Expression profile of microRNAs in bovine lymphocytes infected with Theileria annulata and treated with buparvaquone.

Several miRNA-based studies on Theileria-transformed bovine cells have been conducted; however, the mechanism by which transformed cells exhibit uncontrolled proliferation is not yet fully understood. Therefore, it is necessary to screen more microRNAs that may play a role in the transformation process of host cells infected with Theileria annulata to better understand the transformation mechanisms of Theileria-infected cells. RNA sequencing was used to analyze miRNAs expression in the host bovine lymphocytes infected with T. annulata at different time points after buparvaquone (BW720) treatment and DMSO treatment (control groups). Differential miRNAs related to cell proliferation and apoptosis were identified through comparison with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and a regulatory network of miRNA-mRNA was constructed. In total, 272 differentially expressed miRNAs were found at 36, 60 and 72 h. The miRNAs change of bta-miR-2285t, novel-miR-622, bta-miR-2478, and novel-miR-584 were significant. Analysis of 27 of these co-differential expressed miRNAs revealed that 15 miRNAs were down-regulated and 12 miRNAs were up-regulated. A further analysis of the changes in the expression of each of these 27 miRNAs in the three datasets suggested that bta-miR-2285t, bta-miR-345-5p, bta-miR-34a, bta-miR-150, and the novel-miR-1372 had significantly changed. Predicted target genes for these 27 miRNAs were analyzed by KEGG and the results demonstrated that EZR, RASSF, SOCS1 were mainly enriched in the signaling pathway microRNAs in cancer. MAPKAPK2, RELB, FLT3LG, and GADD45B were mainly enriched in the MAPK signaling pathway, and some genes were enriched in Axon guidance. This study has provided valuable information to further the understanding of the regulatory function of miRNAs in the host microenvironment and host-parasite interaction mechanisms.

Molecular Detection and Characterization of Cryptosporidium spp. and Giardia duodenalis in Dairy Calves from Ningxia, China.

PURPOSE: Cryptosporidium spp. and Giardia duodenalis are two important foodborne human and animal parasites that can be disseminated through both food and water, leading to diarrheal disease. Nevertheless, available information on the circumstances of Cryptosporidium and Giardia duodenalis from Ningxia is limited. METHODS: A total of 208 stool samples of dairy calves derived from large-scale farms (> 1000 heads) of five cities randomly in Ningxia were gathered randomly, were amplified and analyzed by nested PCR based on the three target genes (18S rRNA, gp60 and tpi)and phylogenetic systematics. RESULTS: The prevalence of cryptosporidiosis and giardiasis in dairy calves in Ningxia were 13.0% (27/208 samples, 95% CI 9.1-18.2%) and 1.9% (4/208, 95% CI 0.8-4.9%) respectively. Three Cryptosporidium species appeared in this study which are Cryptosporidium parvum (C. parvum), Cryptosporidium andersoni (C. andersoni) and Cryptosporidium ryanae (C. ryanae) based on the 18S rRNA gene sequence. IIdA15G1 and IIdA13G1 belonging to the subtypes of Cryptosporidium were detected by the gp60 PCR. The genotypes of Giardia duodenalis were only assemblage E through the amplification of the triosephosphate-isomerase gene (tpi gene). CONCLUSION: There is a risk of transmission to humans in Ningxia because of zoonotic genotypes (C. parvum, C. andersoni, assemblage E) and subtypes (IId) of Cryptosporidium spp. and G. duodenalis in dairy calves, and it is necessary to pay attention to the disease to prevent a widespread epidemic of the disease with the purpose to protect human and livestock health.

Molecular Characterization of Tick-borne Pathogens in Bactrian Camels and Ticks from Gansu Province, China.

PURPOSE: Ticks are dangerous ectoparasites for humans and other animals, and tick-borne pathogens of Bactrian camels have been epidemiologically surveyed in Gansu Province, China. We aimed to determine the current distribution of tick-borne pathogens among Bactrian camels in Gansu during August 2013 using molecular tools. METHODS: All ticks underwent morphological identification. We extracted DNA from the blood samples and ticks, screened them for Theileria, Babesia, Anaplasma, and Ehrlichia using standard or nested PCR with specific primers. RESULTS: All ticks collected from the skin were identified as Hyalomma asiaticum. The blood and tick samples harbored similar pathogens, including the Theileria species, T. annulata, T. luwenshuni, T. uilenbergi, and T. capreoli, the Anaplasma species A. bovis and uncultured Anaplasma, the Ehrlichia species E. canis and uncultured Ehrlichia, and a new haplotype of Babesia species. CONCLUSION: Our findings of anaplasmataceae and piroplasmida in Bactrian camels in Gansu provide a theoretical basis for deeper investigation into the epidemiology of tick-borne pathogens in these camels.

