α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells.

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 µmol/L) or pretreated with a p38 inhibitor (50 µmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 µmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 µmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 µmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression significantly increased (P < 0.05) in the 40 µmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 µmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 µmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 µmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). CONCLUSION: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.

RhoA improves cryopreservation of rooster sperm through the Rho/RhoA-associated kinase/cofilin pathway.

Cryopreservation of rooster sperm leads to relatively low semen quality due to cytoskeletal damage during the freeze-thawing process. This study aimed to explore how the addition of RhoA recombinant protein affected the viability and subcellular structure of rooster sperm after freeze-thawing and elucidated the molecular mechanisms of sperm cryopreservation. Semen quality and acrosome integrity testing revealed that the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotectant fluid significantly increased sperm motility, survival rate, linearity, straight-line velocity, and acrosome integrity after freeze-thawing (P < 0.05). Ultrastructure analysis of cryopreserved sperm showed structural damage to the sperm plasma membrane, nuclear membrane, and tail. However, compared to the control, these structural changes were reduced upon the addition of RhoA recombinant protein to the cryoprotective fluid (P < 0.05). Western blotting revealed that the expression of Rho/RhoA-associated kinase and p-cofilin was increased, and cofilin expression was decreased after sperm cryopreservation with recombinant RhoA protein. Treatment with Y-27632, a ROCK antagonist, suppressed ROCK and p-cofilin expression and decreased semen quality, acrosome integrity, and ultrastructure integrity. In summary, we have demonstrated a cryoprotective effect in spermatozoa involving the Rho/ROCK pathway during freeze-thawing. Furthermore, the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotective fluid improved rooster semen quality and subcellular structural homeostasis after freeze-thawing via the Rho/ROCK pathway. This pathway may regulate the dynamic reorganization of the actin cytoskeleton by regulating the cofilin phosphorylation.

Molecular epidemiology of hepatitis B virus genotypes and subgenotypes in ethnic minority populations, Yunnan province, China.

The aim of our study was to determine the distribution of hepatitis B virus (HBV) genotypes and subgenotypes in ethnic minorities in Yunnan province to provide evidence supporting the theoretical basis for hepatitis B prevention and control. We obtained serum samples and demographic data from 765 individuals reported by Yunnan province who had either acute or chronic HBV infection and were from one of 20 ethnic minority populations: Achang, Bai, Brown, Tibetan, Dai, Deang, Dulong, Hani, Hui, Jingpo, Lahu, Yi, Lisu Miao, Naxi, Nu, Pumi, Wa, Yao, or Zhuang people. We sequenced the HBV DNA and determined the genotypes and subgenotypes of the isolated HBVs. We mapped the genotype and subgenotype distribution by ethnic minority population and conducted descriptive analyses. There were four genotypes among the 20 ethnic groups: genotype B (21.3% of samples), C (76.6%), D (1.8%) and I (0.3%). The most common subgenotype was C1. There were no genotype differences by gender (P = 0.954) or age (P = 0.274), but there were differences by region (P < 0.001). There were differences in genotype distribution (P < 0.001) and subgenotype distribution (P = 0.011) by ethnic group. Genotype D was most prominent in Tibet and most HBV isolates were C/D recombinant viruses. The only two genotype I virus isolates were in Zhuang people. Susceptibility and geographic patterns may influence HBV prevalence in different ethnic populations, but additional research is needed for such a determination.

Reduction patterns of Japanese encephalitis incidence following vaccine introduction into long-term expanded program on immunization in Yunnan Province, China.

BACKGROUND: Japanese encephalitis (JE) is a leading cause of childhood viral encephalitis both at global level and in China. Vaccination is recommended as a key strategy to control JE. In China most JE cases have been reported in southwest provinces, which include Yunnan. In this study, we quantify the epidemiological shift of JE in Yunnan Province from 2005 to 2017, covering before and after the introduction of JE vaccination into routine Expanded Program on Immunization (EPI) in 2007. METHODS: We used routinely collected data in the case-based JE surveillance system from 2005 through 2017 in Yunnan. Cases were reported from hospital and county-level Centers for Disease Control in line with the National JE Surveillance Guideline. Epidemiological data were extracted, analysed and presented in appropriate ways. Immunization coverage was estimated from actual JE doses administered and new births for each year. RESULTS: A total 4780 JE cases (3077 laboratory-confirmed, 1266 clinical and 437 suspected) were reported in the study period. Incidence of JE (per 100 000 population) increased from 0.95 in 2005 to 1.69 in 2007. With increase in vaccination coverage, incidence rates decreased steadily from 1.16 in 2009 to 0.17 in 2017. However, seasonality remained similar across the years, peaking in June-September. Banna (bordering Myanmar and Laos), Dehong (bordering Myanmar), and Zhaotong (an inland prefecture) had the highest incidence rates of 2.3, 1.9, and 1.6, respectively. 97% of all cases were among local residents. As vaccination coverage increased (and incidence decreased), proportion of JE cases among children < 10 years old decreased from 70% in 2005 to 32% in 2017, while that among adults ≥20 years old increased from 12 to 48%. There were a large number of JE cases with unknown treatment outcomes, especially in the earlier years of the surveillance system. CONCLUSIONS: The 13-year JE surveillance data in Yunnan Province showed dramatic decrease of total incidence and a shift from children to adults. Improving vaccination coverage, including access to adults at risk, and strengthening the JE surveillance system is needed to further control or eliminate JE in the province.

