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  1. Article ; Online: Impact of Genetic Diversity and Genome Plasticity of

    Santi, Ana Maria Murta / Murta, Silvane Maria Fonseca

    Frontiers in cellular and infection microbiology

    2022  Volume 12, Page(s) 826287

    Abstract: Leishmaniasis is one of the major public health concerns in Latin America, Africa, Asia, and Europe. The absence of vaccines for human use and the lack of effective vector control programs make chemotherapy the main strategy to control all forms of the ... ...

    Abstract Leishmaniasis is one of the major public health concerns in Latin America, Africa, Asia, and Europe. The absence of vaccines for human use and the lack of effective vector control programs make chemotherapy the main strategy to control all forms of the disease. However, the high toxicity of available drugs, limited choice of therapeutic agents, and occurrence of drug-resistant parasite strains are the main challenges related to chemotherapy. Currently, only a small number of drugs are available for leishmaniasis treatment, including pentavalent antimonials (Sb
    MeSH term(s) Amphotericin B/pharmacology ; Antiprotozoal Agents/pharmacology ; Antiprotozoal Agents/therapeutic use ; Genetic Variation ; Humans ; Leishmania ; Leishmaniasis/drug therapy
    Chemical Substances Antiprotozoal Agents ; Amphotericin B (7XU7A7DROE)
    Language English
    Publishing date 2022-01-24
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2619676-1
    ISSN 2235-2988 ; 2235-2988
    ISSN (online) 2235-2988
    ISSN 2235-2988
    DOI 10.3389/fcimb.2022.826287
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Antioxidant defence system as a rational target for Chagas disease and Leishmaniasis chemotherapy.

    Santi, Ana Maria Murta / Murta, Silvane Maria Fonseca

    Memorias do Instituto Oswaldo Cruz

    2022  Volume 117, Page(s) e210401

    Abstract: Chagas disease and leishmaniasis are neglected tropical diseases caused by the protozoan parasites Trypanosoma cruzi and Leishmania spp., respectively. They are among the most important parasitic diseases, affecting millions of people worldwide, being a ... ...

    Abstract Chagas disease and leishmaniasis are neglected tropical diseases caused by the protozoan parasites Trypanosoma cruzi and Leishmania spp., respectively. They are among the most important parasitic diseases, affecting millions of people worldwide, being a considerable global challenge. However, there is no human vaccine available against T. cruzi and Leishmania infections, and their control is based mainly on chemotherapy. Treatments for Chagas disease and leishmaniasis have multiple limitations, mainly due to the high toxicity of the available drugs, long-term treatment protocols, and the occurrence of drug-resistant parasite strains. In the case of Chagas disease, there is still the problem of low cure rates in the chronic stage of the disease. Therefore, new therapeutic agents and novel targets for drug development are urgently needed. Antioxidant defence in Trypanosomatidae is a potential target for chemotherapy because the organisms present a unique mechanism for trypanothione-dependent detoxification of peroxides, which differs from that found in vertebrates. Cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of trypanothione, involving different enzymes that act in concert. This study provides an overview of the antioxidant defence focusing on iron superoxide dismutase A, tryparedoxin peroxidase, and ascorbate peroxidase and how the enzymes play an important role in the defence against oxidative stress and their involvement in drug resistance mechanisms in T. cruzi and Leishmania spp.
    MeSH term(s) Animals ; Antioxidants/therapeutic use ; Chagas Disease/drug therapy ; Humans ; Leishmania ; Leishmaniasis/drug therapy ; Trypanosoma cruzi
    Chemical Substances Antioxidants
    Language English
    Publishing date 2022-02-28
    Publishing country Brazil
    Document type Journal Article ; Review
    ZDB-ID 953293-6
    ISSN 1678-8060 ; 0074-0276
    ISSN (online) 1678-8060
    ISSN 0074-0276
    DOI 10.1590/0074-02760210401
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Deletion of the lipid droplet protein kinase gene affects lipid droplets biogenesis, parasite infectivity, and resistance to trivalent antimony in Leishmania infantum.

