RESUMO
Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron- and calcium-rich conditions. qRT-PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up-regulation of the iNOS and NOX-2 genes as well as the generation of NO and H2 O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro-inflammatory cytokines and a downregulation of anti-inflammatory cytokines, as well as up-regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway.
Assuntos
Doenças dos Bovinos , Tritrichomonas foetus , Animais , Bovinos , Feminino , Camundongos , Gravidez , Cálcio/metabolismo , Doenças dos Bovinos/parasitologia , Citocinas/metabolismo , Ferro/metabolismo , Macrófagos , Proteínas de Membrana/metabolismo , Fator 88 de Diferenciação Mieloide , Receptor 4 Toll-Like , Tritrichomonas foetus/genética , Tritrichomonas foetus/metabolismoRESUMO
Tritrichomonas foetus is a flagellated parasite that primarily infects the reproductive tissues of livestock, causing bovine trichomoniasis. The cytoplasmic membrane of T. foetus contains various compounds that contribute to adherence, colonization, and pathogenicity. Metronidazole (MTZ) is the main treatment for trichomoniasis, but the emergence of drug-resistant strains is a concern due to improper use and dosing. T. foetus infection induces inflammation, and macrophages are key players in the immune response. However, our understanding of the host's immune response to T. foetus is limited, and the specific mechanisms underlying these responses are not well understood. This study aimed to investigate the impact of T. foetus surface proteins from trophozoites cultured under different sublethal MTZ conditions (MTZ-treated T. foetus MPs) on macrophage activation. By analyzing cytokine levels and gene expression in murine macrophages, we demonstrated that MTZ-treated T. foetus MPs induce a specific proinflammatory response. MTZ-treated T. foetus MPs-exposed macrophages exhibited a higher NO and H2 O2 production and overexpression of iNOS and NOX-2 genes in comparison to untreated T. foetus. Additionally, MTZ-treated T. foetus MPs triggered a significant induction of the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and IFN-γ, as well as the overexpression of the TLR4, MyD88, and NF-κB genes on murine macrophages. The study aimed to unravel the immunological response and potential proinflammatory pathways involved in T. foetus infection and MTZ stress. Understanding the immune responses and mechanisms through which T. foetus surface proteins activate macrophages can contribute to the development of new therapeutic strategies for controlling bovine trichomoniasis.
Assuntos
Tricomoníase , Tritrichomonas foetus , Animais , Bovinos , Camundongos , Metronidazol/farmacologia , Citocinas , Macrófagos , Proteínas de MembranaRESUMO
BACKGROUND: Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). METHODS AND RESULTS: 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. CONCLUSIONS: The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.
Assuntos
Impressões Digitais de DNA , Genética Populacional , Humanos , Frequência do Gene/genética , México , Impressões Digitais de DNA/métodos , Repetições de Microssatélites/genéticaRESUMO
Testicular cancer is the most prevalent tumor among males aged 15 to 35, resulting in a significant number of newly diagnosed cases and fatalities annually. Non-coding RNAs (ncRNAs) have emerged as key regulators in various cellular processes and pathologies, including testicular cancer. Their involvement in gene regulation, coding, decoding, and overall gene expression control suggests their potential as targets for alternative treatment approaches for this type of cancer. Furthermore, epigenetic modifications, such as histone modifications, DNA methylation, and the regulation by microRNA (miRNA), have been implicated in testicular tumor progression and treatment response. Epigenetics may also offer critical insights for prognostic evaluation and targeted therapies in patients with testicular germ cell tumors (TGCT). This comprehensive review aims to present the latest discoveries regarding the involvement of some proteins and ncRNAs, mainly miRNAs and lncRNA, in the epigenetic aspect of testicular cancer, emphasizing their relevance in pathogenesis and their potential, given the fact that their specific expression holds promise for prognostic evaluation and targeted therapies.