[Molecular epidemiological analysis of ECHO7 virus isolated from sewage water in Yunnan Province, China].

To investigate the epidemic and evolutionary trends of enterovirus (EV) in the external environment of Yunnan Province, China, molecular typing was performed on 4 EV strains that were isolated from environmental sewage in Yunnan. The VP1 region of isolates was amplified by RT-PCR using universal enterovirus primers, and the amplified VP1 region was sequenced for GenBank BLAST search and genotype analysis. The 4 EV strains were identified as ECHO7. Their nucleotide and amino acid homologies with the VP1 sequences of 68 ECHO7 strains retrieved from GenBank were measured by Mega software analysis. Our findings showed that ECHO7 strains from environmental sewage and population samples were in different evolutionary branches. These strains showed typical geographical and temporal differences; In addition, there were different transmission chains at the same time and in the same area. ECHO7 strains isolated from sewage water and patients with acute flaccid paralysis during the same period in Yunnan belonged to different clusters and evolved at different speeds. Special concerns are needed for this problem. Continuous molecular biological surveillance of human EV in the external environment of Yunnan will provide strong support for early warning of EV diseases.

[Study on the molecular typing and epidemiology of non-polio enterovirus in Yunnan Province, China during 2006-2010].

In order to explore the genotype distribution and molecular evolution of non-polio enterovirus (NPEVs)in Yunnan Province,the People's Republic of China, we sequenced and analyzed the partial VP1 coding region of 105 NPEVs isolated from acute flaccid paralysis (AFP) surveillance in Yunnan province during a 5- year study period from 2006 to 2010. The viral genomes of 105 NPEVs were translated to corresponding amino acid sequences and compared with those of the prototype strains, and the phylogenetic tree was constructed among these VP1 nucleotide sequences and other prototype strains from GenBank. Analysis showed that 18 isolates were classified into 7 serotypes of human enterovirus A species, while 77 isolates into 22 serotypes of B and 10 isolates into 4 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under AFP surveillance, human enterovirus B species accounted for 73. 3% of the 105 isolates and was considered as the predominant one,followed by human enterovirus A(17. 1%) and human enterovirus C(9. 5%). Phylogenetic analysis showed that various serotypes of the virus and the corresponding prototype strains or other representative strains clustered into the same grooup, however, Yunnan strains and prototype strains were located in the different branches (except CA2,EV90 and EV76). The degree of variation was different even among the same genotype strains. This report showed that different genotype strains spread widely in Yunnan Province.

Gene chip array for differentiation of mycobacterial species and detection of drug resistance.

BACKGROUND: Gene chip array can differentiate isolated mycobacterial strains using various mycobacterium specific probes simultaneously. Gene chip array can evaluate drug resistance to isoniazid and rifampin of tuberculosis strains by detecting drug resistance related gene mutation. This technique has great potential for clinical application. We performed a retrospective study to investigate the capability of gene chip array in the rapid differentiation of species and detection of drug resistance in mycobacterium, and to evaluate its clinical efficacy. METHODS: We selected 39 patients (54 clinical mycobacterium isolates), used gene chip array to identify the species of these isolates and detect drug resistance to isoniazid and rifampin in Mycobacterium tuberculosis isolates. Meanwhile, these patients' clinical data were analyzed retrospectively. RESULTS: Among these 39 patients whose mycobacterium culture were positive, 32 patients' isolates were identified as Mycobacterium tuberculosis, all of them were clinical infection. Seven patients' isolates were identified as non-tuberculosis mycobacterium. Analyzed with their clinical data, only two patients were considered as clinical infection, both of them were diagnosed as hematogenous disseminated Mycobacterium introcellulare infection. The other five patients' isolates were of no clinical significance; their clinical samples were all respiratory specimens. Clinical manifestations of tuberculosis and non-tuberculous mycobacterial infections were similar. Isoniazid resistance was detected in two tuberculosis patients, while rifampin resistance was detected in one tuberculosis patient; there was another patient whose Mycobacterium tuberculosis isolate was resistant to both isoniazid and rifampin (belongs to multidrug resistance tuberculosis). The fact that this patient did not respond to routine anti-tuberculosis chemotherapy also confirmed this result. CONCLUSIONS: Gene chip array may be a simple, rapid, and reliable method for the identification of most mycobacterial species and detection of drug resistance in Mycobacterium tuberculosis. It is useful in diagnosis, treatment, and hospital infection control of mycobacterial infections, and it may have a great potential for clinical application.