Changes in TFG gene expression in bovine leucocytes transformed by Theileria annulata.

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

Metabolomic profiling of bovine leucocytes transformed by Theileria annulata under BW720c treatment.

BACKGROUND: When Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear. METHODS: The transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC-QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found. RESULTS: A total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids. CONCLUSION: BW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and L-carnitine, and induces host cell apoptosis.

Development and application of an indirect ELISA to detect antibodies to Neospora caninum in cattle based on a chimeric protein rSRS2-SAG1-GRA7.

Neospora caninum is an important apicomplexan parasite causing neosporosis in cattle. The disease is recognized as one of the most important cause of reproductive problems and abortion in cattle worldwide. In this context, we developed an indirect enzyme-linked immunosorbent assays (ELISA) with chimeric protein rSRS2-SAG1-GRA7 to diagnose antibodies to Neospora-infection. This indirect ELISA was compared to indirect fluorescent antibody test (IFAT) and western blotting (WB), and the sensitivity and specificity results of ELISA were calculated to be 86.7 and 96.1%, respectively. The overall coincidence rate was 92.6% using IFAT and WB. Additionally, 329 aborting dairy cattle serum samples were tested using this ELISA to evaluate the prevalence of N. caninum in Ningxia, China. The positive rate of N. caninum in these farms was from 19.05 to 57.89%, and the mean rate was 41.64% (±11.01%), indicating that infection with N. caninum may be one of the important causes of cattle abortion in this region. This established rSRS2-SAG1-GRA7 indirect ELISA is capable for detecting the antibodies against N. caninum, and it could be a useful screening tool for monitoring the epidemiology of neosporosis in cattle.

Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs).

BACKGROUND: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. METHODS: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. RESULTS: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. CONCLUSIONS: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ) and asthenozoospermia (AZ) usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control), with only complete methylation and mild hypomethylation(<50% unmethylated CpGs) identified, and there was no significant difference in the occurrence of these two methylation patterns between AZ and NZ (P>0.05). However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs ) and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05%) and AZ (0.58±1.77%) (P<0.001).Additional, our data indicated the occurrence of >20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05). In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

The human leukocyte antigen G promotes trophoblast fusion and ß-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines.

The human leukocyte antigen G (HLA-G) is expressed on the fetal-maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell-cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (ß-hCG) were elevated. HLA-G up-regulates ß-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in ß-hCG compared with control cells. The defect in ß-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating ß-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

[Construction of eukaryotic expression vector and package of lentivirus vector encoding human leukocyte antigen-G5].

AIM: To construct a lentiviral expression vector carrying HLA-G5. METHODS: The CDs region of HLA-G5 gene was cloned into the lentiviral vector by restriction endonuclease Nhe I/Not I digestion and T4DNA ligase ligation. After transformation into completent E.coli cells, the candidate clones were identified by PCR and kidney cell line 293T cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. RESULTS: The lentiviral vector pCDH-CMV-MCS-EF1-copGFP Vector for HLA-G5 was constructed successfully, and the virus in the supernatant reached a titer of TU/mL. CONCLUSION: This research completed the package of lentivirus vector encoding HLA-G5 as a tool for further study.

Rapid and efficient reprogramming of human amnion-derived cells into pluripotency by three factors OCT4/SOX2/NANOG.

