OBJECTIVE: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications.
METHODS: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA).
RESULTS: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells.
CONCLUSIONS: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
［摘要］ 目的 制备并鉴定鼠抗刚地弓形虫致密颗粒蛋白24 (dense granule protein 24, GRA24) 多克隆抗体, 并探索其初 步应用。方法 利用MEGA-X软件比对弓形虫不同虫株GRA24编码区序列, 使用Protean软件分析GRA24蛋白优势肽 段, 通过PCR反应扩增编码该肽段的碱基序列, 并连接至pET-28a载体中。将获得的GRA24截短蛋白原核表达质粒转化 于大肠埃希菌BL21感受态细胞中, 异丙基-β-D-硫代半乳糖苷 (isopropyl-beta-D-thiogalactopyranoside, IPTG) 诱导后采用 十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 检测蛋白表达 与纯化。使用纯化的GRA24截短蛋白皮下注射免疫BALB/c小鼠获得GRA24截短蛋白多克隆抗体, 采用酶联免疫吸附 试验 (enzyme-linked immunosorbent assay, ELISA) 检测抗体效价, 采用Western blotting检测抗体特异性, 并将该抗体应用 于免疫荧光试验 (immunofluorescence assay, IFA) 。结果 SDS-PAGE结果表明成功构建重组质粒, 考马斯亮蓝染色结果 显示获得高纯度重组GRA24截短蛋白。ELISA结果显示, GRA24 截短蛋白多克隆抗体效价在 1:208 400 以上; Western blotting检测发现, 该抗体可识别弓形虫不同虫株内源性 GRA24 蛋白, 特异性识别重组 GRA24 截短蛋白; 间接IFA检测发 现, 弓形虫入侵宿主细胞 16 h 后分泌的GRA24蛋白定位于宿主细胞核中。结论 成功制备广适性、高效价、强特异性的 抗弓形虫 GRA24 多克隆抗体, 可应用于 Western blotting 与 IFA, 为进一步研究 GRA24 功能奠定了基础。.

The global malaria epidemic is still severe. Because of simple procedures, rapid detection and accuracy results, rapid diagnostic test (RDT) has become the most important and the most widely used diagnostic tool for malaria prevention and control. However, deletions in the RDT target Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) genes may cause false-negative results of RDT, which has been included as one of the four biological threats to global malaria elimination. This article reviews the applications of RDT in the global malaria diagnosis, analyzes the threats and challenges caused by Pfhrp2/3 gene deletion, proposes methods for monitoring Pfhrp2/3 gene deletion, and summarizes the causes and countermeasures of negative RDT detections, so as to provide insights into consolidation of malaria elimination achievements in China and contributions to global malaria elimination.
［摘要］ 全球疟疾流行依然严峻, 疟疾快速诊断试纸条 (rapid diagnostic test, RDT) 操作简便、检测快速、结果准确, 已成为当前疟 疾防控中最重要和最广泛使用的诊断工具。但RDT靶标恶性疟 原虫富组氨酸蛋白2/3 (Plasmodium falciparum histidine-rich protein 2/3, Pfhrp2/3) 基因缺失可导致RDT产生假阴性检测结果, 被 WHO列为全球消除疟疾的四大生物学挑战之一。本文通过回顾 RDT在全球疟疾诊断中的应用, 分析Pfhrp2/3 基因缺失带来的威 胁与挑战、提出Pfhrp2/3 基因缺失的监测方法、总结RDT检测阴性 的原因与对策, 为巩固我国消除疟疾成果、助力全球消除疟疾提 供参考。.

