Effect of Ivermectin on the Expression of P-Glycoprotein in Third-Stage Larvae of Haemonchus contortus Isolated from China.

Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

Analyses of mitogenomic markers shed light on the divergence, population dynamics, and demographic history of Pakistani chickens.

Pakistan is one of a few sites, associated with the earliest known independent domestication event in the evolutionary history of chicken, which is socio-economically and historically the most important poultry bird in the country. However, the divergence, past population dynamics, and demographic history of Pakistani chickens have not been addressed so far. Therefore, we herein investigated the indigenous Pakistani chickens using mitogenomic markers. We first prepared individual DNA samples from the chicken feathers, and generated nucleotide sequence data, which was then subjected to various population genetics analyses. In molecular phylogenetic analysis, the Pakistani chickens were clustered under nine different clades. Among the wild fowls, the Indian red jungle fowl (IRJF) shared very close affinities to Pakistani chickens. The Bayesian skyline plot showed an increase in the effective population size of Pakistani chickens during the last 50 years. Finally, a time-calibrated phylogeny inferred molecular divergence of the Pakistani chickens. A molecular rate of 3.6 × 10-6 mutations/site/year (95% HPD interval: 2.28 × 10-8 to 9.32 × 10-6) was estimated for the data set. In a rooted tree with root-age of 12058 years (95% HPD interval: 1161-38411), the Pakistani chicken haplotypes showed divergence from IRJF haplotypes around 6987 years (95% HPD interval: 1132-20746) ago, and they shared their most recent common ancestor with Gallus gallus spadiceus, and G. g. jabouillei at the root of the tree. Overall, these results suggest that Pakistani chicken haplotypes share their ancestral gene pool with the IRJF as compared to other red jungle fowl subspecies.

Suppression of hyaluronidase reduces invasion and establishment of Haemonchus contortus larvae in sheep.

Haemonchus contortus is a hematophagous endoparasite of small ruminants, which is responsible for huge economic losses in livestock sector. Hyaluronidase produced by infective larvae of H. contortus can degrade hyaluronic acid present in the host's abomasal tissue. Thus, it facilitates larval tissue invasion and early establishment. We herein explored this ability of hyaluronidase in H. contortus, and tested whether hyaluronidase is utilized as a virulence factor by H. contortus while establishing the infection. We first successfully blocked the hyaluronidase gene in L3 larvae by RNA interference (RNAi), which was subsequently confirmed by qPCR, enzymatic activity, and immunohistochemistry assays. Using these larvae we then conducted in vivo and in vitro assays on sheep to assess the effects of hyaluronidase suppression on larval invasion and establishment of infection. The in vivo assay showed a significant drop in worm burden in siRNA treated group in comparison to control group. During in vitro assay we applied an ovine ex vivo model where siRNA treated group of larvae showed significantly reduced invasion of the abomasal tissue explants as compared to control group. These findings indicate that hyaluronidase plays a key role in host's tissue invasion and larval establishment, and it is used as a virulence factor by H. contortus while establishing the infection. As an invasive virulence molecule, its functional research is thus conducive to the prevention of haemonchosis.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus.

The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin (IVM). Resistance against IVM has emerged in parasites, and preserving its efficacy is now becoming a serious issue. The parasitic nematode Haemonchus contortus (Rudolphi, 1803) is economically an important parasite of small ruminants across the globe, which has a successful track record in IVM resistance. There are growing evidences regarding the multigenic nature of IVM resistance, and although some genes have been proposed as candidates of IVM resistance using lower magnification of genome, the genetic basis of IVM resistance still remains poorly resolved. Using the full magnification of genome, we herein applied a population genomics approach to characterize genome-wide signatures of selection among pooled worms from two susceptible and six ivermectin-resistant isolates of H. contortus, and revealed candidate genes under selection in relation to IVM resistance. These candidates also included a previously known IVM-resistance-associated candidate gene HCON_00148840, glc-3. Finally, an RNA-interference-based functional validation assay revealed the HCON_00143950 as IVM-tolerance-associated gene in H. contortus. The possible role of this gene in IVM resistance could be detoxification of xenobiotic in phase I of xenobiotic metabolism. The results of this study further enhance our understanding on the IVM resistance and continue to provide further evidence in favor of multigenic nature of IVM resistance.

Analysis of genome-wide SNPs based on 2b-RAD sequencing of pooled samples reveals signature of selection in different populations of Haemonchus contortus.

