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.

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.

The development of new compounds to treat Chagas disease is imperative due to the adverse effects of current drugs and their low efficacy in the chronic phase. This study aims to investigate nitroisoxazole derivatives that produce oxidative stress while modifying the compounds\' lipophilicity, affecting their ability to fight trypanosomes. The results indicate that these compounds are more effective against the epimastigote form of T. cruzi, with a 52 ± 4% trypanocidal effect for compound 9. However, they are less effective against the trypomastigote form, with a 15 ± 3% trypanocidal effect. Additionally, compound 11 interacts with a higher number of amino acid residues within the active site of the enzyme cruzipain. Furthermore, it was also found that the presence of a nitro group allows for the generation of free radicals; likewise, the large size of the compound enables increased interaction with aminoacidic residues in the active site of cruzipain, contributing to trypanocidal activity. This activity depends on the size and lipophilicity of the compounds. The study recommends exploring new compounds based on the nitroisoxazole skeleton, with larger substituents and lipophilicity to enhance their trypanocidal activity.

Background and Objectives: Malaria continues to be a significant global health challenge. The efficacy of artemisinin-based combination therapies (ACTs) has declined in many parts of the Greater Mekong Subregion, including Vietnam, due to the spread of resistant malaria strains. This study was conducted to assess the efficacy of the Dihydroartemisinin (DHA)-Piperaquine (PPQ) regimen in treating uncomplicated falciparum malaria and to conduct molecular surveillance of antimalarial drug resistance in Binh Phuoc and Dak Nong provinces. Materials and Methods: The study included 63 uncomplicated malaria falciparum patients from therapeutic efficacy studies (TES) treated following the WHO treatment guidelines (2009). Molecular marker analysis was performed on all 63 patients. Methods encompassed Sanger sequencing for pfK13 mutations and quantitative real-time PCR for the pfpm2 gene. Results: This study found a marked decrease in the efficacy of the DHA-PPQ regimen, with an increased rate of treatment failures at two study sites. Genetic analysis revealed a significant presence of pfK13 mutations and pfpm2 amplifications, indicating emerging resistance to artemisinin and its partner drug. Conclusions: The effectiveness of the standard DHA-PPQ regimen has sharply declined, with rising treatment failure rates. This decline necessitates a review and possible revision of national malaria treatment guidelines. Importantly, molecular monitoring and clinical efficacy assessments together provide a robust framework for understanding and addressing detection drug resistance in malaria.

The high-throughput proteomics data generated by increasingly more sensible mass spectrometers greatly contribute to our better understanding of molecular and cellular mechanisms operating in live beings. Nevertheless, proteomics analyses are based on accurate genomic and protein annotations, and some information may be lost if these resources are incomplete. Here, we show that most proteomics data may be recovered by interconnecting genomics and proteomics approaches (i.e., following a proteogenomic strategy), resulting, in turn, in an improvement of gene/protein models. In this study, we generated proteomics data from Leishmania donovani (HU3 strain) promastigotes that allowed us to detect 1908 proteins in this developmental stage on the basis of the currently annotated proteins available in public databases. However, when the proteomics data were searched against all possible open reading frames existing in the L. donovani genome, twenty new protein-coding genes could be annotated. Additionally, 43 previously annotated proteins were extended at their N-terminal ends to accommodate peptides detected in the proteomics data. Also, different post-translational modifications (phosphorylation, acetylation, methylation, among others) were found to occur in a large number of Leishmania proteins. Finally, a detailed comparative analysis of the L. donovani and Leishmania major experimental proteomes served to illustrate how inaccurate conclusions can be raised if proteomes are compared solely on the basis of the listed proteins identified in each proteome. Finally, we have created data entries (based on freely available repositories) to provide and maintain updated gene/protein models. Raw data are available via ProteomeXchange with the identifier PXD051920.

BACKGROUND: Malaria results in more than 550,000 deaths each year due to drug resistance in the most lethal Plasmodium (P.) species P. falciparum. A full P. falciparum genome was published in 2002, yet 44.6% of its genes have unknown functions. Improving the functional annotation of genes is important for identifying drug targets and understanding the evolution of drug resistance.
RESULTS: Genes function by interacting with one another. So, analyzing gene co-expression networks can enhance functional annotations and prioritize genes for wet lab validation. Earlier efforts to build gene co-expression networks in P. falciparum have been limited to a single network inference method or gaining biological understanding for only a single gene and its interacting partners. Here, we explore multiple inference methods and aim to systematically predict functional annotations for all P. falciparum genes. We evaluate each inferred network based on how well it predicts existing gene-Gene Ontology (GO) term annotations using network clustering and leave-one-out crossvalidation. We assess overlaps of the different networks\' edges (gene co-expression relationships), as well as predicted functional knowledge. The networks\' edges are overall complementary: 47-85% of all edges are unique to each network. In terms of the accuracy of predicting gene functional annotations, all networks yielded relatively high precision (as high as 87% for the network inferred using mutual information), but the highest recall reached was below 15%. All networks having low recall means that none of them capture a large amount of all existing gene-GO term annotations. In fact, their annotation predictions are highly complementary, with the largest pairwise overlap of only 27%. We provide ranked lists of inferred gene-gene interactions and predicted gene-GO term annotations for future use and wet lab validation by the malaria community.
CONCLUSIONS: The different networks seem to capture different aspects of the P. falciparum biology in terms of both inferred interactions and predicted gene functional annotations. Thus, relying on a single network inference method should be avoided when possible.
METHODS: Attached.

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here, we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and for responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

UNASSIGNED: Trypanosoma cruzi is a protozoan parasite that causes the tropical ailment known as Chagas disease, which has its origins in South America. Globally, it has a major impact on health and is transported by insect vector that serves as a parasite. Given the scarcity of vaccines and the limited treatment choices, we conducted a comprehensive investigation of core proteomics to explore a potential reverse vaccine candidate with high antigenicity.
UNASSIGNED: To identify the immunodominant epitopes, T. cruzi core proteomics was initially explored. Consequently, the vaccine sequence was engineered to possess characteristics of non-allergenicity, antigenicity, immunogenicity, and enhanced solubility. After modeling the tertiary structure of the human TLR4 receptor, the binding affinities were assessed employing molecular docking and molecular dynamics simulations (MDS).
UNASSIGNED: Docking of the final vaccine design with TLR4 receptors revealed substantial hydrogen bond interactions. A server-based methodology for immunological simulation was developed to forecast the effectiveness against antibodies (IgM + IgG) and interferons (IFN-g). The MDS analysis revealed notable levels of structural compactness and binding stability with average RMSD of 5.03 Aring;, beta-factor 1.09e+5 Å, Rg is 44.7 Aring; and RMSF of 49.50 Aring;. This is followed by binding free energies calculation. The system stability was compromised by the complexes, as evidenced by their corresponding Gibbs free energies of -54.6 kcal/mol.
UNASSIGNED: Subtractive proteomics approach was applied to determine the antigenic regions of the T cruzi. Our study utilized computational techniques to identify B- and T-cell epitopes in the T. cruzi core proteome. In current study the developed vaccine candidate exhibits immunodominant features. Our findings suggest that formulating a vaccine targeting the causative agent of Chagas disease should be the initial step in its development.