In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice\'s splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

The large-scale proteomic analysis of Dictyostelium discoideum has contributed to our understanding of intracellular as well as secreted proteins in this versatile model eukaryote. Mass spectrometry-based proteomic analysis is a robust, sensitive, and rapid analytical method for identification and characterization of proteins extracted from tissues, cells, cell fractions, or pull-down assays. The availability of core facilities which make proteomics inexpensive and easy to do has facilitated a wide range of research projects. In this chapter, we present a simple standard methodology to extract proteins and prepare samples from D. discoideum for mass spectrometry and methods to analyze the identified proteins.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium\'s proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.

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.