Geraldo Santana Magalhães

The salivary glands of hematophagous arthropods possess a remarkable molecular diversity, reflecting evolutionary adaptations to blood feeding lifestyle and vector-host interactions. In this study, we characterized the salivary-gland transcriptome of adult female Simulium pertinax , a medically important black fly species widely distributed in South America. Through RNA sequencing and bioinformatics analyses, we identified 15,708 high-quality transcripts, of which 1947 clusters were related to secreted proteins, distributed across 23 distinct functional families, including digestive enzymes, protease inhibitors, antimicrobial peptides, and hemostasis modulators. Phylogenetic analyses of selected hematophagy-associated families, including Kunitz-domain inhibitors, Apyrases, Kazal-type inhibitors, Serpins, and SVEP homologs, revealed a combination of conserved evolutionary relationships and lineage-specific diversification among simuliids. Notably, the SVEP ( Simulium Vasodilator Erythema Protein) family was highly represented and appears to be restricted to black flies. Given that its biological role remains largely uncharacterized, we selected a novel SVEP variant for heterologous expression and functional characterization, which was designated as recombinant Simulium pertinax Vasoactive Protein (rSpVaP). This recombinant protein elicited vasodilator activity in the arterioles of the mouse cremaster muscle, as well as significant paw edema. These findings support rSpVaP as a biologically active SVEP-related salivary vasoactive protein in simuliids, underscoring its contribution to facilitating blood-feeding. Taken together, these results highlight the value of combining transcriptomic profiling with functional characterization to uncover the molecular adaptations underlying hematophagy in black flies, enhancing our understanding of vector-host interactions and the evolutionary pressures shaping the sialotranscriptome.

Arapaima gigas growth hormone (ag-GH) cDNA was previously cloned from A. gigas pituitaries. In this work ag-GH has been synthesized using human embryonic kidney 293 cells (HEK293) transiently transfected with the 3.4-TOPO^® vector carrying ag-GH cDNA. The 4th day after transfection, the presence of putative ag-GH was detected via SDS-PAGE and Western blotting in comparison with human GH. Ion exchange purification exhibited a clearly symmetric peak, absent in the control medium. The purified fraction, submitted to high-performance size-exclusion chromatography (HPSEC), SDS-PAGE, and Western blotting, contained an immunoreactive molecule, slightly smaller than hGH as expected. MALDI-TOF-MS determined a high-resolution molecular mass of 21,220 Da versus a theoretical value of 21,150. A phylogenetic analysis positioned ag-GH within basal teleost lineages, consistent with earlier analyses of A. gigas gonadotrophic hormones, reinforcing the structural and functional conservation relevant for its biologic activity. An in vivo bioassay based on the body weight increase of dwarf “little” mice demonstrated a biological activity for ag-GH comparable to that of the international reference preparation of rec-hGH. For two species (H. sapiens and A. gigas) separated by an evolutionary period of >100 million years, such a positive biological correlation is remarkable.

Background/Objective: Cationic polymers were shown to assemble with negatively charged proteins yielding nanoparticles (NPs). Poly-diallyl-dimethyl-ammonium chloride (PDDA) combined with ovalbumin (OVA) yielded a stable colloidal dispersion (OVA/PDDA-NPs) eliciting significant anti-OVA immune response. Dendritic cells (DCs), as sentinels of foreign antigens, exert a crucial role in the antigen-specific immune response. Here, we aimed to evaluate the involvement of DCs in the immune response induced by OVA/PDDA. Methods: In vivo experiments were used to assess the ability of OVA/PDDA-NPs to induce anti-OVA antibodies by ELISA, as well as plasma cells and memory B cells using flow cytometry. Additionally, DC migration to draining lymph nodes following OVA/PDDA-NP immunization was evaluated by flow cytometry. In vitro experiments using bone marrow-derived DCs (BM-DCs) were used to analyze the binding and uptake of OVA/PDDA-NPs, DC maturation status, and their antigen-presenting capacity. Results: Our data confirmed the potent effect of OVA/PDDA-NPs inducing anti-OVA IgG1 and IgG2a antibodies with increased CD19⁺CD138⁺ plasma cells and CD19⁺CD38⁺CD27⁺ memory cells in immunized mice. OVA/PDDA-NPs induced DC maturation and migration to draining lymph nodes. The in vitro results showed higher binding and the uptake of OVA/PDDA-NPs by BM-DCs. In addition, the NPs were able to induce the upregulation of costimulatory and MHC-II molecules on DCs, as well as TNF-α and IL-12 production. Higher OVA-specific T cell proliferation was promoted by BM-DCs incubated with OVA/PDDA-NPs. Conclusions: The data showed the central role of DCs in the induction of antigen-specific immune response by OVA-PDDA-NPs, thus proving that these NPs are a potent adjuvant for subunit vaccine design.

