Parasite diseases pose a significant threat due to the serious harm they cause to hosts, and developing affordable drugs for treatment presents enormous challenges. "Target repurposing" has emerged as a promising strategy to overcome this difficulty by leveraging the drug targets for human diseases and matching them with homologs in parasites. The PI3K/AKT signaling pathway, known for its crucial role in regulating malignant cell proliferation, migration, and invasion, has been validated as a drug target for cancer. Interestingly, the PI3K/AKT signaling pathway is also involved in parasite development, infection, and survival within the hosts. During infection, parasites can exploit the host's PI3K/AKT signaling, which influences immune cell apoptosis, macrophage activation, regulatory T cell functions, and the communication between immune cells, thus facilitating chronic infection. The immunoregulatory functions of the PI3K/AKT signaling make its inhibitors promising candidates for the control of parasite diseases. This review mainly outlines the characteristics and functions of the PI3K/AKT signaling pathway during parasite infection and further provides an overview of the drugs targeting the PI3K/AKT signaling pathway for treating parasite diseases.

Cyst echinococcosis, caused by Echinococcus granulosus, remains a zoonotic disease posing a great threat to public health and meat production industry. Sheep infected with E. granulosus show relatively high abundance of egr-miR-71 in the sera, but its role is unknown. Using bioinformatics and cell migration and Transwell assays, we comparatively analyzed the proteomes and cell invasion of sheep PBMCs in response to egr-miR-71 overexpression. The results showed that the egr-miR-71 induced a total of 157 proteins being differentially expressed and mainly involved in immune responses. In sheep PBMCs, egr-miRNA-71 overexpression induced significant downregulation of macrophage migration inhibitory factor (MIF) and accordingly promoted cell migration and invasion compared with the control. The results will provide a clue for further investigation of a role of circulating egr-miR-71 in immune responses during E. granulosus infection.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1β and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Taenia hydatigena, a globally distributed parasite, is a canine tapeworm and causes huge economic losses in the food industry. Using LC-MS/MS, the proteomes of T. hydatigena cyst scolex, designated as CS, and the cyst without the scolex, designated as CWS, were profiled and a total of 764 different proteins were identified, 664 of which were identified in CS, 412 identified in CWS, and 312 in both. Comparative analysis revealed that CS had more abundant proteins associated with growth and development, while CWS had more abundant proteins constituting a scaffolding and protective extracellular matrix. Consistent with the sequencing data, the abundance of the five selected proteins was validated to be higher in CWS than CS by Western blotting. The current data will provide a clue for further pinpointing a role of these proteins in the biology of T. hydatigena.

为了探究细粒棘球绦虫来源的miR-71(egr-miR-71)对绵羊外周血单核细胞(PBMCs)的免疫调节作用,利用egr-miR-71模拟物和阴性对照分别转染绵羊PBMCs,通过qPCR分析了对绵羊PBMCs中细胞因子和LPS/TLR4信号通路中关键基因表达的影响,并且还利用Griess试剂测定了NO的分泌水平.结果 显示,egr-miR-71的过表达会显著上调CD14,为阴性对照组的2.21倍(P＜0.01),但IL-1α、IL-1β、TNF-α的表达则显著下调,分别是阴性对照组的74％、59％和76％(P＜0.05).NO的测定结果还显示,egr-miR-71能明显促进NO的生成(P＜0.05).以上结果提示,egr-miR-71对绵羊PBMCs具有免疫调节作用.

为了探究细粒棘球绦虫(Echinococcus granulosus)来源的miR-71(egr-miR-71)对绵羊外周血单核细胞(PBMCs)蛋白组的影响.本研究使用密度梯度离心法分离得到绵羊PBMCs,并使用电穿孔法将egr-miR-71转染至绵羊PBMCs,用qPCR检测转染效率.基于iTRAQ方法,对比分析egr-miR-71过表达对绵羊PBMCs蛋白表达的影响,通过qPCR和Western-blotting对骨髓源性生长因子(MYDGF)转录水平和翻译水平进行验证.结果 显示,绵羊PBMCs转染egr-miR-71模拟物后,miR-71的相对表达量极显著上调(P＜0.01).利用iTRAQ共鉴定出7 642个蛋白质,其中157个呈差异表达,与对照组相比,68个蛋白显著上调,89个蛋白显著下调.下调的蛋白主要包括巨噬细胞迁移抑制因子(MIF)、MYDGF、酰基辅酶A结合蛋白(ACBP)和碳酸酐酶(CAs)等.这些差异蛋白主要参与丝裂原活化蛋白激酶(MAPK)、转化生长因子-β(TGF-β)等信号通路.qPCR和Western-blotting结果显示,与对照组相比,模拟物转染组中MYDGF转录和翻译水平均显著下调(P＜0.05).通过对转染egr-miR-71后绵羊PBMCs的蛋白进行组学分析,初步了解了egr-miR-71对绵羊PMBCs表达蛋白的调控,鉴定出的差异蛋白可能在细粒棘球蚴与宿主互作中发挥重要作用.这为深入研究细粒棘球绦虫的感染机制提供了思路,也为新型抗包虫病药物的研发提供了理论依据.