The parasitic nematode Haemonchus contortus is one of the world's most important parasites of small ruminants that causes significant economic losses to the livestock sector. The population structure and selection in its various strains are poorly understood. No study so far compared its different populations using genome-wide data. Here, we focused on different geographic populations of H. contours from China (Tibet, TB; Hubei, HB; Inner Mongolia, IM; Sichuan, SC), UK and Australia (AS), using genome-wide population-genomic approaches, to explore genetic diversity, population structure and selection. We first performed next-generation high-throughput 2b RAD pool sequencing using Illumina technology, and identified single-nucleotide polymorphisms (SNPs) in all the strains. We identified 75,187 SNPs for TB, 82,271 for HB, 82,420 for IM, 79,803 for SC, 83,504 for AS and 78,747 for UK strain. The SNPs revealed low-nucleotide diversity (pi= 0.0092-0.0133) within each strain, and a significant differentiation level (average Fst = 0.34264) among them. Chinese populations TB and SC, along with the UK strain, were more divergent populations. Chinese populations IM and HB showed affinities to the Australian strain. We then analysed signature of selection and detected 44 (UK) and 03 (AS) private selective sweeps containing 49 and 05 genes, respectively. Finally, we performed the functional annotation of selective sweeps and proposed biological significance to signature of selection. Our data suggest that 2b-RAD pool sequencing can be used to assess the signature of selection in H. contortus.

Microsatellite analysis reveals extensive gene flow, and lack of population structure in the farm populations of Haemonchus contortus in northern China.

The parasitic nematode Haemonchus contortus is economically an important parasite of small ruminants across the globe. China is the world's largest producer, consumer, and importer of mutton. With ubiquitous distribution across the country H. contortus is one of the potential candidates to cause huge economic losses to small ruminant farming industry in China. We herein investigated genetic diversity and population structure of six farm populations of H. contortus in northern China, and also compared them to H. contortus isolates from UK and Australia. We first prepared individual DNA samples from 240 adult worms, and generated genotyping data using eight microsatellite markers. Obtained data was then subjected to allelic frequency and population genetic analyses. The overall allelic richness (mean/locus/pop = 7.375 ±â€¯0.844-10.125 ±â€¯1.109), and expected heterozygosity (mean/locus/pop = 0.646 ±â€¯0.040-0.735 ±â€¯0.025) indicated high degree of population genetic variation across the Chinese isolates. Low level of genetic differentiation (Fst = 0.010-0.066) was observed across all the populations. AMOVA results showed high level of variation (93%) within the populations. PCA analysis revealed mixed clustering of all the populations with no visible geographical sub-structuring. Finally the population admixture analysis resulted in extensive admixing of genotypes across all the populations. With these findings we conclude that there is no obvious population genetic structure with extensive gene flow across all the farm populations of H. contortus in northern China.

Ethnogenetic analysis reveals that Kohistanis of Pakistan were genetically linked to west Eurasians by a probable ancestral genepool from Eurasian steppe in the bronze age.

Despite the unique geographic, ethnic, social and cultural features of Kohistan in Pakistan, the origin and descent of Kohistanis remain still obscure. In an effort to address questions concerning the genetic structure, origin and genetic affinities of Kohistanis, we herein applied an ethnogenetic approach consisting on mitochondrial DNA (mtDNA) analysis and dental morphology analysis. We sequenced HVS1 of mtDNA, observed 14 haplotypes and assigned a total of 9 haplogroups belonging to macrolineages M (17%) and N (83%). Genetic diversity estimates in Kohistanis (Hd = 0.910 ±â€¯0.014; Pi = 0.019 ±â€¯0.001; θw = 0.019 ±â€¯0.006) were similar to that of previous studies in other Pakistani populations. Overall, the analyses of dental morphology and mtDNA profile of Kohistanis resulted in similar findings. All the analyses indicate that Kohistanis share affinities to populations from Europe, Near East, Central Asia and South Asia. The Kohistani HVS1 haplotype 2 shares 100% identity to HVS1 haplotypes across the Europe. These results in light of recent insights into ancient genomics lead us to conclude that ancestry from Eurasian Steppe genetically linked Kohistanis to all these populations in the Bronze Age. This is consistent with linguistic evidence and also with the Indo-Aryan migration model for the peopling of South Asia.