Assuntos
MicroRNAs , Neoplasias Embrionárias de Células Germinativas , Neoplasias Testiculares , Masculino , Humanos , Neoplasias Testiculares/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Epigênese Genética , Neoplasias Embrionárias de Células Germinativas/genéticaRESUMO
BACKGROUND: Macrophages play an important role in the inflammatory response towards pathogens and their effector functions depend on the mode of activation which is mediated by recognition of pathogen-associated molecular patterns, as peptides. Trichomonas vaginalis provokes an inflammatory response in the host in which macrophages are the first line of defense. This study aimed to analyze the effect of a specific peptide derived from the transporter TvZIP8 of T. vaginalis on the activation of macrophages. METHODS: An in silico approach based on computational prediction of epitopes was applied to detect potential murine MHC class II-restricted peptides from TvZIP8 that can activate macrophages and the immunomodulatory activity was evaluated by in vitro stimulation of murine macrophages. RESULTS: Based on binding scores, one peptide denominated TvZIP8-pep was selected for further analysis. In vitro stimulation with synthetic TvZIP8-pep triggered on murine macrophages the NO and H2O2 production and an overexpression of iNOS and NOX-2 genes. Also, a significant increase of pro-inflammatory cytokines: IL-1ß, IL-6, and TNF-α, as well as, overexpression of the TLR4, MyD88, and NF-κB genes and NF-κB activation were observed on macrophages after stimulation with TvZIP8-pep in vitro. Moreover, higher levels of IFN-γ were detected in co-cultures using CD4 + T cells with TvZIP8-pep-stimulated macrophages. CONCLUSION: These results support the potential of TvZIP8 as a promising antigen to stimulates a specific macrophage response against T. vaginalis, but further analyses are required to evaluate their possible potential as a novel antigen for immunodiagnosis and/or vaccine against trichomoniasis.
Assuntos
Antígenos de Bactérias/imunologia , Ativação de Macrófagos , Tricomoníase , Animais , Citocinas , Camundongos , Trichomonas vaginalisRESUMO
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most prevalent non-viral sexually transmitted infection that affects over 170 million people worldwide. The only type of drug recommended for the therapeutic control of trichomoniasis is the 5-nitroimidazoles, although there have been reports of some undesirable side effects and clinical resistance. Hence, the need for the search for new tricomonicidal agents is necessary. In a previous work, we demonstrated that two 2-amino-4-aryl thiazole derivatives (ATZ-1 and ATZ-2) possess a portent antigiardial effect. In the current paper, we investigated the in vitro antitrichomonal activity of these thiazole compounds. Both ATZ-1 and ATZ-2 reduced the viability and growth of parasites in a dose-dependent manner, with an IC50 value of 0.15 µg/mL and 0.18 µg/mL, respectively. Furthermore, both thiazole compounds were able to decrease the proteolytic activity in T. vaginalis trophozoites compared with untreated parasites. Interestingly, a full proteolytic inhibition profile was observed in the 50-kDa region which was associated with the decreased expression of the gene that codes for the trichomonad protease TvMP50. The docking simulations predicted strong interactions of the thiazole compounds in the TvMP50 protease's active site, suggesting a possible role as protease inhibitors. Our results demonstrate the potential of 2-amino-4-aryl thiazole derivatives as trichomonicidal compounds and could be, mechanistically, involved in the inhibition of key trichomonad proteases.
Assuntos
Antitricômonas/farmacologia , Inibidores de Proteases/farmacologia , Tiazóis/farmacologia , Tricomoníase/tratamento farmacológico , Trichomonas vaginalis/efeitos dos fármacos , Humanos , Testes de Sensibilidade Parasitária , Tricomoníase/parasitologiaRESUMO
Zinc is an essential micronutrient that plays an important role as a co-factor to several proteins, including zinc-responsive transcription factors. Trichomonas vaginalis is able to survive in the presence of high zinc concentrations in the male urogenital tract. Several genes in T. vaginalis have been shown to respond to changes in zinc concentrations, however, the zinc-dependent mechanism remains undetermined. Recently, we identified in T. vaginalis the zinc finger protein, TvZNF1, which is an ortholog of the mammal metal transcription factor (MTF1). We searched for several of the zinc-responsive genes in T. vaginalis to determine whether if they contain metal response elements (MRE), cis-acting DNA elements that specifically bind MTF1. Six highly conserved over-represented sequence motifs (TvMREs), which share similarity with other eukaryotic MREs, were identified in the zinc-responsive genes in T. vaginalis. We also demonstrated that some of the TvMREs assemble as divalent complexes either as two closely spaced TvMREs or as two overlapping TvMREs forming a palindromic-like sequence: TGCC(N3)GGCA. Electrophoretic mobility shift assays were used to detect the zinc-dependent binding of TvZNF1 and nuclear proteins from T. vaginalis to this specific palindromic motif. Our results support a novel mechanism used by T. vaginalis for the transcriptional regulation of associated zinc-responsive genes through a MTF1/MRE-like system.