Reprogramming human somatic cells to pluripotency represents a valuable resource for research aiming at the development of in vitro models for human diseases and regenerative medicines to produce patient-specific induced pluripotent stem (iPS) cells. Seeking appropriate cell resources for higher efficiency and reducing the risk of viral transgene activation, especially oncogene activation, are of significance for iPS cell research. In this study, we tested whether human amnion-derived cells (hADCs) could be rapidly and efficiently reprogrammed into iPS cells by the defined factors: OCT4/SOX2/NANOG. hADCs from normal placenta were isolated and cultured. The 3rd passage cells were infected with the lentiviral vectors for the delivery of OCT4, SOX2, and NANOG. Afterwards, the generated iPSCs were identified by morphology, pluripotency markers, global gene expression profiles, and epigenetic status both in vitro and in vivo. The results showed that we were able to reprogram hADCs by the defined factors (OCT4/SOX2/NANOG). The efficiency was significantly high (about 0.1%), and the typical colonies appeared on the 9th day after infection. They were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and the epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into various cell types of all three germ layers both in vitro and in vivo. These results demonstrate that hADCs were an ideal somatic cell resource for the rapid and efficient generation of iPS cells by OCT4/SOX2/NANOG.

[Expression of fms-like tyrosine kinase receptor 1 in placenta of pre-eclampsia].

OBJECTIVE: To investigate the expression of fms-like tyrosine kinase receptor 1 (Flt-1) in placentas of pre-eclampsia. METHODS: The expression of Flt-1 mRNA in the placentas from 20 pre-eclampsia patients and 20 pregnant women with normal blood pressure was detected by semi-quantitative reverse transcription-polymerase chain reaction. The protein expression of Flt-1 was analyzed using western blot in 18 pre-eclampsia patients and 18 normotensive pregnant women. RESULTS: Placental Flt-1 mRNA level in pre-eclampsia was 2.25 +/- 0.19 (intensity ratios of Flt-1 mRNA to beta-actin mRNA), significantly higher than in normotensive pregnant women 1.23 +/- 0.29 (P < 0.05). Western blot showed that Flt-1 protein level in pre-eclamptic placenta was 2.67 +/- 1.19 [western blot signal intensity ratios of Flt-1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], significantly higher than in pregnant women with normal blood pressure 0.94 +/- 0.51 (P < 0.05). CONCLUSION: Increased Flt-1 expression in pre-eclamptic placenta may be involved in pathogenesis of pre-eclampsia.

[Alteration and potential role of soluble fms-like tyrosine kinase receptor 1 in preeclampsia].

OBJECTIVE: To investigate the alteration of serum soluble fms-like tyrosine kinase receptor 1 (sFlt-1) and the possible source in preeclampsia, and the relationship between sFlt-1 and the pathogenesis of preeclampsia. METHODS: (1) Semi-quantitative RT-PCR was carried out to detect the level of sFlt-1 mRNA in placental tissue of 10 preeclampsia (preeclampsia group) and 10 normotensive pregnancies (normotensive pregnancy group). (2) Enzyme linked immunosorbent assay (ELISA) was used to detect the serum level of sFlt-1 in peripheral venous blood in preeclampsia group 1 (n = 35) and normotensive pregnancies group 1 (n = 35); the serum level of sFlt-1 of uterine vein blood in preeclampsia group 2 (n = 20) and normotensive pregnancies group 2 (n = 20); and the volume of peripheral venous blood sFlt-1 in 10 early (early pregnancy group) and 10 middle pregnancies (middle pregnancy group). RESULTS: (1) sFlt-1 mRNA of placental tissue was significantly higher in preeclampsia group (0.95 +/- 0.04) than that in normal pregnancy group (0.64 +/- 0.15). (2) The serum level of sFlt-1 of peripheral vein in preeclampsia group 1 (5640 +/- 3191) ng/L was higher than that in normal pregnancy group 1 (2194 +/- 635) ng/L. (3) The serum sFlt-1 of uterine vein in preeclampsia group 2 (7673 +/- 2296) ng/L was higher than that in normotensive pregnancy group 2 (3057 +/- 785) ng/L, indicating that the volume of sFlt-1 of uterine vein blood was significantly higher than that of peripheral venous blood (P < 0.01). (4) The serum levels of sFlt-1 in early and middle pregnancy groups were (32 +/- 20) ng/L and (994 +/- 302) ng/L, respectively, showing that the level of sFlt-1 in peripheral venous blood increasingly elevated with the development of pregnancy. CONCLUSIONS: (1) The placenta may be the major source of elevated sFlt-1. (2) The serum level of sFlt-1 is related with the development of pregnancy. The alteration of sFlt-1 may contribute to the pathogenesis of preeclampsia.