Spatiotemporal expression pattern of Sjfz7 and its expression comparison with other frizzled family genes in developmental stages of Schistosoma japonicum.

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.

Population structure, genetic diversity and phylogenetic analysis of different rural and commercial chickens of Pakistan using complete sequence of mtDNA D-loop.

Chicken is the most important poultry bird in Pakistan that not only provides nutrition but also contributes to country's economy. The Pakistani chicken and its germplasm resources are not genetically characterized and explored. Here, we focused at rural and commercial chickens of Pakistan and explored genetic diversity, population structure and phylogeny. We first collected feather samples from Rural and Broiler populations of Pakistani chickens, isolated DNA and sequenced complete D-loop of mtDNA. The length of complete D-loop ranged from 1231 to 1234 bp in Pakistani chickens. The GC content was 39%. Hotspots of mutations were three hypervariable sites (HVS). Most of the variations (77%) were in HVS1. In a total, 26 polymorphic sites defined 12 haplotypes and all major haplogroups (A-I) in genetic structure of Pakistani chickens. Genetic diversity remained relatively very low in Broiler (Pi = 0.00212 ± 0.00136). There was a low sharing of matrilineages between the two populations (Fst = 0.170). With high Hd value (0.825 ± 0.051) and presence of all nine major haplogroups the rural chicken population showed relatively rich genepool. Finally we did molecular phylogenetic analysis and inferred phylogeny. Presence of subcontinent specific haplogroups E3 and I and clustering of Indian red junglefowl closely with Pakistani chickens in Bayesian inference tree, provide further evidence for an independent domestication event of chicken in subcontinent.

SjCRT, a recombinant Schistosoma japonicum calreticulin, induces maturation of dendritic cells and a Th1-polarized immune response in mice.

BACKGROUND: It is well known that immunization of radiation-attenuated (RA) schistosoma cercariae or schistosomula can induce high levels of protective immunity against schistosoma cercariae reinfection in many animals. Many studies have shown that the Th1 cellular immune response is crucial for the protective effect elicited by RA schistosomula. However, the molecular mechanism of this strong protective immunity remains unclear. METHODS: The expression profiles of Schistosoma japonicum calreticulin (SjCRT) in RA and normal schistosoma-derived cells were investigated by flow cytometry. The effect of recombinant SjCRT (rSjCRT) on mouse dendritic cells (DCs) was determined by FACS, ELISA and RT-PCR analysis. We also analyzed the effects of SjCRT on the activation of spleen cells from mice immunized with rSjCRT by detecting lymphocyte proliferation and the cytokine profiles of splenocytes. RESULTS: We found that the expression level of SjCRT in the cells from RA larvae was significantly higher than that in cells from normal schistosomula at early stages of development (day 4). The results of effect of rSjCRT on mouse DCs showed that rSjCRT could induce phenotypic and functional maturation of DCs, and SjCRT bound to the surface of DCs through the CD91 receptor and could be engulfed by DCs. The results of activation of splenocytes from mice immunized with rSjCRT also demonstrate that rSjCRT can effectively stimulate the proliferative response of splenic lymphocytes, elicit splenocytes from immunized mice to secrete high levels of IFN-γ, TNF-α and IL-4, and activate CD4+ T cells to produce high levels of IFN-γ. CONCLUSION: SjCRT is one of the immunostimulatory molecules released from RA schistosomula cells, might play a crucial role in conferring a Th1-polarized immune response induced by RA cercariae/schistosomula in mice, and is a candidate molecule responsible for the high levels of protective immunity induced by RA schistosomula.

Characterization and expression pattern of a novel Frizzled 8 receptor gene in Schistosoma japonicum.

Wnt signaling as mediated by the Frizzled family receptors plays a vital role in the early development of animal embryos, organ formation, tissue regeneration and other physiological processes. In the present study, a novel Frizzled member, SjFz8, was isolated and characterized in Schistosoma japonicum. SjFz8 encodes an 1162-amino-acid protein with typical characteristics of Frizzled proteins. Quantitative real-time polymerase chain reaction analysis indicated that SjFz8 transcript level was highest in 7-day-old schistosomula. In adult stages, SjFz8 mRNA expression remained at a low level after male-female pairing. The immunohistochemical localization of the Fz8 protein revealed that it existed in almost all tissues of S. japonicum, including subtegumental muscle, parenchyma, oral suckers, ventral suckers, testes of the male and ovaries of the female. We speculated that the Wnt signaling pathway that was mediated by Fz8 might take part in regulating histogenesis and organogenesis during the schistosomulum period, and play an important role in regulating further growth and development of male and female worms.

