Bis (2-butoxyethyl) Phthalate Delays Puberty Onset by Increasing Oxidative Stress and Apoptosis in Leydig Cells in Rats.

Objective: This study investigated the effects of bis (2-butoxyethyl) phthalate (BBOP) on the onset of male puberty by affecting Leydig cell development in rats. Methods: Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles; Leydig cell morphological metrics; mRNA and protein levels; oxidative stress; and AKT, mTOR, ERK1/2, and GSK3ß pathways were assessed. Results: BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day (P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1, and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland (P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly down-regulated, by BBOP treatment at 250-500 mg/kg bw per day (P < 0.05). Furthermore, BBOP at 500 mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3ß and SIRT1 levels mg/kg bw per day (P < 0.05). Finally, BBOP at 100 or 500 µmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro (P < 0.05). Conclusion: BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats.The graphical abstract is available on the website www.besjournal.com.

Predicting the spatial distribution of Biomphalaria straminea, a potential intermediate host for Schistosoma mansoni, in China.

Schistosomiasis is one of the most prevalent parasitic diseases impacting human health in the tropics and sub-tropics. The geographic distribution of Schistosoma mansoni, the most widespread species, includes areas in Africa, the Middle East, South America and the Caribbean. Snails of the genus Biomphalaria act as intermediate host for S. mansoni. Biomphalaria straminea is not indigenous in China but was accidentally introduced to Hong Kong from South America and has spread to other habitats in the southern parts of the country. This species is known for its great dispersal capacity that highlights the importance of the snail as a potential host for S. mansoni in China. In this connection, although no such infection has been recorded in the field so far, the continuous expansion of China's projects in endemic areas of Africa and import of the infection via returning workers or visitors deserve attention. The purpose of this study was to map and predict the spatial distribution of B. straminea in China. Snail occurrence data were assembled and investigated using MaxEnt software, along with climatic and environmental variables to produce a predictive risk map. Of the environmental variables tested, the precipitation of warmest quarter was the most contribution factor for snail's spatial distribution. Risk areas were found in southeastern China and it is expected that they will guide policies and control programmes to potential areas area of snail abundance and used for spatial targeting of control interventions for this invasive species.

[Diagnosis and Treatment of the First Imported Case of Plasmodium knowlesi Infection in China].

Objective: To diagnose and treat the first imported active case of Plasmodium knowlesi infection in China. Methods: The clinical information of the patient was collected. Microscopy of blood smear was conducted after Giemsa staining. Genomic DNA was extracted from blood, and PCR was conducted to amplify rDNA. The PCR products were sequenced and analyzed with BLAST Results: The patient returned from a one-week tour in a tropical rain forest in Malaysia. The first disease attack occurred in Guangzhou on Oct. 16, 2014, with fever, shivering and sweating. The patient was initially diagnosed as malaria and hospitalized on Oct. 26, 2014. Microscopic observation revealed typical forms of P. knowlesi in blood smear. The red blood cells became enlarged, with big trophozoites appearing as a ring with dual cores and dark brown malaria pigment. The trophozoites were slightly bigger and thicker than P. falciparum. The schizont had 6-8 merozoites, with obvious brown malaria pigment. PCR resulted in a specific band of 1 099 bp. BLAST analysis showed that the sequence of the PCR product was 99% homologous to P. knowlesi (acession No. AM910985.1, L07560.1 and AY580317.1). The patient was diagnosed as P. knowlesi infection, and was then given an 8-day treatment with chloroquine and primaquine, together with dihydroartemisinin piperaquine phosphate tablet. The patient was discharged after recovery on Oct. 28, 2014. Conclusion: According to the clinical symptoms, epidemiological history and laboratory test, the patient has been confirmed as P. knowlesi infection. It may also be the first active case of knowlesi malaria reported in China.

[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.

[Distribution and schistosomiasis transmission risks of Biomphalaria straminea in inland China].

Biomphalaria straminea, the intermediary host of Schistosoma mansoni, was found by survey in local areas of Shenzhen City in 1981, which was the first finding of the snail in inland China. By 2013, the snail had spread in large range of Shenzhen City and overspread to the surrounding regions, Dongguan City and Huizhou City. Due to the facts that Shenzhen City has many international communications and is a key area of labor export, with high population mobility, while the reports of S. mansoni infection in the returnees of our country from Africa have been increasing recently, it must be paid a high attention to whether the epidemic or transmission of schistosomiasis mansoni may happen under the present circumstance. This paper initially discusses the finding, distribution, spreading and overspreading and transmission risk of B. straminea in inland China and puts forward some related control suggestions.

Adverse effects of di-(2-ethylhexyl) phthalate on Leydig cell regeneration in the adult rat testis.

