Genetic diversity and structure of Oncomelania hupensis hupensis in two eco-epidemiological settings as revealed by the mitochondrial COX1 gene sequences.

BACKGROUND: Oncomelania hupensis hupensis is the only intermediate host of Schistosoma japonicum, the causative agent of schistosomiasis in China and is therefore of significant medical and veterinary health importance. Although tremendous progress has been achieved, there remains an understudied area of approximately 2.06 billion m2 of potential snail habitats. This area could be further increased by annual flooding. Therefore, an understanding of population genetics of snails in these areas may be useful for future monitoring and control activities. METHODS AND RESULTS: We sampled snails from Hexian (HX), Zongyang (ZY) and Shitai (ST) in Anhui (schistosomiasis transmission control), and from Hengtang (HT), Taicang (TC), Dongsan (DS) and Xisan (XS) in Jiangsu (schistosomiasis transmission interrupted), downstream of Anhui. ST, DS and XS are classified as hilly and mountainous areas, and HX, ZY, TC and HT as lake and marshland areas. The mitochondrial cytochrome c oxidase subunit I gene were sequenced. Out of 115 snails analyzed, 29 haplotypes were identified. We observed 56 (8.72%) polymorphic sites consisting of 51 transitions, four transversions and one multiple mutational change. The overall haplotype and nucleotide diversity were 0.899 and 0.01569, respectively. Snail populations in Anhui had higher genetic diversity than in Jiangsu. 73.32% of total variation was distributed among sites and 26.68% within sites. Snails were significantly separated according to eco-epidemiological settings in both network and phylogenetic analyses. CONCLUSION: Our results could provide important guidance towards assessing coevolutionary interactions of snails with S. japonicum, as well as for future molluscan host monitoring and control activities.

Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine.

O-linked ß-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy-D-gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism.

Oocyte IVM or vitrification significantly impairs DNA methylation patterns in blastocysts as analysed by single-cell whole-genome methylation sequencing.

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Knockdown of bone morphogenetic protein 4 gene induces apoptosis and inhibits proliferation of bovine cumulus cells.

The expression and function of bone morphogenetic protein 4 (BMP4) gene in bovine cumulus cells (CCs) was investigated to reveal the mechanisms by which it regulated cell apoptosis and proliferation. The mRNA and protein expression of BMP4 were detected using quantitative PCR (qPCR) and immunofluorescence staining in CCs. The effective siRNAs against BMP4 gene were screened using qPCR and western blotting. The mRNA expression levels of apoptosis-related genes and proliferation-related genes were estimated by qPCR after knocking-down the BMP4 gene in bovine CCs. Cell apoptosis, proliferation and cell cycle were measured with Annexin V-FITC, CCK-8 and propidium iodide staining by flow cytometry. Results showed that the BMP4 gene was expressed and its protein was in the cytoplasm and nuclei of bovine CCs. The BMP4 knockdown increased the cell apoptosis rate and upregulated the mRNA levels of apoptosis genes CASPASE-3 and BAX with downregulation of the anti-apoptosis gene BCL-2 (P < 0.05). The proliferation rate declined and the mRNA expression levels of proliferation-related genes PCNA, CDC42 and CCND2 were downregulated in the bovine CCs with BMP4 low expression (P < 0.05). The BMP4 knockdown significantly increased the percentage of G0/G1 phase cells while decreased that of S phase cells. Therefore, the expression of BMP4 and its biological functions on the cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. BMP4 knockdown induced cell apoptosis, cell cycle arrest and inhibited proliferation of bovine CCs.

In vivo praziquantel efficacy of Schistosoma japonicum over time: A systematic review and meta-analysis.

