Proteins selected in Leishmania (Viannia) braziliensis by an immunoproteomic approach with potential serodiagnosis applications for tegumentary leishmaniasis

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationaryphase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-0496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, APQ-482976/2012-8, and APQ-488237/2013-0). In addition, this study was partially funded in Madrid by a Spanish grant from Ministerio de Economía y Competitividad-FEDER (FISPI14/00366 from the Instituto de Salud Carlos III)Peer Reviewe

Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad

Funding Information: This work received financial support from PT national funds ( FCT/MCTES , Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project CIRCNA/BRB/0281/2019 . Funding Information: This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project CIRCNA/BRB/0281/2019.The authors further thank FCT/MCTES for supporting Research Units LAQV-REQUIMTE (UIDB/50006/2020), GHTM (UID/Multi/04413/2020). Publisher Copyright: © 2023 The AuthorsMalaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.publishersversionepub_ahead_of_prin

Endo- and exometabolome crosstalk in mesenchymal stem cells undergoing osteogenic differentiation

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.publishe

A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis

Background: LiHyV is an antigenic hypothetical protein present in both promastigote and amastigote stages of Leishmania infantum, which was recently identified by an immunoproteomic approach. A recombinant version of this protein (rLiHyV) was evaluated as a diagnostic marker for canine VL (CVL). In addition, the prophylactic efficacy of the rLiHyV protein, and two of its CD8+ T cell epitopes, has been analyzed in a murine model of visceral leishmaniasis (VL). Methods: Initially, the rLiHyV protein was evaluated by an ELISA technique for the serodiagnosis of CVL. Secondly, vaccines composed of the recombinant protein and both chemically synthesized peptides, combined with saponin as an adjuvant; were administered subcutaneously into BALB/c mice. The cellular and humoral responses generated by vaccination were evaluated. In addition, the parasite burden and immune response were studied 10 weeks after L. infantum infection. Results: The rLiHyV protein was recognized by antibodies of VL dogs. No cross-reactivity was obtained with sera from dogs vaccinated with a Brazilian commercial vaccine, with sera from animals infected with Trypanosoma cruzi, Babesia canis and Ehrlichia canis, or those from non-infected animals living in an endemic area for leishmaniasis. After challenge with L. infantum, spleen cells of BALB/c mice vaccinated with rLiHyV/saponin stimulated with parasite antigens showed a higher production of IFN-γ, IL-12 and GM-CSF, than the same cells obtained from mice vaccinated with the individual peptides, or mice from control (inoculated with saline or saponin) groups. This Th1-type cellular response observed in rLiHyV/saponin vaccinated mice was accompanied by the induction of parasite-specific IgG2a isotype antibodies. Animals immunized with rLiHyV/saponin showed significant reductions in the parasite burden in the liver, spleen, bone marrow and in the lymph nodes draining the paws relative to control mice. Conclusions: The present study showed for the first time that the L. infantum LiHyV protein could be considered as a vaccine candidate against L. infantum infection, as well as a diagnostic marker for CVL.This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8, and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and APF are grant recipient of CNPq.Peer Reviewe

Estudo do complexo valvar atrioventricular cardíaco esquerdo em búfalos (Bubalus bubalis) da raça Jafarabadi

Atrioventricular valve complex of 30 Jafarabadi water buffaloes, adult males were studied in this research with no heart diseases. The animals were obtained from a slaughterhouse in Brazilian State of Parana. The hearts were opened at the third portion affording access to the valve complex. The complexes had its area, number and type of tendinous cords submitted to analysis. The results showed that the complex is composed by two cusps and four accessory cusps, two or three papillary muscles in which 10-25 tendinous cords fix on the cusps that face the ventricle wall. The total area of the complex was on average 38.56cm², with a minimum of 24.96cm² and a maximum of 55.54cm². Statistically, no relation between the number of cords and the cusps' area where they are inserted or with the number of papillary muscle where they originated from was observed.Foram estudados os complexos valvares atrioventricular esquerdo de 30 búfalos da raça Jafarabadi, machos e adultos, sem alterações cardíacas, provenientes de abatedouros do Estado do Paraná. Os corações foram examinados em seu terço médio para acesso ao complexo valvar, que foi submetido a estudos de área, número e tipificação de cordas tendíneas. Os resultados demonstram que este complexo é formado por duas cúspides principais e quatro cúspides acessórias, apresentam em sua formação de 2-3 músculos papilares, nos quais se inserem de 10-25 cordas tendíneas, que se fixam em cúspides voltadas para a parede do ventrículo. A área total deste complexo apresenta uma média de 38,56cm² com um mínimo de 24,96cm² e um máximo de 55,54cm². Estatisticamente não há relação entre número de cordas e a área da cúspide onde estas estão inseridas, nem com o número de músculos papilares dos quais elas provem

Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads' draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti-Leishmania IgG2a antibodies and a predominant IL-12-driven IFN-γ production (mainly produced by CD4 T cells) against parasite proteins, whereas unprotected controls showed anti-Leishmania IgG1 antibodies and parasite-mediated IL-4 and IL-10 responses. Vaccinated mice showed an anti-LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD-specific IFN-γ, IL-12 and GM-CSF cytokines before and after infection. The protection was correlated with the Leishmania-specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.Instituto Nacional de Ci^encia e Tecnologia em Nano-biofarmac^eutica (INCT-NanoBiofar), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-482976/2012-8, APQ-488237/2013-0 and APQ-467640/2014-9). In addition, this study was partially funded by the Spanish grant from Ministerio de Economía y Competitividad-FEDER (FIS PI14/00366 from the Instituto de Salud Carlos III)Peer Reviewe

Dog in sheep’s clothing: livestock depredation by free-ranging dogs may pose new challenges to wolf conservation

Livestock depredation is a common cause of human-carnivore conflicts. In Portugal, free-ranging dogs are increasingly abundant and overlap endangered Iberian wolf territories, with reports of livestock depredation. However, the lack of awareness about dogs’ possible role as predators leads to bias against wolves in cases of damages. Our goal was to assess and compare wolf and free-ranging dog’s diet composition at southern wolf range in Portugal, to offer insights on dogs’ predatory role on livestock and its implications for the conservation of an endangered wolf subpopulation. We assessed diet composition from 107 to 95 genetically confirmed wolf and dog scats, respectively, and complemented the analysis with data from 40 attacks on livestock with successful genetic predator assignment. Scat analysis highlighted goats as the most consumed dog prey in all analysed regions, with lagomorphs, small mammals, and wild boars as second most consumed in each region, respectively. Wolves mainly relied on goats and wild boars in the west, whereas in the central region they mostly fed on birds. The dietary overlap between both canids was very high (Pianka’s index O = 0.93), showing potential for competition. Additionally, we found that dogs were the sole predators detected in most attacks (62%). Our findings highlight dogs’ role as predators of livestock, and possibly also wild species, posing a further challenge to wolf conservation. Alongside adequate husbandry practices, we emphasise the need for a stronger enforcement of the legislation on dog ownership and an effective management of the stray population to reduce human-wolf conflict.info:eu-repo/semantics/publishedVersio

Potential application for antimicrobial and antileukemic therapy of a flavonoid-rich fraction of Camellia sinensis

The antimicrobial and antileukemic effect of a purified fraction of flavonoids from the leaves of Camellia sinensis was evaluated. An extraction yield of 9.77 mg.g-1 total flavonoids was recovered through a pressurized liquid extraction associated with solid-phase extraction. This fraction was tested against pathogenic microorganisms (Staphylococcus, Salmonella, and Enterococcus), considering the minimum inhibitory concentration. In addition, the human monocyte cell line THP-1, derived from a patient with acute monocytic leukemia, was used for the antitumor assay. The results show that the flavonoid-rich fraction obtained by coupling a Pressurized Liquids Extraction in-line with a Solid Phase Extraction (PLE-SPE) has a high antimicrobial effect and resulted in cell cycle blockage G0 / G1, increased DNA fragmentation, and altered leukemic cell morphology. These results suggest that a flavonoid-rich fraction obtained from Camellia sinensis can be applied as potential adjuvants in chemotherapy treatment to mitigate the side effects caused by chemotherapy or even as a supplement to cancer therapy9 página

Proteins selected in Leishmania (Viannia) braziliensis by an immunoproteomic approach with potential serodiagnosis applications for tegumentary leishmaniasis

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationaryphase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TLThis work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ- 472090/2011-9, APQ-482976/2012-8, and APQ-488237/2013-0). In addition, this study was partially funded in Madrid by a Spanish grant from Ministerio de Economía y Competitividad-FEDER (FISPI14/00366 from the Instituto de Salud Carlos III). M.A.C.F. is a grant recipient of FAPEMIG/CAPES. E.A.F.C., A.P.F., and M.O.C.R. are recipients of grants from CNP

Fighting HIV/AIDS in a developing country: lessons from a small cohort from the largest Brazilian city

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