[Molecular typing of enteroviruses from healthy children in the border areas of Yunnan Province and Myanmar and the genetic characteristics of ECHO7 and ECHO13 in 2009].

To explore the enteroviruses surveillance among healthy children under 15 years old in the border areas of Yunnan Province and Myanmar in 2009. The stool samples were collected from the healthy children under 15 years old who came from the border areas of Myanmar and Yunnan Province, virus isolation and sequencing were conducted for all the 271 samples. 6 strains of polioviruses (PVs) were detected from 271 stools with an isolation rate of 2.8%, which belonged to vaccine strains and 24 non-polioviruses (NPVs) were detected with an isolation rate of 8.9%. 24 NPVs belonged to human enterovirus group B (HEV-B) with 6 serotypes, HEV-A, HEV-C and HEV-D viruses were not isolated. Among them, 13 NPVs were E7 (54.17%) and 5 NPVs were E13 (20.83%). Our results showed that the enterovirus carrying rate in the border areas of Yunnan province was higher than the rate of routine acute flaccid paralysis (AFP) detection system. The HEV-B viruses were the only enteroviruses isolated. The phylogenetical analysis showed that Echovirus 7(E7) and 13 (E13) exhibited genetic polymorphism.

[Distribution of hepatitis B virus genotypes and serotypes in people who had a physical examination in Yunnan Province].

OBJECTIVE: To know genotypes and serotypes of hepatitis B virus (HBV) detected from hepatitis B infected people in Yunnan Province. METHODS: Serum samples were collected from HBsAg carriers detected from people who had a physical examination at Yunnan Provincial Center for Disease Control and Prevention. The S genes of HBV were amplified by nested PCR and the PCR products were sequenced. The viral genotype was identified by phylogenetic analysis. 27 reference sequences corresponding to HBV genotype A to I were obtained from GenBank. According to the amino acid sequences deduced from the nucleotide sequences of S gene, the dominant serotype of HBV detected from these people were confirmed. RESULTS: 39 HBsAg positive serum samples were detected from 2216 people who had a physical examination. The results shows that 76.9% were C genotype; 15.4% were B genotype; 5.1% were D genotype; 2.5% were I genotype. Three serotypes were found. The rates of adw2, adrq+ and ayr serotypes are 71.8%, 17.9% and 10.3% respectively. All of adw2 subtype specimens are C genotype. Among the serum specimens in which both HBsAg and HBeAg are positive, 75% were C genotype and adw2 subtype. CONCLUSION: It is determined that the main genotype and subtype of HBV prevailed in Yunnan province is C genotype and adw2 subtype.

[Molecular identification of hepatitis B virus genotype I in Yunnan Province, the People's Republic of China].

Molecular typing was conducted according to the reported method for one HBsAg positive carrier who had a physical examination in Yunnan Province. The S gene of this HBV sample was amplified by nested PCR and the PCR products were directly sequenced. Blast searching was done on the Genbank database and the sequence were compared with the HBV reference sequences in database. The phylogenetic tree was constructed. Homology analysis of nucleotide and smino acid were performed between the sequences from the sample and the reference sequences corresponding to HBV genotype A to I. Analysis of nucleotide and amino acid identities suggested that the sample belonged to HBV genotype I. The HBV genotype I is the first reported in China.

[Status of enterovirus infection and molecular identification among healthy children at the areas bordering Myanmar, in Yunnan province, China].

OBJECTIVE: To explore the enterovirus infection status among healthy children under 15 years old in the border areas of Yunnan province that connecting Myanmar. METHODS: A total of 319 stool samples were collected from healthy children in the 10 entrance ports. Enterovirus was isolated from these stool samples and then poliovirus and adenovirus were serotyped by neutralization test using specific anti-sera. All the non-polio enteroviruses (NPEVs) were identified by partial sequencing of VP1 gene. RESULTS: All 53 enterovirus were isolated from 319 stool samples and 16.6% of them carried the virus. 23 polio virus (PVs) and 30 NPEVs were isolated with rates of carrying the virus were 7.2% and 9.4% respectively. 4 adenovirus were also isolated with a rate as 1.25%. 1 isolate could not be amplified by any Pan-enterovirus primers or by RT-PCR so was not able to be sequenced. The results of NPEVs sequencing showed that:1 isolate (3.3%) was classified into 1 serotype of HEV-A while 20 isolates (66.7%) were classified into 11 serotypes of HEV-B and 8 isolates (26.7%) were classified into 3 serotypes of HEV-C. However, we could not isolate any viruses that belong to HEV-D. nt. Result from the aa identify calculation showed that the nt and aa identification between isolates and corresponding standard strains were more than 75% and 85% respectively. The findings were similar to the international standards. CONCLUSION: Our results showed that the rate of carrying the enterovirus especially poliovirus in some areas of Yunnan province that bordering Myanmar was higher than that of rate through the routine acute flaccid paralysis detection system. Of the enterovirus isolated, HEV-B group appeared the predominant with the wide spread of enterovirus serotype. Some newer enterovirus were also detected such as EV73 (2 strains), EV75 (1 strain), EV80 (1 strain) and EV96 (4 strains).