Genome-wide SNP analysis using 2b-RAD sequencing identifies the candidate genes putatively associated with resistance to ivermectin in Haemonchus contortus.

BACKGROUND: The excessive and uncontrolled use of anthelmintics, e.g. ivermectin (IVM) for the treatment of livestock parasites has led to widespread resistance in gastrointestinal nematodes, such as Haemonchus contortus. There is an urgent need for better management of drug-use in nematode control and development of novel anthelmintics. Discovery and identification of anthelmintic resistance-associate molecules/markers can provide a basis for rational anthelmintics-use and development of novel drugs. Recent studies have shown that ivermectin resistance in H. contortus is likely to be multi-genic in nature except for several genes coding for IVM target and efflux pump. However, no other IVM resistance-associated genes were characterized by conventional methods or strategies. In the present study we adopted a new strategy, i.e. using genome-wide single nucleotide polymorphism (SNP) analysis based on 2b-RAD sequencing, for discovering SNPs markers across the genomes in both IVM susceptible and resistant isolates of H. contortus and identifying potential IVM resistance-associated genes. RESULTS: We discovered 2962 and 2667 SNPs within both susceptible and resistant strains of H. contortus, respectively. A relative lower and similar genetic variations were observed within both resistant and susceptible strains (average π values were equal to 0.1883 and 0.1953, respectively); whereas a high genetic variation was found across both strains (average π value was equal to 0.3899). A significant differentiation across 2b-RAD tags nucleotide sites was also observed between the two strains (average FST value was equal to 0.3076); the larger differences in average FST were observed at SNPs loci between coding and noncoding (including intronic) regions. Comparison between resistant and susceptible strains revealed that 208 SNPs loci exhibited significantly elevated FST values, 24 SNPs of those loci were located in the CDS regions of the nine genes and were likely to have signature of IVM directional selection. Seven of the nine candidate genes were predicted to code for some functional proteins such as potential IVM target and/or efflux pump proteins, component proteins of receptor complex in membrane on neuromuscular cells, and transcriptional regulation proteins. Those genes might be involved in resistance to IVM. CONCLUSIONS: Our data suggest that candidate genes putatively associated with resistance to IVM in H. contortus may be identified by genome-wide SNP analysis using 2b-RAD sequencing.

Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions.

Secreted extracellular vesicles play an important role in pathogen-host interactions. Increased knowledge of schistosome extracellular vesicles could provide insights into schistosome-host interactions and enable the development of novel intervention strategies to inhibit parasitic processes and lessen disease transmission. Here, we describe biochemical characterization of Schistosoma japonicum exosome-like vesicles (S. japonicum EVs). A total of 403 proteins were identified in S. japonicum EVs, and bioinformatics analyses indicated that these proteins were mainly involved in binding, catalytic activity, and translation regulatory activity. Next, we characterized the population of small RNAs associated with S. japonicum EVs. Further studies demonstrated that mammalian cells could internalize S. japonicum EVs and transfer their cargo miRNAs to recipient cells. Additionally, we found that a specific miRNA, likely originating from a final host, ocu-miR-191-5p, is also associated with S. japonicum EVs. Overall, our findings demonstrate that S. japonicum EVs could be implicated in the pathogenesis of schistosomiasis via a mechanism involving the transfer of their cargo miRNAs to hosts. Our findings provide novel insights into the mechanisms of schistosome-host interactions.

The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection.

Water buffalo are less susceptible to Schistosoma japonicum infection than yellow cattle. The factors that affect such differences in susceptibility remain unknown. A Bos taurus genome-wide gene chip was used to analyze gene expression profiles in the peripheral blood of water buffalo and yellow cattle pre- and post-infection with S. japonicum. This study showed that most of the identified differentially expressed genes (DEGs) between water buffalo and yellow cattle pre- and post-infection were involved in immune-related processes, and the expression level of immune genes was lower in water buffalo. The unique DEGs (390) in yellow cattle were mainly associated with inflammation pathways, while the unique DEGs (2,114) in water buffalo were mainly associated with immune-related factors. The 83 common DEGs may be the essential response genes during S. japonicum infection, the highest two gene ontology (GO) functions were associated with the regulation of fibrinolysis. The pathway enrichment analysis showed that the DEGs constituted similar immune-related pathways pre- and post-infection between the two hosts. This first analysis of the transcriptional profiles of natural hosts has enabled us to gain new insights into the mechanisms that govern their susceptibility or resistance to S. japonicum infections.