Assuntos
Fatores de Transcrição/genética , Trichomonas vaginalis/genética , Zinco/análise , Elementos de Resposta , Zinco/metabolismoRESUMO
Epigenomics refers to the study of genome-wide changes in epigenetic mechanisms including DNA methylation, histone modifications and non-coding RNAs expression. The alterations in normal DNA methylation and histone acetylation/deacetylation patterns lead to deregulated transcription and chromatin organization resulting in altered gene expression profiles that facilitates tumor development and progression. In consequence, novel therapeutic strategies aimed at reversing aberrant epigenetic marks in cancer cells have been developed and used in recent molecular studies and clinical trials. Pharmaco-epigenomics is a research area, which refers to the study of epigenome changes in cancer development and how chemotherapeutic agents can reverse these aberrant epigenetic marks by targeting the epigenetic machinery. Besides, the effects of genome-wide polymorphisms in populations leading to variations in drug response are also study subject of pharmaco-epigenomics and are being studied extensively in cancer. Recent findings showed that drug response could be largely influenced by the presence of aberrant epigenetic marks of the whole genome. This implies that biological pathways and cellular processes are under the impact of epigenome status. However, data about the relationship between drug response and the epigenomic variations is still scarce mainly because the epigenome is highly variable between individuals. The present chapter reviewed the advances on the epigenetics changes mainly DNA methylation and histones modifications on cervical and breast human cancers. A special emphasis in how they could be used as targets for the development and use of novel drugs in cancer therapy is delineated.
Assuntos
Epigenômica , Farmacogenética , Pesquisa Translacional Biomédica , Metilação de DNA , Epigenômica/tendências , Humanos , Neoplasias/fisiopatologia , Neoplasias/terapia , Farmacogenética/tendências , Pesquisa Translacional Biomédica/tendênciasRESUMO
Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common nonviral sexually transmitted infection worldwide. Although drug treatment is available, unpleasant side effects and increased resistance to the nitroimidazole family have been documented. Hence, there is a need for the identification of new and safe therapeutic agents against T. vaginalis. Antimicrobial activity of anthraquinone compounds has been reported by a number of authors. The genus Morinda is well known for the diversity of anthraquinones with numerous biological activities. A new anthraquinone, lucidin-ω-isopropyl ether, was isolated from the roots of Morinda panamensis Seem. The structure of the compound was determined by 1 H and 13 C Nuclear Magnetic Resonance (NMR) analyses, in addition to comparison with literature reports. Using in vitro susceptibility assay, the half inhibitory concentration (IC50 ) of lucidin-ω-isopropyl ether for T. vaginalis (1.32 µg/mL) was found similar to that of metronidazole concentration tested (6 µM = 1.03 µg/mL). In addition, this anthraquinone was capable of inhibiting the parasite's ability to kill HeLa cells and decreased proteolytic activity of the proteinase TvMP50 from T. vaginalis. This was associated with the decreased expression of the mp50 gene. These results demonstrate the trichomonicidal potential by lucidin-ω-isopropyl ether. Further action-mode studies are necessary to elucidate the antiparasitic mechanism of this new anthraquinone to develop a more potent antitrichomonal agent.