[Combined application of enzyme-linked immunospot assay, positron emission tomography, and gene chip assay in the diagnosis of a case of chronic disseminated tuberculosis].

OBJECTIVE: To highlight the clinical features and diagnosis of chronic disseminated tuberculosis, with emphasizing the usefulness of several recently available diagnostic technologies in this setting. METHOD: We presented a case of chronic disseminated tuberculosis diagnosed with the combined application of interferon-gamma release assay T-SPOT. TB, 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET), and gene chip assay. RESULTS: A 53-year-old gentleman who had chronic cough for 7 years and fever for 2 weeks was referred to our hospital for further evaluation. 18F-FDG-PET/CT scan showed increased FDG uptake in multiple lesions involving bilateral lungs, supraclavicular, mediastinal and intro-abdominal lymph nodes and bones, mimicking metastatic malignancy. T-SPOT. TB assay revealed significant responses [ early secreting antigen target 6 (ESAT-6): 3 908 spot forming cells (SFCs)/10(6) peripheral blood mononuclear cells (PBMCs), culture filtrate protein (CFP-10): 3 400 SFCs/10(6) PBMCs]. Subsequent biopsy of supraclavicular lymph node, lung, and ilium revealed granulomas, while culture of the obtained tissue yeilded mycobacteria. Gene chip testing identified M. tuberculosis sensitive to isoniazid and rifampin. After 10 weeks of treatment for tuberculosis, the patient's condition was improved and a second T-SPOT. TB assay showed significantly reduced responses (ESAT-6: 1528 SFCs/10(6) PBMCs; CFP-10: 1460 SFCs/10(6) PBMCs). CONCLUSIONS: Timely diagnosis of chronic disseminated tuberculosis requires high index of suspicion. T-SPOT. TB assay, PET/CT, and gene chip assay may provide valuable information that facilitates further diagnostic procedures and treatment decision.

[Molecular identification of human enterovirus 73 in Yunnan Province, the People's Republic of China].

Molecular typing was conducted according to the reported methods for 2 enteroviruses that were isolated from healthy children in the border areas of Yunnan Province with Myanmar. RT-PCR and sequencing were performed with 292/222 primers according to the Oberste's methods. The resulting sequences were blasted against the Genbank database and compared with all available enterovirus database. Analysis of homology at nucleotide and amino acid level identically suggested that the two enteroviruses are human enterovirus 73.

[Genetic association of vitamin D receptor polymorphisms with primary biliary cirrhosis and autoimmune liver diseases on Chinese].

OBJECTIVE: To study the association between the polymorphisms of vitamin D receptor (VDR) gene and autoimmune liver diseases and (AIH) and primary biliary cirrhosis (PBC) in Chinese. METHODS: PCR-RELP was used to investigate the polymorphisms in exon 2, and exon 7 to exon 9 of VDR among 49 AIH patients, 58 PBC patients, and 160 healthy controls, all Chinese. VDR polymorphisms were assessed by FokI, BsmI, ApaI, and TaqI endonucleases restriction fragment length polymorphism analysis after specific polymerase chain reaction (PCR) amplification. RESULTS: The distribution of VDR gene polymorphism in Chinese was similar to those in Koreans and Japanese, and different from those in the Germans and Spaniards. The percentage of ff phenotype carriers was significantly higher in the AIH patients than in the healthy controls (34.7% vs. 48.1%, chi(2) = 5.47, P = 0.019) and the percentage of Ff phenotype carriers was lower in the AIH patients than in the healthy controls (34.7% vs. 48.1%, P = 0.057). The percentage of Bb phenotype carriers was significantly lower in the PBC patients than in the healthy controls (5.2% vs. 17.5%, P = 0.021) and. the percentage of bb phenotype carriers was significantly higher in the PBC patients than in the healthy controls (94.8% vs. 80.6%, chi(2) = 6.52, P = 0.01). We also detected a significant association of the BsmI polymorphisms in PBC patients in comparison with controls (P = 0.01). Furthermore we found the difference in the FokI, BsmI, ApaI, and TaqI genotype distribution between Chinese health controls and Caucasian health controls. CONCLUSION: There is a significant association between FokI polymorphism and AIH and a significant association between the BsmI polymorphisms and PBC in Chinese.