Characterization and expression analysis of Wnt5 in Schistosoma japonicum at different developmental stages.

Wnt signaling is a key pathway involving the regulation of cell development and growth in metazoa. An analysis of Wnt signaling in Schistosoma japonicum might provide information regarding the molecular mechanisms underlying parasite development, which might be useful for vaccine screening and identification of pharmaceutical targets. The SjWnt5 gene, a member of the Wnt gene family, contained an 1149-bp open reading frame that encoded a 382-aa protein. Analysis of the SjWnt5 amino acid sequence revealed a domain that was conserved among members of the Wnt protein family. Expression of SjWnt5 was observed at all of the developmental stages in definitive hosts, and the highest level of SjWnt5 messenger RNA (mRNA) was detected at the schistosomula stage. Higher levels of SjWnt5 mRNA and protein were observed in mature male worms, compared with those in mature females. SjWnt5 mRNA was expressed at higher levels in maldeveloped worms from nonpermissive host or single-sex infection than in normal worms from permissive host and mixed-sex infection. The immunohistochemical analysis showed that SjWnt5 protein was expressed in the subtegumental musculature and acetabulum musculature of schistosomulum and adult worms, suggesting that SjWnt5 may play a role in regulation of parasite muscle development. Furthermore, SjWnt5 was found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female, suggesting that SjWnt5 may involve in the development of the reproductive organs of both sexes.

Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein.

Schistosomiasis is a worldwide parasitic disease, and while it can be successfully treated with chemotherapy, this does not prevent reinfection with the parasite. Adenovirus vectors have been widely used for vaccine delivery, and a vaccination approach has the potential to prevent infection with Schistosoma. Here, we developed a recombinant adenoviral vector that expresses Schistosoma japonicum inhibitor apoptosis protein (Ad-SjIAP) and assessed its immunoprotective functions against schistosomiasis in mice. Murine immune responses following vaccination were investigated using enzyme-linked immunosorbent assays (ELISA), lymphocyte proliferation, and cytokine assays. The protective immunity in mice was evaluated by challenging with S. japonicum cercariae. Our results indicated that immunization with the Ad-SjIAP in mice induced a strong serum IgG response against IAP including IgG1, IgG2a, and IgG2b. In addition, lymphocyte proliferation experiments showed that mice treated with Ad-SjIAP significantly increased the lymphocyte response upon stimulation with recombinant Schistosoma japonicum inhibitor apoptosis protein (rSjIAP). Moreover, cytokine assays indicated that vaccination of Ad-SjIAP significantly increased the production of interferon (IFN)-γ and IL-2 as compared to the corresponding control group. Furthermore, following the challenge with S. japonicum cercariae, the vaccine conferred moderate protection, with an average rate of 37.95% for worm reduction and 31.7% for egg reduction. Taken together, our preliminarily results suggested that schistosoma IAP may be a potential vaccine against S. japonicum and that adenoviral vectors may serve as an alternative delivery vehicle for schistosome vaccine development.

Microarray analysis of gene expression profiles of Schistosoma japonicum derived from less-susceptible host water buffalo and susceptible host goat.

BACKGROUND: Water buffalo and goats are natural hosts for S. japonicum in endemic areas of China. The susceptibility of these two hosts to schistosome infection is different, as water buffalo are less conducive to S. japonicum growth and development. To identify genes that may affect schistosome development and survival, we compared gene expression profiles of schistosomes derived from these two natural hosts using high-throughput microarray technology. RESULTS: The worm recovery rate was lower and the length and width of worms from water buffalo were smaller compared to those from goats following S. japonicum infection for 7 weeks. Besides obvious morphological difference between the schistosomes derived from the two hosts, differences were also observed by scanning and transmission electron microscopy. Microarray analysis showed differentially expressed gene patterns for parasites from the two hosts, which revealed that genes related to lipid and nucleotide metabolism, as well as protein folding, sorting, and degradation were upregulated, while others associated with signal transduction, endocrine function, development, immune function, endocytosis, and amino acid/carbohydrate/glycan metabolism were downregulated in schistosomes from water buffalo. KEGG pathway analysis deduced that the differentially expressed genes mainly involved lipid metabolism, the MAPK and ErbB signaling pathways, progesterone-mediated oocyte maturation, dorso-ventral axis formation, reproduction, and endocytosis, etc. CONCLUSION: The microarray gene analysis in schistosomes derived from water buffalo and goats provide a useful platform to disclose differences determining S. japonicum host compatibility to better understand the interplay between natural hosts and parasites, and identify schistosome target genes associated with susceptibility to screen vaccine candidates.