Praziquantel (PZQ), the only choice of chemotherapy for schistosomiasis recommended by World Health Organization (WHO), has been widely used over 40 years. The long-term, and rapid expansion of, PZQ use for disease control across a large populations continues to raise concern regarding the potential for emergence and establishment of drug resistance. Recent research has also proposed that the long survival and low sensitivity of unpaired worms, derived from either incomplete treatment cure rates or single-sex schistosome infections within final hosts, could exacerbate the risk of PZQ resistance (PZQ-R) emerging. With the aim of assessing whether PZQ efficacy amongst S. japonicum may have changed over time in China, we performed a unique systematic review and meta-analyses on datasets which evaluated the efficacy of PZQ via laboratory assays of field S. japonicum isolates on experimental mice over time. Relevant published literatures from four electronic bibliographic databases and lists of article references were searched. Two indexes, d, a measure used in meta-analyses for worm burden difference between two groups, and r, a traditional measure for worm reduction percentage after treatment but without considering sample size were calculated for each study. A total of 25 papers including 127 experimental studies with eligible data on 2230 mice were retrieved. The pooled d (D) was 3.91 (3.56-4.25) and pooled r (R) was 54.52% (52.55%-56.52%). D significantly increased over time, whereas R non-significantly decreased; both estimates were significantly associated with the total drug dose. Such findings suggested no evidence of PZQ-R emergence S. japonicum to date. However, we consider the potential role of parasite origins, PZQ dosage, and single versus mixed gender infections of the results published to date, and the avenues now needed for further research.

Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells.

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P < 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3, BAX and BAX/BCL-2 ratio (P < 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12, two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P < 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed.

Meta-analyses of Schistosoma japonicum infections in wild rodents across China over time indicates a potential challenge to the 2030 elimination targets.

China once suffered greatly from schistosomiasis japonica, a major zoonotic disease. Nearly 70 years of multidisciplinary efforts have achieved great progress in disease control, with infections in both humans and bovines significantly reduced to very low levels. However, reaching for the target of complete interruption of transmission at the country level by 2030 still faces great challenges, with areas of ongoing endemicity and/or re-emergence within previously 'eliminated' regions. The objectives of this study were, by using meta-analytical methods, to estimate the overall prevalence of Schistosoma japonicum infections in abundant commensal rodent species in mainland China after the introduction of praziquantel for schistosomiasis treatment in humans and bovines in 1980s. In doing so we thereby aimed to further assess the role of wild rodents as potential reservoirs in ongoing schistosome transmission. Published studies on infection prevalence of S. japonicum in wild rodents in mainland China since 1980 were searched across five electronic bibliographic databases and lists of article references. Eligible studies were selected based on inclusion and exclusion criteria. Risks of within and across study biases, and the variations in prevalence estimates attributable to heterogeneities were assessed. The pooled infection prevalence and its 95% confidence intervals (CIs) were calculated with the Freeman-Tukey double arcsine transformation. We identified a total of 37 relevant articles involving 61 field studies which contained eligible data on 8,795 wild rodents across mainland China. The overall pooled infection prevalence was 3.86% (95% CI: 2.16-5.93%). No significant change in the overall pooled prevalence was observed between 1980-2003 (n = 23 studies) and 2004-current (n = 38 studies). However, whilst the estimated prevalence decreased over time in the marshland and lake regions, there was an apparent increase in prevalence within hilly and mountainous regions. Among seven provinces, a significant prevalence reduction was only seen in Jiangsu where most endemic settings are classified as the marshland and lakes. These estimates changed over season, ranging from 0.58% in spring to 22.39% in winter, in association with increases in rodent density. This study systematically analyzed S. japonicum infections in wild rodents from the published literature over the last forty years after the introduction of praziquantel for schistosomiasis treatment in humans and bovines in 1980s. Although numbers of schistosomiasis cases in humans and bovines have been greatly reduced, no such comparable overall change of infection prevalence in rodents was detected. Furthermore, there appeared to be an increase in S. japonicum prevalence in rodents over time within hilly and mountainous regions. Rodents have been projected to become the dominant wildlife in human-driven environments and the main reservoir of zoonotic diseases in general within tropical zones. Our findings thus suggest that it is now necessary to include monitoring and evaluation of potential schistosome infection within rodents, particularly in hilly and mountainous regions, if we are ever to reach the new 2030 elimination goals and to maximize the impact of future public, and indeed One Health, interventions across, regional, national and international scales.

A meta-analysis of infection rates of Schistosoma japonicum in sentinel mice associated with infectious waters in mainland China over last 40 years.

BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease. After nearly 70 years of control efforts in China, Schistosomiasis transmission has been reduced to a much lower level. The absence or near absence of infections in humans or livestock, based on traditional fecal and serological tests, has made the targets and priorities of future control efforts difficult to determine. However, detection of schistosome cercariae in waters using sentinel mice could be an alternative way of identifying remaining foci of infection, or even serve as a tool for evaluation of control efficacy. This method has been employed in China over last forty years. We therefore performed a meta-analysis of the relevant research to investigate if infections in sentinel mice mirror the ongoing trend of schistosomiasis transmission in China. METHODS: We conducted a meta-analysis of studies reporting infection rates of S. japonicum in sentinel mice in China before Sep 1, 2018 in accordance with the PRISMA guidelines. We retrieved all relative studies based on five databases (CNKI, WanFang, VIP, PubMed and Web of Science) and the reference lists of resulting articles. For each individual study, the infection rate in sentinel mice is presented together with its 95% confidence interval (CI). Point estimates of the overall infection rates and their 95% CIs were calculated. Subgroup analyses were performed according to study periods, seasons or regions. RESULTS: We identified 90 articles, including 290 studies covering eight endemic provinces. The overall rate in sentinel mice was 12.31% (95% CI: 10.14-14.65%) from 1980 to 2018. The value of 3.66% (95% CI: 2.62-4.85%) estimated in 2004 to 2018 was significantly lower than in 1980 to 2003 (22.96%, 95% CI: 19.25-26.89%). The estimate was significantly higher in the middle and lower reaches than in the upper reaches of the Yangtze River. The highest estimates were obtained in Hunan (30.11%, 95% CI: 25.64-34.77%) followed by Anhui (26.34%, 95% CI: 12.88-42.44%) and then Jiangxi (13.73%, 95% CI: 6.71-22.56%). Unlike the other provinces in the middle and lower reaches, no significant reduction was seen in Hubei after 2003. Even in Hubei two studies carried out after 2014 reported infections in sentinel mice, although no infected snails were reported across the province. Infections were most found in April (17.40%, 95% CI: 1.13-45.49%), July (24.98%, 95% CI: 15.64-35.62%) and October (17.08%, 95% CI 5.94-32.05%). High degrees of heterogeneity were observed. CONCLUSION: This meta-analysis provides a comprehensive analysis of schistosome infection in sentinel mice across China. The estimates largely mirror the ongoing trends of transmission in terms of periods and regions. Infections were most likely to occur in April, July and October. In areas where no infected snails were reported infections in sentinel mice were still observed. Due to the presence of snails and infected wildlife, detection of schistosomes in waters using such a highly sensitive method as the deployment of sentinel mice, remains of importance in schistosomiasis monitoring. We would suggest the current criteria for transmission interruption or elimination of schistosomiasis in China be adjusted by integrating the results of sentinel mice based surveys.

Population genetics of Oncomelania hupensis snails, intermediate hosts of Schistosoma japonium, from emerging, re-emerging or established habitats within China.

Schistosomiasis remains one of the world's most significant neglected tropical diseases, second only to malaria in terms of socioeconomic impact. In 2014, China proposed the goal of schistosomiasis japonicum elimination by 2025. However, one major challenge is the widely distributed, and in certain cases potentially increasing, habitats of Oncomelania hupensis, the snail intermediate hosts of S. japonicum. Therefore, an understanding of population genetics of O. hupensis in new or re-emerged habitats, together with that of the established habitats with snail persistence, would be valuable in controlling and predicting the future transmission dynamics of schistosomiasis in China. Using nine microsatellite loci, we conducted population genetic analyses of snails sampled from one habitat where snails were detected for the first time, one (previously eliminated) habitat with re-emerged snails, and one habitat with established snail persistence. Results showed lower diversities, in terms of number of observed alleles per locus (Na), number of effective alleles per locus (NeA), observed (Ho) and expected heterozygosity (He), in snails from new or re-emerged snail habitats than from the habitat with snail persistence. The smallest effective population size was inferred in the re-emerged snail habitat, but the largest was in the new habitat rather than in the habitat with snail persistence. No bottleneck effects were detected in new or re-merged habitats. No or low sub-structure was inferred in new and persistent snail habitats. Snails from the three sites were clearly separated and low gene flow was estimated between sites. We propose that snails at the new habitat may have been introduced through immigration, whereas snails at the re-emerged habitat may be the consequence of those few snails remaining subsequently expanding through reproduction. We discuss our results in terms of their theoretical and applied implications.