BACKGROUND: Bone defects, resulting from substantial bone loss that exceeds the natural self-healing capacity, pose significant challenges to current therapeutic approaches due to various limitations. In the quest for alternative therapeutic strategies, bone tissue engineering has emerged as a promising avenue. Notably, excretory proteins from Toxoplasma gondii (TgEP), recognized for their immunogenicity and broad spectrum of biological activities secreted or excreted during the parasite\'s lifecycle, have been identified as potential facilitators of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Building on our previous findings that TgEP can enhance osteogenic differentiation, this study investigated the molecular mechanisms underlying this effect and assessed its therapeutic potential in vivo.
METHODS: We determined the optimum concentration of TgEP through cell cytotoxicity and cell proliferation assays. Subsequently, hBMSCs were treated with the appropriate concentration of TgEP. We assessed osteogenic protein markers, including alkaline phosphatase (ALP), Runx2, and Osx, as well as components of the BMP/Smad signaling pathway using quantitative real-time PCR (qRT-PCR), siRNA interference of hBMSCs, Western blot analysis, and other methods. Furthermore, we created a bone defect model in Sprague-Dawley (SD) male rats and filled the defect areas with the GelMa hydrogel, with or without TgEP. Microcomputed tomography (micro-CT) was employed to analyze the bone parameters of defect sites. H&E, Masson and immunohistochemical staining were used to assess the repair conditions of the defect area.
RESULTS: Our results indicate that TgEP promotes the expression of key osteogenic markers, including ALP, Runx2, and Osx, as well as the activation of Smad1, BMP2, and phosphorylated Smad1/5-crucial elements of the BMP/Smad signaling pathway. Furthermore, in vivo experiments using a bone defect model in rats demonstrated that TgEP markedly promoted bone defect repair.
CONCLUSIONS: Our results provide compelling evidence that TgEP facilitates hBMSC osteogenic differentiation through the BMP/Smad signaling pathway, highlighting its potential as a therapeutic approach for bone tissue engineering for bone defect healing.

BACKGROUND: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed.
METHODS: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 102 live oocysts of coccidia. A total of 5 × 104 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection.
RESULTS: The results showed that the proportion of CD4+ and CD8+ T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group.
CONCLUSIONS: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis.

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.

BACKGROUND: Malaria transmission-blocking vaccines (TBVs) aim to inhibit malaria parasite development in mosquitoes and prevent further transmission to the human host. The putative-secreted ookinete protein 25 (PSOP25), highly conserved in Plasmodium spp., is a promising TBV target. Here, we investigated PvPSOP25 from P. vivax as a TBV candidate using transgenic murine parasite P. berghei and clinical P. vivax isolates.
RESULTS: A transgenic P. berghei line expressing PvPSOP25 (TrPvPSOP25Pb) was generated. Full-length PvPSOP25 was expressed in the yeast Pichia pastoris and used to immunize mice to obtain anti-rPvPSOP25 sera. The transmission-blocking activity of the anti-rPvPSOP25 sera was evaluated through in vitro assays and mosquito-feeding experiments. The antisera generated by immunization with rPvPSOP25 specifically recognized the native PvPSOP25 antigen expressed in TrPvPSOP25Pb ookinetes. In vitro assays showed that the immune sera significantly inhibited exflagellation and ookinete formation of the TrPvPSOP25Pb parasite. Mosquitoes feeding on mice infected with the transgenic parasite and passively transferred with the anti-rPvPSOP25 sera showed a 70.7% reduction in oocyst density compared to the control group. In a direct membrane feeding assay conducted with five clinical P. vivax isolates, the mouse anti-rPvPSOP25 antibodies significantly reduced the oocyst density while showing a negligible influence on mosquito infection prevalence.
CONCLUSIONS: This study supported the feasibility of transgenic murine malaria parasites expressing P. vivax antigens as a useful tool for evaluating P. vivax TBV candidates. Meanwhile, the moderate transmission-reducing activity of the generated anti-rPvPSOP25 sera necessitates further research to optimize its efficacy.

