ABSTRACT
Abstract Leishmaniasis is a zoonotic disease caused by over 20 species of protozoan parasites of the genus Leishmania. Infection is commonly spread by sandflies and produces a wide spectrum of clinical signs and symptoms. Therefore, from an epidemiological and therapeutic standpoint, it is important to detect and differentiate Leishmania spp. The objective of this study was to combinate in silico and in vitro strategies to evaluate the analytical specificity of primers previously described in the literature. According to electronic PCR (e-PCR) analysis, 23 out of 141 pairs of primers selected through literature search matched their previously reported analytical specificity. In vitro evaluation of nine of these primer pairs by quantitative PCR (qPCR) confirmed the analytical specificity of five of them at the level of Leishmania spp., L. mexicana complex or Leishmania and Viannia subgenera. Based on these findings, the combination of e-PCR and qPCR is suggested to be a valuable approach to maximize the specificity of new primer pairs for the laboratory diagnosis of infections with Leishmania spp.
Resumo As leishmanioses são zoonoses causadas por mais de 20 espécies de protozoários do gênero Leishmania. As infecções são comumente disseminadas por flebotomíneos e causam um amplo espectro de manifestações clínicas. Portanto, a detecção e diferenciação de espécies de Leishmania são importantes do ponto de vista epidemiológico e terapêutico. O objetivo deste estudo foi combinar estratégias in silico e in vitro para avaliar a especificidade analítica dos primers descritos anteriormente na literatura. De acordo com a PCR eletrônica (e-PCR), 23 dos 141 pares de primers selecionados por meio de pesquisa da literatura estavam de acordo com a especificidade analítica anteriormente relatada. A avaliação in vitro de nove desses pares de primers, por PCR quantitativa (qPCR), confirmou a especificidade analítica de cinco deles ao nível de espécie de Leishmania, do complexo L. mexicana ou dos subgêneros Leishmania e Viannia. Com base nos resultados, sugere-se que a combinação de e-PCR e qPCR é uma abordagem valiosa para a validação e maximização da especificidade de novos pares de primers para o diagnóstico laboratorial de infecções com Leishmania spp.
Subject(s)
Animals , Psychodidae , Leishmaniasis/veterinary , Leishmania/genetics , Computer Simulation , Leishmaniasis/diagnosis , DNA, Protozoan , Real-Time Polymerase Chain Reaction/veterinaryABSTRACT
Purpose To investigate the effect of EPCR on the proliferation and migration, and to explore the molecular mechanism of EPCR affecting the tumor growth and metastasis in human breast cancer cell line MCF-7. Methods MCF-7 cell was transfected with EPCR siRNA and treated with anti-PAR-1 antibody. Then CCK-8 assay was performed to determine the proliferation of MCF-7 cell. Transwell migration assay was employed to determine the cell's migration. Cell-ELISA was used to detect the activation of PAR-1 on the membranes of MCF-7. Result After EPCR siRNA transfection, the proliferation and migration ability of the MCF-7 in the interference of EPCR gene group was significantly decreased compared with the negative control and untreated control group. After treated with anti-PAR-1 antibody, the proliferation and migration of ability of MCF-7 were decreased significantly compared with the negative control group and the untreated control group. Cell-ELISA assay indicated that the activation of PAR-1 in the cells surface of MCF-7 cell in the EPCR gene interference group was mitigated versus the negative control and untreated control group. Conclusion EPCR may promote the proliferation and migration of MCF-7 cell by activating PAR-1.
ABSTRACT
Objective To investigate the genetic variants in the protein C (PC) and endothelial protein C receptor (EPCR) genes associated with the risk and outcome of acute respiratory distress syndrome (ARDS) patients in Chinese Han race.Methods Five tagSNPs (single nucleotide polymorphism,SNP) in the PC and EPCR genes were genotyped in patients with ARDS (n =275) and non-ARDS (n =337) in order to find the association between them in this case-control study.The SNPs were genotyped by SNPstream Beckman platform.Then,the correlation between the associated SNPs and plasma levels of activated protein C (APC) in patients with ARDS was investigated.The APC levels were measured using enzyme linked immunosorbent assay (ELISA) method.Results Association analysis rcvealed that two PC SNPs in perfect linkage disequilibrium,rs1799809 and rs1158867,were significantly associated with susceptibility to ARDS.T allele frequency of rs1799809 in ARDS patients was significantly higher than that in non-ARDS patients (OR =1.569,95％ CI:1.192-2.066).And the genotype frequencies of rs1799809 were also significantly different between these two groups (P =0.007).The association remained significant after adjustment for multiple comparisons.Haplotype consisting of three SNPs in the PC gene was also associated with susceptibility to ARDS.The frequency of haplotype CCC in the ARDS samples was significantly lower than that in the non-ARDS group (P ＜ 0.01).Moreover,ARDS patients canrying rs1799809 TT genotype showed lower serum levels of APC than patients with TC and CC genotypes (Padj =0.02).However,genotype and allele analyses of EPCR did not show any significant difference between ARDS and non-ARDS patients.Conclusions These findings indicated that common genetic variation in the PC gene was significantly associated with susceptibility to ARDS in Chinese Han race.The PC genetic variation influenced plasma concentration of APC in patients with ARDS.
ABSTRACT
Purpose To observe the effects of midkine ( MK) on human breast cancer cell line MDA-MB-231 angiogenesis in vitro, and to explore its mechanism. Method shRNA interference was performed to silence the expression of MK in MDA-MB-231 cells, and Western blot was used to identify the expression of MK and EPCR. After MK and EPCR knockdown, or treated with anti protease-activated receptor 1 (PAR1) antibody, the culture medium of MDA-MB-231 cells were collected and the conditioned medium were pre-pared. Human umbilical vein endothelial cells ( HUVECs) were cultured with conditioned medium, and the endothelial cells prolifera-tion was detected by CCK-8 assay, cell migration was detected by transwell method, vasculogenic activity was assessed by Matrigel-based tube formation assay. Results After knockdown of MK, the protein level of EPCR was decreased in MDA-MB-231 cells. Com-pared with control, knockdown of MK and EPCR decreased the proliferation, migration and angiogenesis ability of HUVECs significant-ly (P<0. 05), and the effect of EPCR knockdown group was stronger than MK knockdown group (P<0. 05). After treated with anti-PAR1 antibody, the proliferation, migration and angiogenesis ability of HUVECs were decreased compared with control and EPCR knockdown group (P<0. 05). Conclusion MK promotes human breast cancer cell line MDA-MB-231 angiogenesis through EPCR /PAR1 signaling pathway in vitro.
ABSTRACT
Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10-4 to 10-6 M. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.