Lipophosphoglycans from \u3cem\u3eLeishmania amazonensis\u3c/em\u3e Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly

Sr ISOTOPES BY LA-MC-ICP-MS PROCEDURES COUPLED WITH THE MACS3 REFERENCE MATERIAL IN A CORAL SAMPLE: A RECORD OF ENVIRONMENTAL CHANGES: Isótopos de Sr analisados por LA-MC-ICP-MS como material de referência MACS3 em uma amostra de coral: um registro de mudanças ambientais

The main aim of this work is to demonstrate that the Laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) is a powerful tool for the analysis of strontium (Sr) isotopes in corals. This work discusses certification strategies for Sr isotopes determination, using reference material (RM) analyses and the results treatment based on detailed data acquired in biological materials, a coral sample. To obtain reliable results, it is essential to properly adjust the mass spectrometer and laser ablation system. Adjusting the equipment to its maximum intensity does not always result in correct 87Sr/86Sr ratios. Therefore, the optimization of the mass spectrometer was performed using the reference material NIST SRM-987 (solution) and adjusting the correct Sr isotope ratio to the reference material (USGS MACS3 and NIST-612, solids) before each analytical session. The protocol applied the solid reference material USGS MACS3 with an isotopic ratio 87Sr/86Sr of 0.72000. The values obtained for this RM varied between 0.7012 and 0.7014, with a correction factor calculated between 0.990 and 0.988. In order to account for potential drifts in the mass spectrometer during an analytical session, the application of bracketing correction and the use of the most convenient reference material are suggested. The analytical uncertainty of Sr data obtained by LA-MC-ICP-MS is comparable to studies carried out on other carbonate materials. The results of ablation techniques are reproducible within the analytical error, which implies that this technique produces robust results when applied to coral carbonates. In addition, several comparative measurements of different reference materials (e.g. USGS MACS3 and NIST 612) and the comparison of the 87Sr/86Sr ratios highlight the robustness of the method. The results along the coral growth axes showed a decrease in the 87Sr/86Sr ratio from the inner to the outer layer of the coral (from 0.70920 to 0.70627), which indicate variations in the availability of particulate matter during the coral growth, probably related to local marine environmental changes.- O objetivo principal deste trabalho é demonstrar que a espectrometria de massa com plasma indutivamente acoplado e ablação a laser (LA-MC-ICP-MS) é uma ferramenta poderosa para a análise de isótopos de Sr em corais. Este trabalho discute estratégias de certificação para determinação de isótopos de Sr, usando análises de material de referência e o detalhamento do tratamento dos resultados adquiridos em materiais biológicos (coral). Para obter resultados confiáveis, é essencial ajustar adequadamente o espectrômetro de massas e o sistema de ablação a laser de forma a obter a intensidade máxima e em seguida promover as correções para obter as razões 87Sr / 86Sr corretas. Nestes termos, a otimização do espectrômetro de massa foi realizada usando o material de referência NIST SRM-987 (em solução) que em sequência foi migrado para a ablação a laser com ajuste da razão isotópica Sr correta para o material de referência (NIST-612, vidro), antes de cada sessão analítica. O protocolo incluiu a utilização material de referência sólido USGS MACS3 com razão isotópica 87Sr / 86Sr de 0,72000. Os valores obtidos para esta RM variaram entre 0,7012 e 0,7014, com fator de correção calculado entre 0,990 e 0,988. A fim de contabilizar possíveis desvios no espectrômetro de massa durante uma sessão analítica, sugere-se a aplicação de correção de bracketing e o uso do material de referência com a mesma matriz. Os resultados das técnicas de ablação são reproduzíveis dentro do erro analítico, o que indica que esta técnica produz resultados robustos quando aplicada a carbonatos de coral. Além disso, várias medições comparativas de diferentes materiais de referência (por exemplo, USGS MACS3 e NIST 612) e a comparação das suas respectivas razões 87Sr / 86Sr destacam a robustez do método. Os resultados ao longo dos eixos de crescimento do coral mostraram uma diminuição na razão 87Sr / 86Sr da camada interna para a externa do coral (de 0,70920 para 0,70627). Estes resultados sugerem variações na disponibilidade de material particulado durante o crescimento do coral, provavelmente relacionado a mudanças ambientais marinhas locais. Palavras-chave: Carbonato biológico. Isótopos de Sr. LA-MC-ICP-MS. Materiais de referência. Metodologia. Ambiente marinho. &nbsp

Blood-stage antiplasmodial activity and oocyst formation-blockage of metallo copper-cinchonine complex

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes’ midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite

It Takes Two to Tango, Part II : Synthesis of A-Ring Functionalised Quinones Containing Two Redox-Active Centres with Antitumour Activities

In 2021, our research group published the prominent anticancer activity achieved through the successful combination of two redox centres (ortho-quinone/para-quinone or quinone/seleniumcontaining triazole) through a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The combination of two naphthoquinoidal substrates towards a synergetic product was indicated, but not fully explored. Herein, we report the synthesis of 15 new quinone-based derivatives prepared from click chemistry reactions and their subsequent evaluation against nine cancer cell lines and the murine fibroblast line L929. Our strategy was based on the modification of the A-ring of paranaphthoquinones and subsequent conjugation with different ortho-quinoidal moieties. As anticipated, our study identified several compounds with IC50 values below 0.5 µM in tumour cell lines. Some of the compounds described here also exhibited an excellent selectivity index and low cytotoxicity on L929, the control cell line. The antitumour evaluation of the compounds separately and in their conjugated form proved that the activity is strongly enhanced in the derivatives containing two redox centres. Thus, our study confirms the efficiency of using A-ring functionalized para-quinones coupled with ortho-quinones to obtain a diverse range of two redox centre compounds with potential applications against cancer cell lines. Here as well, it literally takes two for an efficient tango

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.Centro Regional de Estudios Genómico

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

<p>Abstract</p> <p>Background</p> <p><it>Leishmania (Viannia) shawi </it>parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from <it>L. (V.) shawi </it>promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained.</p> <p>Methods</p> <p>F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated.</p> <p>Results</p> <p>The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8⁺T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4⁺central memory T lymphocytes and activation of both CD4⁺and CD8⁺T cells. In addition, F1-immunized groups showed an increase in IgG2a levels.</p> <p>Conclusions</p> <p>The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.</p