流产衣原体(Chlamydiaabortus)广泛分布于世界各地,能感染猪、牛、羊等多种家畜,引起孕畜流产,是危害畜牧业最严重的衣原体病原.针对流产衣原体抗体检测的血清学方法如间接血凝试验(IHA)、酶联免疫吸附试验(ELISA)等在兽医临床检测中广泛使用.以聚合酶链反应(PCR)为代表的病原分子生物学检测技术敏感性和特异性高,有效地弥补了血清学方法的不足.在流行病学研究中,进一步对流行菌株的分型分析在阐释流产衣原体遗传进化、毒力演变和跨物种传播以及疫情预警和疫苗研制等方面具有重要意义.论文就当前国内外广泛应用的流产衣原体检测技术和分型方法进行综述,可为我国动物衣原体病的诊断和防控提供参考.

目的 分析在胰岛素、阿苯达唑和人工胃液刺激下,细粒棘球蚴原头节miR-71的分泌表达特征. 方法 从新疆屠宰场采集绵羊肝、肺细粒棘球蚴包囊,经固定、包埋及切片后,用miR-71探针进行杂交.与异硫氰酸荧光素(FITC)标记的抗Digoxin抗体(Anti-Digoxin-FITC)作用,经4',6-二脒基-2-苯基吲哚(DAPI)核酸染料染色后,在荧光显微镜下观察miR-71在包囊壁和原头节中的定位,分析miR-71在原头节中的分布,确定miR-71在原头节中的表达情况.在人工胃液(人工胃液组)、胰岛素(胰岛素组)或阿苯达唑(阿苯达唑组)等不同条件下培养原头节,收集上清并用差速离心法分离外泌体,采用透射电镜观察外泌体形态结构,利用纳米粒径分析仪测定外泌体的粒径分布;利用外泌体生物标志分子烯醇化酶和14-3-3蛋白的抗体蛋白质免疫印迹(Western blotting)鉴定外泌体;采用实时荧光定量PCR (qPCR)检测外泌体中miR-71的丰度. 结果 原位杂交结果显示,miR-71在囊壁生发层和原头节中均有表达.人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体粒径分别为48.9、49.7和65.4 nm,均在40～120 nm内;外泌体形态呈由膜包裹的球形.Western blotting 从外泌体中检出烯醇化酶和14-3-3蛋白,表明外泌体提取成功.qPCR结果显示,人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体中,miR-71的表达量分别是其对照组的1.84、1.87和2.38倍(t=12.8、26.7、29.3,均P＜ 0.01). 结论 miR-71在细粒棘球蚴囊壁和原头节中广泛表达,且可以通过外泌体进行分泌;经胰岛素、人工胃液和阿苯达唑刺激后其分泌表达水平升高.

[目的]对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.[方法]以绵羊痘病毒甘肃古浪株DNA为模板,通过PCR扩增RING finger基因.利用Pfam数据库、DNAstar等软件进行序列及遗传进化分析;将RING finger基因片段插入原核表达载体pGEX-4T-1中,构建pGEX-SPPVRFP重组表达载体,在大肠杆菌BL21 (DE3)中诱导表达,并进行SDS-PAGA和Western-blot分析.[结果]绵羊痘病毒RING finger基因由723个核苷酸组成,编码的240个氨基酸的分子量约为28.5 ku,具有RING finger结构域.不同羊痘病毒株间RING finger基因核苷酸序列同源性高达99.2％,氨基酸序列同源性高达98.3％.不同的RING finger蛋白都含有8个保守的半胱氨酸和组氨酸.SDS-PAGA分析显示,重组SPPVRFP大小约为55 ku,Westem-blot分析显示,重组SPPVRFP不能与绵羊痘病毒阳性血清反应.[结论]成功克隆、表达并纯化了绵羊痘病毒RING finger基因,对SPPV GS-GL株RING-finger蛋白进行序列分析,推测其可能具有E3泛素连接酶活性.