Assuntos
Antraquinonas/farmacologia , Antitricômonas/farmacologia , Morinda , Extratos Vegetais/farmacologia , Raízes de Plantas , Trichomonas vaginalis/efeitos dos fármacos , Antraquinonas/isolamento & purificação , Antitricômonas/isolamento & purificação , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Extratos Vegetais/isolamento & purificação , Trichomonas vaginalis/metabolismoRESUMO
Trichomoniasis is a common sexually transmitted infection caused by Trichomonas vaginalis, which actually does not exist a vaccine for control or prevention. Thus, the identification of new and potent immunogens in T. vaginalis, which can contribute to the development of a vaccine against this parasite, is necessary. Therefore, the aim of this work was to evaluate the potential of a recombinant Transient Receptor Potential-like channel of T. vaginalis (TvTRPV), as a promising immunogen in BALB/c mice. First, TvTRPV was cloned and expressed as a recombinant protein in Escherichia coli BL21 cells and purified by nickel affinity. Next, BALB/c mice were immunized and the antibody levels in mice serum and cytokines from the supernatant of macrophages and from co-culture systems were evaluated. Recombinant TvTRPV triggered high levels of specific total IgG in sera from the immunized mice. Also, a statistically significant increase of cytokines: IL-1ß, IL-6, and TNF-α after stimulation with the corresponding antigens in vitro, was identified. Moreover, co-cultures using CD4+ T cells from immunized mice were able to identify higher levels of IL-10 and IFN-γ. These results were useful to validate the immunogenicity of TvTRPV in BALB/c mice, where IL-10-IFN-γ-secreting cells could play a role in infection control, supporting the potential of TvTRPV as a promising target for vaccine against T. vaginalis.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Vacinas Protozoárias/imunologia , Canais de Cátion TRPV/imunologia , Trichomonas vaginalis/enzimologia , Animais , Antígenos de Protozoários/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPV/genética , Tricomoníase/prevenção & controle , Trichomonas vaginalis/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5 kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7 M urea with 38% α-helix and 14% ß-sheet in solution and a molecular weight of 40.5 kD. Tv-PSP1 models were used to perform dynamic simulations over 100 ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.
Assuntos
Percloratos/metabolismo , Proteínas de Protozoários/análise , Proteínas de Ligação a RNA/análise , Ribonucleases/análise , Trichomonas vaginalis/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Proteínas de Protozoários/genética , Proteínas de Ligação a RNA/genética , Ribonucleases/genéticaRESUMO
Infection with Trichomonas vaginalis produces a malodorous seropurulent vaginal discharge due to several chemicals, including polyamines. The presence of 1,4-diamino-2-butanone (DAB) reduces the amount of intracellular putrescine by 90%, preventing the cotransport of exogenous spermine. DAB-treated parasites present morphological changes, which are restored by adding exogenous putrescine into the culture medium. However, the effect of polyamines over the trichomonad proteomic profile is unknown. In this study, we used a proteomic approach to analyze the polyamine-depletion and restoration effect by exogenous putrescine on T. vaginalis proteome. In the presence of inhibitor DAB, we obtained 369 spots in polyamine-depleted condition and observed 499 spots in the normal culture media. With DAB treatment, the intensity of 43 spots was increased but was found to be reduced in 39 spots, as compared to normal conditions. Interestingly, in DAB-treated parasites restored with a medium with added exogenous putrescine, 472 spots were found, of which 33 were upregulated and 63 were downregulated in protein intensity. Some of these downregulated proteins in DAB-treated parasites are involved in several cellular pathways such as glycolysis, glycolytic fermentation, arginine dihydrolase pathway, redox homeostasis, host cell binding mediated by carbohydrate, chaperone function, and cytoskeletal remodeling. Interestingly, the intensity of some of the proteins was restored by adding exogenous putrescine. In conclusion, the presence of DAB altered the proteomic profile of T. vaginalis, resulting in a decrease in the intensity of 130 proteins and an increase in the intensity of 43 proteins that was restored by the addition of putrescine.
Assuntos
Proteoma/efeitos dos fármacos , Putrescina/análogos & derivados , Putrescina/metabolismo , Espermina/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Meios de Cultura/metabolismo , Regulação para Baixo , Feminino , Proteômica/métodos , Putrescina/farmacologia , Vagina/química , Vagina/parasitologiaRESUMO
The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.