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

OBJECTIVE: To perform a bibliometric analysis of researches on the Plasmodium falciparum repetitive interspersed families of polypeptides (RIFIN) protein from 1993 to 2022 and identify the hot topics in the RIFIN protein research, so as to provide insights into future researches on RIFIN protein.
METHODS: RIFIN protein-associated publications were retrieved in the Web of Science Core Collection from 1993 to 2022 and all bibliometric analyses were performed using the software CiteSpace 6.2.4.0. The annual number of RIFIN protein-associated publications was analyzed from 1993 to 2022, and country, author and institution collaboration networks were created. Keywords were extracted from RIFIN protein-associated publications for plotting keyword co-occurrence, clustering, burst and timeline maps to identify the hot topics in the RIFIN protein research.
RESULTS: A total of 745 English RIFIN protein-associated publications were included in the final bibliometric analysis, and there were 18 to 36 publications each year from 1993 to 2022. The top three countries with the highest activity in the RIFIN protein research included the United States, the United Kingdom and France, universities and research institutes were highly active in the RIFIN protein research; however, no authors were identified with a high activity in the RIFIN protein research. There were three keyword clusters in the RIFIN protein-associated publications, including repetitive DNA sequence, molecular epidemiology and antigenic variation. Keyword co-occurrence, burst and timeline analyses showed that previous RIFIN protein-associated publications mainly focused on gene properties and functions, involving keywords of repetitive DNA sequence and evolution, and recent hot topics for the RIFIN protein research shifted to genetic diversity and immune response, involving keywords of genetic diversity, antigenic variation and binding.
CONCLUSIONS: The annual number of RIFIN protein-associated publications was relatively stable from 1993 to 2022. This bibliometric analysis may provide insights into future researches on the RIFIN protein.
［摘要］ 目的 对 1993—2022 年恶性疟原虫重复散布蛋白家族 (repetitive interspersed families of polypeptides, RIFIN) 研 究文献进行文献计量学分析, 总结研究热点, 为 RIFIN 蛋白后续研究提供参考。方法 在 Web of Science 核心合集数据 库中检索 1993—2022 年RIFIN蛋白研究相关论文, 采用 CiteSpace 6.2.4.0 软件分析 1993—2022 年各年发文量并构建国 家、作者、机构合作网络图谱; 提取文献的关键词绘制关键词共现、聚类、突现和时间线图谱, 分析 RIFIN 蛋白研究热点。结果 共纳入 745 篇英文文献进行文献计量学分析, 1993—2022 年年发文量为 18~36 篇。RIFIN蛋白研究领域活跃度 居前 3 位的国家为美国、英国、法国, 活跃度较高的机构是高校和研究机构, 未发现活跃度特别高的作者。关键词共形成 重复DNA序列、分子流行病学、抗原变异等 3 个聚类。关键词共现、突现和时间线分析结果显示, 既往 RIFIN 蛋白研究主 要集中在基因性质、功能等方面, 涉及的关键词包括重复DNA序列、进化等; 近年来研究热点转向基因多样性和免疫反 应等方面, 涉及的关键词包括基因多样性、抗原变异、结合等。结论 1993—2022 年RIFIN蛋白研究相关论文逐年发文 量相对稳定, 本研究结果可望为RIFIN蛋白后续研究提供借鉴。.

Toxoplasma gondii is an opportunistic and pathogenic obligate intracellular parasitic protozoan that is widespread worldwide and can infect most warm-blooded animals, seriously endangering human health and affecting livestock production. Toxoplasmosis caused by T. gondii infection has different clinical manifestations, which are mainly determined by the virulence of T. gondii and host differences. Among the manifestations of this condition, abortion, stillbirth, and fetal malformation can occur if a woman is infected with T. gondii in early pregnancy. Here, we discuss how the T. gondii rhoptry protein affects host pregnancy outcomes and speculate on the related signaling pathways involved. The effects of rhoptry proteins of T. gondii on the placental barrier are complex. Rhoptry proteins not only regulate interferon-regulated genes (IRGs) to ensure the survival of parasites in activated cells but also promote the spread of worms in tissues and the invasive ability of the parasites. The functions of these rhoptry proteins and the associated signaling pathways highlight relevant mechanisms by which Toxoplasma crosses the placental barrier and influences fetal development and will guide future studies to uncover the complexity of the host-pathogen interactions.