vein before inoculation. 4,6 and 8 weeks after inoculation, spleen was moved and blood was taken from orbit vein of 3 mice from each group for the detection of cytokines, IgG and IgM antibodies, T lymphocyte subgroups and transformation efficiency. 3 weeks after the last inoculation, 9 mice from each group were challenged intraperitoneally with 50 tachyzoites of T. gondii RH strain and their survival time was observed. RESULTS: rBCG vaccine of T. gondii induced immune response. The value of CD3+ CD4+/CD3+CD8+ of group BCG-GRA4+SAG2 was the highest (14.06+/-1.17) in the 4th week; the IgG titer in the BCG-GRA4+SAG2 group was the highest (0.18+/-0.02) in the 6th week and the IgM titer in the BCG-SAG2 group was the highest (0.82+/-0.05) in the 8th week. The average survival time of the mice in BCG-SAG2 group was about 8.61 days after challenged with tachyzoites, and that of the PBS control group, 7.33 days. The average survival time in the 3 immunized groups was one day longer than that of the control. CONCLUSION: The rBCG vaccine of T. gondii shows certain immuno-protection in mice.

[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].

OBJECTIVE: To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. METHODS: The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. RESULTS: The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. CONCLUSION: The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.

[Expression and purification of Toxoplasma gondii GRA4 gene in prokaryotic system].

OBJECTIVE: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity. METHODS: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. RESULTS: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. CONCLUSION: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

[Species identification of freshwater snail Planorbella trivolvis and analysis of its potential distribution].

OBJECTIVE: To identify the species classification of an ornamental Planorbidae from a flower market in Shanghai and analyze its potential distribution in China. METHODS: In August 2013, six freshwater snail specimens were collected from the Wanshang flower market. The species was identified by morphology and molecular biology. An ecological niche model was constructed based on the native geographic presence occurrence data, and projected onto the whole of China to predict the potential distribution. RESULTS: Their shell external morphology suggested that the specimens belonged to Planorbella trivolvis (Say 1817) of Planorbidae, which is native in North America. The sequence data of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) confirmed its identification. A total of 2 294 georeferenced occurrence points in North America were carried out from the Global Biodiversity Information Facility databases and 614 records with coordinates were used to produce a North American native niche model by a maximum entropy method (Maxent). The projection on China results suggested high probabilities of occurrence mostly in Henan Province and its borderland with nearby provinces. CONCLUSIONS: P. trivolvis is similarly with Biomphalaria species from shell morphology. It is the first records of the species in China, and the field dispersal is not clear.

[High efficiency expression and antigenicity analysis of the SAG2 gene from Toxoplasma gondii RH strain].

OBJECTIVE: To make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E. coli and study the antigenicity of the expressed product. METHODS: The SAG2 gene fragment of T. gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E. coli DH5alpha. After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E. coli DH5alpha. The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E. coli BL21 (DE3) and induced for expression. The expressed product was studied by SDS-PAGE and Western blot. RESULTS: 502 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about Mr 19,000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSION: The plasmid pET23a-SAG2 was constructed and a high efficiency expression of the SAG2 gene from T. gondii RH strain was made. The expressed product shows a specific antigenicity.

[Status of Clonorchis sinensis infection and its influencing factors among migrant workers in Baoan District, Shenzhen City].

OBJECTIVE: To understand the status of Clonorchis sinensis infection and its risk factors among migrant workers in Baoan District of Shenzhen City, so as to provide the evidence for the development of control strategies. METHODS: A total of 642 migrant workers were chosen as the investigation samples by the stratified cluster sampling method. Their sera were collected and tested for Clonorchis sinensis infection with ELISA, and a questionnaires survey was performed to collect the information of clonorchiasis sinensis related to knowledge and behaviors. The influencing factors were summarized with the case-control study method. RESULTS: A total of 642 subjects were investigated, in which 530 subjects received the serological examinations and the positive rate was 6.6% (35/530). The significant differences were not found between genders (Chi2 = 1.19, P = 0.28) or among the age groups (Chi2 = 0.45, P = 0.80). The awareness rates of knowing infection route, health hazard and prevention knowledge were 50.16%, 33.64% and 27.41%, respectively. The rates of healthy behaviors such as not eating semi-cooked fish, not feeding pets with raw fish or shrimps, and differentiating between the raw and cooked food when using cutting boards were 80.67%, 78.41% and 45.95%, respectively. The awareness rate of prevention knowledge was positively related to the infection (OR = 0.16, P < 0.01). CONCLUSION: The prevention and control of Clonorchis sinensis infection among migrant workers could not be neglected, and the health education should be strengthened.

Seroprevalence of Angiostrongylus cantonensis infection in humans in China.

A seroepidemiological survey was carried out in China during 2009-2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the "general group"), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the "occupational group") from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the "occupational group" was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the "general group" (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.

[Detection of circulating antigen of Angiostrongylus cantonensis by 12D5 and 21B7 monoclonal antibodies].