    Ribeiro, Juliana Martins / Silva, Paula Alves / Costa-Silva, Héllida Marina / Santi, Ana Maria Murta / Murta, Silvane Maria Fonseca

    PLoS neglected tropical diseases

    2024  Volume 18, Issue 1, Page(s) e0011880

    Abstract: The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized ... ...

    Abstract The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized CRISPR/Cas9 technology to generate LDK-knockout lines of Leishmania infantum to investigate its role in this parasite. Our findings demonstrate that LDK is not an essential gene for L. infantum, as its deletion did not impede parasite survival. Furthermore, removing LDK did not impact the growth of promastigote forms of L. infantum lacking LDK. However, a noticeable reduction in LDs occurred during the stationary phase of parasite growth following LDK deletion. In the presence of myriocin, a LD inducer, LDK-knockout parasites displayed reduced LD abundance during both logarithmic and stationary growth phases compared to control parasites. Moreover, an infection analysis involving THP-1 cells revealed that 72 h post-infection, LDK-knockout L. infantum lines exhibited fewer infected macrophages and intracellular amastigotes than control parasites. LDK-knockout L. infantum lines also displayed 1.7 to 1.8 -fold greater resistance to trivalent antimony than control parasites. There were no observed alterations in susceptibility to amphotericin B, miltefosine, or menadione in LDK-knockout L. infantum lines. Our results suggest that LDK plays a crucial role in the biogenesis and/or maintenance of LDs in L. infantum, as well as in parasite infectivity and resistance to trivalent antimony.
    MeSH term(s) Animals ; Leishmania infantum/physiology ; Parasites ; Antimony/pharmacology ; Lipid Droplets ; Parasitic Diseases ; Protein Kinases
    Chemical Substances Antimony (9IT35J3UV3) ; Protein Kinases (EC 2.7.-)
    Language English
    Publishing date 2024-01-18
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2429704-5
    ISSN 1935-2735 ; 1935-2735
    ISSN (online) 1935-2735
    ISSN 1935-2735
    DOI 10.1371/journal.pntd.0011880
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations.

    Lima, Davi Alvarenga / Gonçalves, Leilane Oliveira / Reis-Cunha, João Luís / Guimarães, Paul Anderson Souza / Ruiz, Jeronimo Conceição / Liarte, Daniel Barbosa / Murta, Silvane Maria Fonseca

    Parasites & vectors

    2023  Volume 16, Issue 1, Page(s) 167

    Abstract: Background: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in ... ...

    Abstract Background: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD.
    Methods: All complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c-2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis.
    Results: The analytical pipeline with an adjusted P-value of < 0.05 and fold-change > 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation-reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes.
    Conclusions: The transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD.
    MeSH term(s) Humans ; Trypanosoma cruzi ; Trypanocidal Agents/pharmacology ; Transcriptome ; Gene Expression Profiling ; Chagas Disease/drug therapy ; Nitroimidazoles/pharmacology
    Chemical Substances benzonidazole (YC42NRJ1ZD) ; Trypanocidal Agents ; Nitroimidazoles
    Language English
    Publishing date 2023-05-22
    Publishing country England
    Document type Journal Article
    ZDB-ID 2409480-8
    ISSN 1756-3305 ; 1756-3305
    ISSN (online) 1756-3305
    ISSN 1756-3305
    DOI 10.1186/s13071-023-05775-4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Perspectives From Systems Biology to Improve Knowledge of

    Horácio, Elvira Cynthia Alves / Hickson, Jéssica / Murta, Silvane Maria Fonseca / Ruiz, Jeronimo Conceição / Nahum, Laila Alves

    Frontiers in cellular and infection microbiology

    2021  Volume 11, Page(s) 653670

    Abstract: Neglected Tropical Diseases include a broad range of pathogens, hosts, and vectors, which represent evolving complex systems. Leishmaniasis, caused by ... ...