Jararhagin-C (JarC) is a protein from the venom of Bothrops jararaca consisting of disintegrin-like and cysteine-rich domains. JarC shows a modulating effect on angiogenesis and remodeling of extracellular matrix constituents, improving wound healing in a mouse experimental model. JarC is purified from crude venom, and the yield is less than 1%. The aim of this work was to obtain the recombinant form of JarC and to test its biological activity. For this purpose, the bicistronic vector pSUMOUlp1 was used. This vector allowed the expression of the recombinant toxin JarC (rJarC) in fusion with the small ubiquitin-related modifier (SUMO) as well as the SUMO protease Ulp1. After expression, this protease was able to efficiently remove SUMO from rJarC inside the bacteria. rJarC free from SUMO was purified at the expected molecular mass and recognized by polyclonal anti-jararhagin antibodies. In terms of biological activity, both the native and recombinant forms showed no toxicity to the HUVEC cell line CRL1730 and were effective in modulating cell migration activity in the experimental in vitro model. These results demonstrate the successful production of rJarC and the preservation of its biological activity, which may facilitate further investigations into the therapeutic potential of this snake venom-derived protein.

Cationic polymers were previously shown to assemble with negatively charged proteins yielding nanoparticles (NPs). Poly-diallyl-dimethyl-ammonium chloride (PDDA) is a cationic polymer able to combine with ovalbumin (OVA) yielding a stable colloidal dispersion of OVA/PDDA NPs eliciting significant anti-OVA immune response. Dendritic Cells (DC), as sentinels of foreign antigens, exert a crucial role in the induction of antigen-specific T cell activation and consequent adaptive immune response. Objective: The present study aimed at evaluating the involvement of DCs in the adaptive immune response induced by OVA/PDDA. Methods/Results: Confirming the potent induction of adaptive immune response against OVA/PDDA, the data showed increased CD19+CD138+ plasma cells and CD19+CD38+CD27+ memory cells in spleens of mice immunized with OVA/PDDA-NPs 28 days before. OVA/PDDA-NPs also induced the migration and maturation of DCs to draining lymph nodes on days 3 and 4 of mice immunization. The in vitro results with bone-marrow differentiated DCs (iBM-DCs) showed an increase of the binding and uptake of OVA/PDDA NPs by these cells compared with soluble OVA. In addition, OVA/PDDA NPs were able to induce DC maturation and upregulation of costimulatory and MHC-II molecules, TNF- and IL-12 production. iBM-DCs incubated with OVA/PDDA NPs promoted high OVA-specific T cell proliferative response. Conclusion: Altogether, the data demonstrated the central role of DCs in the induction of antigen-specific immune response by OVA-PDDA-NPs, thus proving these NPs as potent adjuvants for subunit vaccine design.

Jararhagin-C (JarC) is a protein from the venom of Bothrops jararaca consisting of disintegrin-like and cysteine-rich domains. JarC shows a modulating effect on angiogenesis and remodeling of extracellular matrix constituents improving wound healing in mice experimental model. JarC is purified from crude venom and the yield is less than 1 %. The aim of this work was to obtain the recombinant form of JarC and to test its biological activity. For this purpose, the bicistronic vector pSUMOUlp1 was used. This vector allowed the expression of the recombinant toxin JarC (rJarC) in fusion with the small ubiquitin-related modifier (SUMO) as well as the SUMO protease Ulp1. After expression, this protease was able to efficiently remove SUMO from rJarC inside the bacteria. rJarC free from SUMO was purified at the expected molecular mass, and recognized by polyclonal anti-Jararhagin antibodies. In terms of biological activity, both the native and recombinant forms showed no toxicity to the HUVEC cell line CRL1730 and were effective in modulating cell migration activity in experimental in vitro model. These results demonstrate the successful production of rJarC and the preservation of its biological activity, which may facilitate further investigations into the therapeutic potential of this snake venom-derived protein.

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.