OBJECTIVE: To detect the rate of Angiostrongylus cantonensis infection and to study the effects of treatment so as to prepare monoclonal antibodies (McAbs), and gold immunochromatography assay (GICA) with 12D5 and 21B7 McAbs could be prepared in advance. METHODS: Two McAbs (12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA. Either 12D5 or 21B7 McAbs was used as antibody and protein A was conjugated with colloid gold as the detection marker. A special pad for GICA was designed according to the reaction procedure, and CAg were detected by GICA in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively. RESULTS: 12D5 McAb was identified as IgG1 and 21B7 McAb was IgM. Results from Western blotting showed that two McAbs could be used to identified 55 KD protein of adult worms of A. cantonensis. The detection rates of CAg in the sera of infected rats was 100% (48/48) and the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonorchiasis, fasciolopsiasis, ancylostomiasis, ancylostomiasis, anisakiasis as well as schistosomiasis wee seen and normal sera did not react with 12D5 and 21B7 McAbs. CONCLUSION: Results from sandwich ELISA and GICA with 12D5 and 21B7 McAbs showed high specificity and acting as detecting CAg of A. cantonensis in sera of infected animals and patients. We noticed that GICA with 12D5 and 21B7 was not only rapid and simple that without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98 kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

[Study on the epidemiological characteristics and natural infectious focus of Angiostrongylus cantonensis in Shenzhen area of Zhujiang Delta in China].

OBJECTIVE: To delimit the natural infectious focus, including the distribution of wildlife, species, ecology of intermediate hosts and final host of Angiostrongylus cantonensis, as well as the routes of transmission and epidemiological characteristics and wildlife of human Angiostrongylus cantonensis, based on human diverging cases identified in Shenzhen, southern area of China. METHODS: Data including rate of infection and density of Angiostrongylus cantonensis among different hosts in 12 different areas in Shenzhen was collected, using microscope to inspect homogenate liquids of snails. Wild mice were captured with mouse cage to examine the adult Angiostrongylus cantonensis. Using larva isolated from wild-snails-infected rats to observe the life cycle of Angiostrongylus cantonensis. RESULTS: Wild life of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with its majority intermediate hosts as Achatina fulica. The overall rate of infection was 31% in wildlife and final host was found to be Rattus andersoni, Achatina fulica which were extensively distributed in the shrub region of Shenzhen because of suitable climate, humidity and vegetation for generating the life cycle of Achatina fulica. Human infected Angiostrongylus cantonensis was mainly due to eating raw snails or vegetables contaminated by larva of Angiostrongylus cantonensis. The peak of infection was seen from April to November in Shenzhen area. CONCLUSION: Wildlife of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with major wildlife reservoir including fresh water snail and wild mouse. The existence of natural focus Angiostrongylus cantonensis was now recognized as an important source of human angiostrongyliasis in Shenzhen area.

[Epidemiological studies on Clonorchis sinensis infection along the Zhujiang River in Lou village of Shenzhen].

OBJECTIVE: To study the transmission route and epidemiological features of Clonorchis sinensis infection in Shenzhen area--the biggest immigration city of Southern China. METHODS: In this study, we examined 1473 individuals (710 males and 763 females) to determine the current status of C. sinensis infection among the people in one village in Zhujiang river region, Guangdong province, China. Blood samples were detected on antibody of C. sinensis with enzyme linked immunosorbent assay,and stool specimens from sera positive cases were examined by modified Kato-Katz thick smear to confirm the density of infection. People were interviewed on their life styles under the structured questionnaire which was administered by trained staff members. Major content of the questionnaire included eating raw fish, using the same utensils for both raw fish and cooked food, using feces of domestic animals and human feces to feed fish and so on. RESULTS: Among 1473 people examined, 70 (4.75%) were found infected with C. sinensis. By counting eggs per gram feces (EPG), it was found that heavy intensities of infection in males was stronger than that of females,and the overall average EPG was 41.87. Of 1473 interviewees, 54% of them did not know about fluke disease or its transmission route, 12% of those who knew about the fluke but believed that the infection caused no harm or only slight harm to their health. 27% of the interviewees ate raw fish at least 1-2 times per months with 5% of the families using the same utensils for both raw fish and cooked food. 40% of the fish ponds owners fed their fish with the feces of domestic animals and human feces. CONCLUSION: Together with these results, unhealthy behaviors, poor knowledge, inappropriate farming/fishery practices, eating raw fish were important factors influencing the C. sinensis prevalence in humans.

Identification of differentially expressed genes in female Culex pipiens pallens.

Culex pipiens pallens is the mosquito vector of a number of human pathogens such as Wuchereria bancrofti, Brugia malayi, and epidemic encephalitis B virus. Female C. pipiens pallens play an important role in transmitting pathogens by sucking blood, which is essential for reproduction. In the present study, a subtractive cDNA library for female C. pipiens pallens was constructed by the suppression subtractive hybridization (SSH) technique and then 100 clones from the female SSH library were sequenced and analyzed. Female-differentially expressed genes in C. pipiens pallens were screened using semiquantitative RT-PCR. The full-length cDNA of an EST sequence (fs68) that was specifically expressed in female C. pipiens pallens was characterized by 3' and 5' rapid amplification of cDNA ends (RACE). The characteristics of the female-specific gene were further analyzed using bioinformatics and Northern blot. It was shown that the female-specific gene was a previously uncharacterized gene and may encode a salivary peptide. This putative salivary peptide could be a very important molecule in the blood feeding of female C. pipiens pallens.