Assuntos
Dedos de Zinco CYS2-HIS2 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Trichomonas vaginalis/genética , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta , Fatores de Transcrição/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/metabolismo , Zinco/metabolismoRESUMO
Trichomonas vaginalis, a human urogenital tract parasite, is capable of surviving in the male microenvironment, despite of the presence of Zn(2+). Concentrations > 1.6 mM of Zn(2+) have a trichomonacidal effect; however, in the presence of ≤1.6 mM Zn(2+), several trichomonad proteins are up- or down-regulated. Herein, we analyzed the proteome of a T. vaginalis male isolate (HGMN01) grown in the presence of Zn(2+) and found 32 protein spots that were immunorecognized by male trichomoniasis patient serum. Using mass spectrometry (MS), the proteins were identified and compared with 23 spots that were immunorecognized in the proteome of a female isolate using the same serum. Interestingly, we found a 50-kDa metallopeptidase (TvMP50). Unexpectedly, this proteinase was immunodetected by the serum of male trichomoniasis patients but not by the female patient serum or sera from healthy men and women. We analyzed the T. vaginalis genome and localized the mp50 gene in locus TVAG_403460. Using an RT-PCR assay, we amplified a 1320-bp mp50 mRNA transcript that was expressed in the presence of Zn(2+) in the HGMN01 and CNCD147 T. vaginalis isolates. According to a Western blot assay, native TvMP50 was differentially expressed in the presence of Zn(2+). The TvMP50 proteolytic activity increased in the presence of Zn(2+) in both isolates and was inhibited by EDTA but not by ptosyl-L-lysine chloromethyl ketone (TLCK), E64, leupeptin, or phenylmethane sulfonyl fluoride. Furthermore, the recombinant TvMP50 had proteolytic activity that was inhibited by EDTA. These data suggested that TvMP50 is immunogenic during male trichomoniasis, and Zn(2+) induces its expression.
Assuntos
Antígenos de Protozoários/metabolismo , Metaloproteases/metabolismo , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/fisiologia , Antígenos de Protozoários/genética , Feminino , Humanos , Masculino , Metaloproteases/genética , Proteômica , Proteínas de Protozoários/genética , Tricomoníase/genética , Tricomoníase/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Zinco/farmacologiaRESUMO
As etiological agents of malaria disease, Plasmodium spp. parasites are responsible for one of the most severe global health problems occurring in tropical regions of the world. This work involved compiling marine cyanobacteria metabolites reported in the scientific literature that exhibit antiplasmodial activity. Out of the 111 compounds mined and 106 tested, two showed antiplasmodial activity at very low concentrations, with IC50 at 0.1 and 1.5 nM (peptides: dolastatin 10 and lyngbyabellin A, 1.9% of total tested). Examples of chemical derivatives generated from natural cyanobacterial compounds to enhance antiplasmodial activity and Plasmodium selectivity can be found in successful findings from nostocarboline, eudistomin, and carmaphycin derivatives, while bastimolide derivatives have not yet been found. Overall, 57% of the reviewed compounds are peptides with modified residues producing interesting active moieties, such as α- and ß-epoxyketone in camaphycins. The remaining compounds belong to diverse chemical groups such as alkaloids, macrolides, polycyclic compounds, and halogenated compounds. The Dolastatin 10 and lyngbyabellin A, compounds with antiplasmodial high activity, are cytoskeletal disruptors with different protein targets.
Assuntos
Alcaloides , Antimaláricos , Cianobactérias , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Malária/tratamento farmacológico , Alcaloides/química , Extratos VegetaisRESUMO
To understand whether protein Tv-PSP1 from Trichomonas vaginalis recognizes mRNA parasite stem-loop structures, we conducted REMSA and intrinsic fluorescence assays. We found the recombinant Tv-PSP1 structure, determined with X-ray crystallography, showed unusual thermal stability of the quaternary structure, associated with a disulfide bridge CYS76-CYS104. To gain deeper insight into the Tv-PSP1 interaction with mRNA stem-loops (mRNAsl) and its relationship with thermal stability, we also used an integrated computational protocol that combined molecular dynamics simulations, docking assays, and binding energy calculations. Docking models allowed us to determine a putative contact surface interaction region between Tv-PSP1 and mRNAsl. We determined the contributions of these complexes to the binding free energy (ΔGb) in the electrostatic (ΔGelec) and nonelectrostatic (ΔGnon-elec) components using the Adaptive Poisson-Boltzmann Solver (APBS) program. We are the first, to the best of our knowledge, to show the interaction between Tv-PSP1 and the stem-loop structures of mRNA.
