In vitro cytotoxicity and genotoxicity assessment of methanolic extracts of vanillas from Brazilian biodiversity with commercial potential.

The Vanilla genus is crucial for global production in food, perfume, and pharmaceutical industries. However, exploitation threatens some species, leading to extinction. Traditional communities use vanilla for medicinal purposes, and there are species like Vanilla chamissonis Klotzsch and Vanilla bahiana Hoehne with potential to occupy the market. For this, methanolic extraction of these two mentioned species was conducted alongside Vanilla planifolia. Analyzes of the cell viability, mutagenic and genotoxic potential were performed. In the Ames test, the assays were performed with concentrations from 0.5 and 5000 µg/ml and on five strains. Only Vanilla planifolia exhibited mutagenicity at the highest concentration in the TA98 strain. Viability tests were performed within a dose range of 0.05-5000 µg/ml and 24, 48, and 72-hour exposures. It was possible to observe a reduction in cell viability observed only at the highest concentration, for all three species and both cell types tested. Genotoxicity induction by the extracts was assessed at concentrations from 0.5 to 500 µg/ml through the cytokinesis-block micronucleus assay. No genotoxic damage or reduction in the Nucleus Division Index (NDI). The study found no mutagenicity, cytotoxicity, or genotoxicity in the species tested, indicating potential human use for food or pharmaceutical purposes.

DNA lesions that block transcription induce the death of Trypanosoma cruzi via ATR activation, which is dependent on the presence of R-loops.

Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.

Vanilla from Brazilian Atlantic Forest: In vitro and in silico toxicity assessment and high-resolution metabolomic analysis of Vanilla spp. ethanolic extracts.

The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and susceptibility to pathogens. There is an increasing demand for natural vanilla, prized for its complex, authentic, and superior quality compared to artificial counterparts. Therefore, there is a necessity for innovative production alternatives to ensure a consistent and stable supply of vanilla flavors. In this context, vanilla crop wild relatives (WRs) emerge as promising natural sources of the spice. However, these novel species must undergo toxicity assessments to evaluate potential risks and ensure safety for consumption. This study aimed to assess the non-mutagenic and non-carcinogenic properties of ethanolic extracts from V. bahiana, V. chamissonis, V. cribbiana, and V. planifolia through integrated metabolomic profiling, in vitro toxicity assays, and in silico analyses. The integrated approach of metabolomics, in vitro assays, and in silico analyses has highlighted the need for further safety assessments of Vanilla cribbiana ethanolic extract. While the extracts of V. bahiana, V. chamissonis, and V. planifolia generally demonstrated non-mutagenic properties in the Ames assay, V. cribbiana exhibited mutagenicity at high concentrations (5000 µg/plate) in the TA98 strain without metabolic activation. This finding, coupled with the dose-dependent cytotoxicity observed in WST-1 (Water Soluble Tetrazolium) assays, a colorimetric method that assesses the viability of cells exposed to a test substance, underscores the importance of concentration in the safety evaluation of these extracts. Kaempferol and pyrogallol, identified with higher intensity in V. cribbiana, are potential candidates for in vitro mutagenicity. Although the results are not conclusive, they suggest the safety of these extracts at low concentrations. This study emphasizes the value of an integrated approach in providing a nuanced understanding of the safety profiles of natural products, advocating for cautious use and further research into V. cribbiana mutagenicity.

Fifteen Years after the Definition of Trypanosoma cruzi DTUs: What Have We Learned?

Trypanosoma cruzi, the protozoan causative of Chagas disease (ChD), exhibits striking genetic and phenotypic intraspecific diversity, along with ecoepidemiological complexity. Human-pathogen interactions lead to distinct clinical presentations of ChD. In 2009, an international consensus classified T. cruzi strains into six discrete typing units (DTUs), TcI to TcVI, later including TcBat, and proposed reproducible genotyping schemes for DTU identification. This article aims to review the impact of classifying T. cruzi strains into DTUs on our understanding of biological, ecoepidemiological, and pathogenic aspects of T. cruzi. We will explore the likely origin of DTUs and the intrinsic characteristics of each group of strains concerning genome organization, genomics, and susceptibility to drugs used in ChD treatment. We will also provide an overview of the association of DTUs with mammalian reservoirs, and summarize the geographic distribution, and the clinical implications, of prevalent specific DTUs in ChD patients. Throughout this review, we will emphasize the crucial roles of both parasite and human genetics in defining ChD pathogenesis and chemotherapy outcome.