    Abstract Neglected Tropical Diseases include a broad range of pathogens, hosts, and vectors, which represent evolving complex systems. Leishmaniasis, caused by different
    MeSH term(s) Animals ; Drug Resistance ; Humans ; Leishmania ; Leishmaniasis ; Psychodidae ; Systems Biology
    Language English
    Publishing date 2021-04-30
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2619676-1
    ISSN 2235-2988 ; 2235-2988
    ISSN (online) 2235-2988
    ISSN 2235-2988
    DOI 10.3389/fcimb.2021.653670
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility.

    Santi, Ana Maria Murta / Silva, Paula Alves / Santos, Isabella Fernandes Martins / Murta, Silvane Maria Fonseca

    Parasites & vectors

    2021  Volume 14, Issue 1, Page(s) 366

    Abstract: Background: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this ... ...

    Abstract Background: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.
    Results: We obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A
    Conclusions: The unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.
    MeSH term(s) Antimony/pharmacology ; Antiprotozoal Agents/pharmacology ; Down-Regulation ; Leishmania infantum/drug effects ; Leishmania infantum/enzymology ; Leishmania infantum/genetics ; Mutation ; Oxidative Stress/drug effects ; Phosphorylcholine/analogs & derivatives ; Phosphorylcholine/pharmacology ; Superoxide Dismutase/genetics
    Chemical Substances Antiprotozoal Agents ; Phosphorylcholine (107-73-3) ; miltefosine (53EY29W7EC) ; Antimony (9IT35J3UV3) ; Superoxide Dismutase (EC 1.15.1.1)
    Language English
    Publishing date 2021-07-15
    Publishing country England
    Document type Journal Article
    ZDB-ID 2409480-8
    ISSN 1756-3305 ; 1756-3305
    ISSN (online) 1756-3305
    ISSN 1756-3305
    DOI 10.1186/s13071-021-04838-8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: New 2-nitroimidazole-N-acylhydrazones, analogs of benznidazole, as anti-Trypanosoma cruzi agents.

    Pitombeira, Marcelly C S R / Júnior, Policarpo A S / Murta, Silvane Maria Fonseca / Romanha, Alvaro / Luccas, Pedro H / Nonato, M Cristina / Rocha, Rafael E O / Ferreira, Rafaela S / da Silveira, Flávia F / Castelo-Branco, Frederico S / Carvalho, Alcione S / Boechat, Nubia

    Archiv der Pharmazie

    2024  , Page(s) e2400059

    Abstract: Chagas disease is a neglected tropical parasitic disease caused by the protozoan Trypanosoma cruzi. Worldwide, an estimated 8 million people are infected with T. cruzi, causing more than 10,000 deaths per year. Currently, only two drugs, nifurtimox and ... ...

    Abstract Chagas disease is a neglected tropical parasitic disease caused by the protozoan Trypanosoma cruzi. Worldwide, an estimated 8 million people are infected with T. cruzi, causing more than 10,000 deaths per year. Currently, only two drugs, nifurtimox and benznidazole (BNZ), are approved for its treatment. However, both are ineffective during the chronic phase, show toxicity, and produce serious side effects. This work aimed to obtain and evaluate novel 2-nitroimidazole-N-acylhydrazone derivatives analogous to BNZ. The design of these compounds used the two important pharmacophoric subunits of the BNZ prototype, the 2-nitroimidazole nucleus and the benzene ring, and the bioisosterism among the amide group of BNZ and N-acylhydrazone. The 27 compounds were obtained by a three-step route in 57%-98% yields. The biological results demonstrated the potential of this new class of compounds, since eight compounds were potent and selective in the in vitro assay against T. cruzi amastigotes and trypomastigotes using a drug-susceptible strain of T. cruzi (Tulahuen) (IC
    Language English
    Publishing date 2024-04-16
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 6381-2
    ISSN 1521-4184 ; 0365-6233 ; 1437-1014
    ISSN (online) 1521-4184
    ISSN 0365-6233 ; 1437-1014
    DOI 10.1002/ardp.202400059
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article: Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility

    Santi, Ana Maria Murta / Silva, Paula Alves / Santos, Isabella Fernandes Martins / Murta, Silvane Maria Fonseca

    Parasites & vectors. 2021 Dec., v. 14, no. 1

    2021  

    Abstract: BACKGROUND: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this ... ...