RESUMO
Controlling Rhipicephalus microplus is among the most significant challenges for livestock production worldwide. The indiscriminate use of acaricides stimulates the selection of resistant tick populations and is therefore ineffective. Understanding the molecular foundations of resistance could help inform the search for new alternatives for tick control. Although the ovary has been suggested as a relevant target organ for tick control, there are few existing studies that focus on tick ovarian tissue. Therefore, we conducted a comparative proteomic analysis on ovaries of R. microplus strains with differential resistance to ivermectin. In resistant ticks, we observed the over-accumulation of proteins involved in several biological processes, including translation, proteolysis, transport, cellular organization, differentiation, and xenobiotic detoxification. We also observed the accumulation of many structural and extracellular proteins such as papilin-like protein, which glycosylation increase its stability-based molecular modeling. Therefore, we propose that ovaries of ivermectin-resistant ticks overcome the negative impact of ivermectin through the activation of detoxification mechanisms and structural proteins associated with the remodeling of the ovary's extracellular matrix. SIGNIFICANCE: Understanding the molecular foundation of ivermectin resistance in Rhipicephalus microplus represents an essential step in cattle farming, which could provide clues and alternatives for tick control. Excessive use of chemicals like ivermectin allows the generation of resistant tick strains in different countries. However, limited molecular information is available concerning the tick's resistance to ivermectin. Detailed proteomics scrutiny in various tick organs will provide more comprehensive molecular information. Thus, we conducted an ovary comparative proteomic-based TMT-SPS-MS3 approach. We highlight in ivermectin-resistant ticks the over-accumulation of structural proteins and enzymes connected to detoxification mechanisms.
Assuntos
Doenças dos Bovinos , Rhipicephalus , Infestações por Carrapato , Feminino , Animais , Bovinos , Ivermectina/metabolismo , Ivermectina/farmacologia , Ovário , Rhipicephalus/metabolismo , Proteômica , Xenobióticos/metabolismo , Xenobióticos/farmacologia , Infestações por Carrapato/veterináriaRESUMO
Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.
RESUMO
The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.
Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Serina/metabolismo , Trichomonas vaginalis/metabolismo , Tirosina/metabolismo , Western Blotting , Eletroforese em Gel Bidimensional , Glicosilação , Ponto Isoelétrico , Fatores de Iniciação de Peptídeos/química , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/química , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Rhipicephalus microplus is the most serious tick parasite for the livestock industry in tropical and subtropical regions. A cost-effective control method to manage the infestation of this parasite involves the use of chemicals such as ivermectin. However, massive overuse of ivermectin over recent decades has selected for ivermectin-resistant populations of R. microplus. Here, we carried out a comparative proteomic analysis of the midgut of ivermectin-susceptible versus ivermectin-resistant ticks using tandem mass tags coupled to synchronous precursor selection. In susceptible ticks, there was an over-representation of proteins associated with blood digestion and anticoagulation. In contrast, resistant ticks exhibited an over-accumulation of proteins involved in phase I and phase II of the detoxification metabolism, including cytochrome P450, glutathione-S-transferase, and ABC transporters, as well as many ribosomal and other translation-related proteins. This information provides new clues about the mechanisms of ivermectin resistance in R. microplus as well as suggesting potential novel molecular targets to cope with ivermectin-resistant populations of R. microplus. SIGNIFICANCE: Cattle farming is an important primary economic activity for food production all over the globe. However, this activity also has detrimental environmental impacts, including the overuse of ivermectin and other chemicals used to control parasite infestations. The overuse of ivermectin selected for parasites with resistance to this chemical, including tick species like R. microplus. There has been extensive to understand the mechanisms that mediate ivermectin resistance in arthropods, but many gaps remain for the full comprehension of this phenomenon. Understanding the biochemistry behind ivermectin resistance could provide new alternatives to fight these parasites. We therefore consider that determining the metabolic mechanisms involved in ivermectin resistance is of great relevance. The comparative proteomic analysis here reported shows the relevance of the active detoxifying metabolism in the midgut of resistant ticks, which may be key for the development of novel control methods.