A non-catalytic function of Rev1 in translesion DNA synthesis and mutagenesis is mediated by its stable interaction with Rad5.

DNA damage tolerance consisting of template switching and translesion synthesis is a major cellular mechanism in response to unrepaired DNA lesions during replication. The Rev1 pathway constitutes the major mechanism of translesion synthesis and base damage-induced mutagenesis in model cell systems. Rev1 is a dCMP transferase, but additionally plays non-catalytic functions in translesion synthesis. Using the yeast model system, we attempted to gain further insights into the non-catalytic functions of Rev1. Rev1 stably interacts with Rad5 (a central component of the template switching pathway) via the C-terminal region of Rev1 and the N-terminal region of Rad5. Supporting functional significance of this interaction, both the Rev1 pathway and Rad5 are required for translesion synthesis and mutagenesis of 1,N(6)-ethenoadenine. Furthermore, disrupting the Rev1-Rad5 interaction by mutating Rev1 did not affect its dCMP transferase, but led to inactivation of the Rev1 non-catalytic function in translesion synthesis of UV-induced DNA damage. Deletion analysis revealed that the C-terminal 21-amino acid sequence of Rev1 is uniquely required for its interaction with Rad5 and is essential for its non-catalytic function. Deletion analysis additionally implicated a C-terminal region of Rev1 in its negative regulation. These results show that a non-catalytic function of Rev1 in translesion synthesis and mutagenesis is mediated by its interaction with Rad5.

Ultrastructural observation and gene expression profiling of Schistosoma japonicum derived from two natural reservoir hosts, water buffalo and yellow cattle.

Water buffalo and yellow cattle are the two of the most important natural reservoir hosts for Schistosoma japonicum in endemic areas of China, although their susceptibility differs, with water buffalo being less conducive to the growth and development of S. japonicum. Results from the current study show that the general morphology and ultrastructure of adult schistosomes derived from the two hosts also differed. Using high-throughput microarray technology, we also compared the gene expression profiles of adult schistosomes derived from the two hosts. We identified genes that were differentially expressed in worms from the two natural hosts. Further analysis revealed that genes associated with protein kinase and phosphatase, the stimulus response, and lipid and nucleotide metabolism were overexpressed, whereas genes associated with reproduction, anatomical structure morphogenesis and multifunctional motif were underexpressed in schistosomes from water buffalo. These differentially expressed genes were mainly involved in nucleotide, energy, lipid metabolism, energy metabolism, transcription, transport and signaling pathway. This suggests that they are key molecules affecting the survival and development of schistosomes in different natural host species. The results of this study add to current understanding of the interplay between parasites and their natural hosts, and provide valuable information for the screening of vaccine candidates or new drug targets against schistosomiasis in the natural reservoir hosts in endemic areas.

[Screening and verification on characteristic differentially expressed genes of Schistosoma japonicum from three reservoir hosts].

OBJECTIVE: To get the characteristic differentially expressed genes of Schistosoma japonicum from three important reservoir hosts: yellow cattle, water buffalo and goat, so as to find the genetic markers to identify the various sources of the parasite reservoir hosts. METHODS: The 49 d worms were collected from artificially infected animals, and the total RNA(s) of worms were extracted and reverse-transcripted to cDNA, and then hybridized with custom-built microarray to screen characteristic differentially expressed genes of every host, and the microarray results were validated by the real-time PCR method. RESULTS: From results of microarray, we got 3 characteristic differentially expressed genes of S. japonicum from yellow cattle, 4 from water buffalo and 7 from goat. We verified schistosome samples from three reservoir hosts in another experiment, the results showed that 2 in yellow cattle, 3 in water buffalo, and 5 in goat were verified to be consistent with microarray results. CONCLUSIONS: The ten characteristic differentially expressed genes of S. japonicum from three reservoir hosts screened by microarray might be used as genetic markers to identify the various sources of reservoir hosts for S. japonicum.