    Abstract BACKGROUND: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids. RESULTS: We obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A⁻/⁻/⁺ L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A⁻/⁻/⁺ L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A⁻/⁻/⁺ mutants tested. The FeSOD-A⁻/⁻/⁺ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A⁻/⁻/⁺ mutant clones than in the WT parasite. CONCLUSIONS: The unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.
    Keywords Leishmania infantum ; Western blotting ; amphotericin B ; aneuploidy ; antimony ; antioxidant activity ; ascorbate peroxidase ; drug development ; gene amplification ; genes ; humans ; hydrogen peroxide ; macrophages ; menadione ; mutants ; oxidative stress ; oxygen ; parasites ; pathogenicity ; reverse transcriptase polymerase chain reaction ; superoxide dismutase
    Language English
    Dates of publication 2021-12
    Size p. 366.
    Publishing place BioMed Central
    Document type Article
    ZDB-ID 2409480-8
    ISSN 1756-3305
    ISSN 1756-3305
    DOI 10.1186/s13071-021-04838-8
    Database NAL-Catalogue (AGRICOLA)

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  9. Article ; Online: Anti-

    Cruz, Mariza Gabriela Faleiro de Moura Lodi / Santi, Ana Maria Murta / Morais-Teixeira, Eliane de / Caldeira, Alisson Samuel Portes / Siqueira, Ezequias Pessoa de / Oliveira, Edward / Alves, Tânia Maria de Almeida / Murta, Silvane Maria Fonseca

    Antimicrobial agents and chemotherapy

    2023  Volume 68, Issue 1, Page(s) e0050923

    Abstract: The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds ... ...

    Abstract The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated
    MeSH term(s) Cricetinae ; Animals ; Mice ; Antiprotozoal Agents/pharmacology ; Antiprotozoal Agents/therapeutic use ; Fluorescence ; Leishmaniasis/drug therapy ; Leishmania infantum/genetics ; Leishmania braziliensis/genetics ; Mesocricetus ; Mice, Inbred BALB C
    Chemical Substances tdTomato ; Antiprotozoal Agents
    Language English
    Publishing date 2023-12-08
    Publishing country United States
    Document type Journal Article
    ZDB-ID 217602-6
    ISSN 1098-6596 ; 0066-4804
    ISSN (online) 1098-6596
    ISSN 0066-4804
    DOI 10.1128/aac.00509-23
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Ascorbate peroxidase overexpression protects Leishmania braziliensis against trivalent antimony effects.

    Moreira, Douglas de Souza / Xavier, Mariana Vieira / Murta, Silvane Maria Fonseca

    Memorias do Instituto Oswaldo Cruz

    2018  Volume 113, Issue 12, Page(s) e180377

    Abstract: Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the ... ...

    Abstract Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.
    MeSH term(s) Antimony/pharmacology ; Antiprotozoal Agents/pharmacology ; Ascorbate Peroxidases/metabolism ; Blotting, Western ; Drug Resistance ; Gene Expression Regulation, Enzymologic ; Leishmania braziliensis/drug effects ; Leishmania braziliensis/enzymology ; Oxidative Stress ; Parasitic Sensitivity Tests ; Phenotype ; Protozoan Proteins/metabolism
    Chemical Substances Antiprotozoal Agents ; Protozoan Proteins ; Antimony (9IT35J3UV3) ; Ascorbate Peroxidases (EC 1.11.1.11)
    Language English
    Publishing date 2018-11-14
    Publishing country Brazil
    Document type Journal Article
    ZDB-ID 953293-6
    ISSN 1678-8060 ; 0074-0276
    ISSN (online) 1678-8060
    ISSN 0074-0276
    DOI 10.1590/0074-02760180377
    Database MEDical Literature Analysis and Retrieval System OnLINE

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