The objective of the present study is to determine whether di-(2-ethylhexyl) phthalate (DEHP) exposure at adulthood affects regeneration of rat Leydig cells. 90-day-old Long-Evans rats received intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate mature Leydig cells, and then were randomly divided into 3 groups, in which rats were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 35 days. Serum testosterone and luteinizing hormone levels were assessed by RIA, Leydig cell numbers and proliferation rate were evaluated, and the mRNA levels of Leydig cell specific genes were measured by qPCR. Both 10 and 750 mg/kg DEHP treatments increased Leydig cell numbers on day 14, 21 and 35 post-EDS, due to significant increase of the number of Leydig cell precursors from day 14 to 21 post-EDS. However, serum testosterone levels were halved in 10 and 750 mg/kg DEHP groups compared to control on day 35 post-EDS despite the increased Leydig cell numbers. Quantitative PCR showed that Leydig cell specific genes including Lhcgr, Cyp11a1, Hsd3b1, and Insl3 were significantly down-regulated in 750 mg/kg DEHP-treated testes on post-EDS day 21 and beyond. The present study suggests that DEHP increases Leydig cell proliferation but inhibits differentiation during the regeneration of Leydig cells.

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.

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.

[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.

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.

[Expression of fibrillin-1 in congenital bicuspid aortic valves].

OBJECTIVE: To detect the expression of fibrillin-1 in congenital bicuspid aortic valves, and to investigate the molecular mechanism of congenital bicuspid aortic valves. METHODS: Specimens of aortic valve were obtained from 12 pediatric patients with congenital bicuspid aortic valve, 11 boys and 1 girl, aged 16.7 (10 - 18), including 5 cases of aortic stenosis (AS), 8 of aortic insufficiency (AI), and 1 of AS and AI, undergoing valve replacement, 8 children who died accidentally without cardiovascular system and collagen system diseases, 6 boys and 2 girls, aged 9.1 (1 - 17), collected in autopsy [normal (tricuspid) aortic valve controls], and 18 pediatric patients of rheumatic valvular heart disease with diseased tricuspid aortic valves who underwent aortic valve replacement, 13 boys and 5 girls, aged 16.5 (12 - 18) (rheumatic valvular heart disease controls). HE staining and light microscopy were conducted. Immunohistochemistry was used to detect the expression of fibrillin-1 in the aortic valves. RESULTS: Microscopy showed that the tissue structure of the congenital bicuspid aortic valves was unclear with hyperplasia of fibrous tissue. The grey degree value of fibrillin-1 of the congenital bicuspid aortic valve group was 170 +/- 10, significantly lower than those of normal aortic valve group and diseased tricuspid aortic valve group (126 +/- 8 and 73 +/- 16 respectively, both P < 0.05). There were not significant difference in the grey degree value of fibrillin-1 among the patients of congenital bicuspid aortic valves with AS, AI, and AS + AI (167 +/- 6, 171 +/- 8, and 168 +/- 6 respectively). CONCLUSION: The expression of fibrillin-1 is significantly reduced in congenital bicuspid aortic valves which may contribute to the morphological changes of the aortic valve leaflets and their resultant functional failure in congenital bicuspid aortic valves.

Effects of chronic multiple stress on learning and memory and the expression of Fyn, BDNF, TrkB in the hippocampus of rats.

BACKGROUND: The effect of chronic stress on cognitive functions has been one of the hot topics in neuroscience. But there has been much controversy over its mechanism. The aim of this study was to investigate the effects of chronic multiple stress on spatial learning and memory as well as the expression of Fyn, BDNF and TrkB in the hippocampus of rats. METHODS: Adult rats were randomly divided into control and chronic multiple stressed groups. Rats in the multiple stressed group were irregularly and alternatively exposed to situations of vertical revolution, sleep expropriation and restraint lasting for 6 weeks, 6 hours per day with night illumination for 6 weeks. Before and after the period of chronic multiple stresses, the performance of spatial learning and memory of all rats was measured using the Morris Water Maze (MWM). The expression of Fyn, BDNF and TrkB proteins in the hippocampus was assayed by Western blotting and immunohistochemical methods. The levels of Fyn and TrkB mRNAs in the hippocampus of rats were detected by RT-PCR technique. RESULTS: The escape latency in the control group and the stressed group were 15.63 and 8.27 seconds respectively. The performance of spatial learning and memory of rats was increased in chronic multiple stressed group (P < 0.05). The levels of Fyn, BDNF and TrkB proteins in the stressed group were higher than those of the control group (P < 0.05). The results of immunoreactivity showed that Fyn was present in the CA3 region of the hippocampus and BDNF positive particles were distributed in the nuclei of CA1 and CA3 pyramidal cells as well as DG granular cells. Quantitative analysis indicated that level of Fyn mRNA was also upregulated in the hippocampus of the stressed group (P < 0.05). CONCLUSIONS: Chronic multiple stress can enhance spatial learning and memory function of rats. The expression of Fyn, BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus. These suggest that Fyn and BDNF/TrkB signal transduction pathways may participate in the process of the enhanced learning and memory during chronic multiple stress.