Recovery of flavor compounds from vanilla bagasse by hydrolysis and their identification through UPLC-MSE.

Vanilla is a globally treasured commodity, and the consequences of its unstable value affect social, environmental, economic, and academic ambits. The extensive range of aroma molecules found in cured vanilla beans is crucial to the complexity of this natural condiment and knowledge about their recovery is of the essence. Many strategies aim on reproducing the chemical intricacies of vanilla flavor, such as biotransformation and de novo biosynthesis. Few studies, however, aim at the exhaustion of the cured pods, of which the bagasse, after the traditional ethanolic extraction, might still bear a highly valued flavor composition. An untargeted liquid chromatography coupled with mass spectrometry (LC-MSE) approach was applied to elucidate if sequential alkaline-acidic hydrolysis was effective in extracting flavor related molecules and chemical classes from the hydro-ethanolic fraction. Important vanilla flavor related compounds present in the hydro-ethanolic fraction were further extracted from the residue through alkaline hydrolysis, such as vanillin, vanillic acid, 3-methoxybenzaldehyde, 4-vinylphenol, heptanoic acid, and protocatechuic acid. Acid hydrolysis was effective on further extracting features from classes such as phenols, prenol lipids, and organooxygen compounds, though representative molecules remain unknown. Finally, sequential alkaline-acidic hydrolysis rendered natural vanilla's ethanolic extraction residues as an interesting supplier of its own products, which could be used as a food additive, and many other applications.

Vanilla beyond Vanilla planifolia and Vanilla × tahitensis: Taxonomy and Historical Notes, Reproductive Biology, and Metabolites.

Vanilla is a worldwide cherished condiment, and its volatile market is associated with the so-called "vanilla crisis". Even though only two species (Vanilla planifolia and V. × tahitensis) are cultivated on a large scale for commercial purposes, the Vanilla genus is comprised of 140 species. The present review article discusses the facets of this crisis, and vanilla crop wild relatives (WRs) are showcased as alternatives to overcome them. Historical, taxonomic, and reproductive biology aspects of the group were covered. Emphasis was given to the metabolic characterization of the vanilla crop WRs, highlighting their main chemical classes and the potential flavor descriptors. Many of these species can produce important flavor compounds such as vanillin, vanillic acid, and acetovanillone, among others. Overall, this review compiles valuable information that can help unravel new chapters of the history of this treasured product by evidencing the biotechnological potential of vanilla crop WRs.

Vanilla flavor: Species from the Atlantic forest as natural alternatives.

The volatility of the vanilla market calls attention to the so-called vanilla crisis. There is a growing worldwide demand for natural vanilla with a concomitant reduction in global supply. However, commercial crops are threatened with extinction due to the lack of gene pool variability, susceptibility to climate change and pandemic diseases. Therefore, there is an urgent need to identify new Vanilla spp. as alternative sources vanilla. Therefore, using undirected LC-MS/MS metabolic profiling and chemometrics, the present study demonstrates the great bioeconomic potential of two Atlantic Forest species - V. bahiana and V. chamissonis - by annotation of important flavor compounds associated with the commercial species and reveals distinct flavor descriptors associated with both wild species. Such similarities and dissimilarities are crucial to the ongoing quest to Vanilla gene pool improvement. Compounds remarkably and frequently associated with vanilla flavor were annotated or identified in this study such as vanillin and p-hydroxybenzaldehyde.

DNA Topoisomerase 3α Is Involved in Homologous Recombination Repair and Replication Stress Response in Trypanosoma cruzi.

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

DNA topoisomerase 3α is involved in homologous recombination repair and replication stress response in Trypanosoma cruzi

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

Differential Modulation of Mouse Heart Gene Expression by Infection With Two Trypanosoma cruzi Strains: A Transcriptome Analysis.

The protozoan Trypanosoma cruzi (T. cruzi) is a well-adapted parasite to mammalian hosts and the pathogen of Chagas disease in humans. As both host and T. cruzi are highly genetically diverse, many variables come into play during infection, making disease outcomes difficult to predict. One important challenge in the field of Chagas disease research is determining the main factors leading to parasite establishment in the chronic stage in some organs, mainly the heart and/or digestive system. Our group previously showed that distinct strains of T. cruzi (JG and Col1.7G2) acquired differential tissue distribution in the chronic stage in dually infected BALB/c mice. To investigate changes in the host triggered by the two distinct T. cruzi strains, we assessed the gene expression profiles of BALB/c mouse hearts infected with either JG, Col1.7G2 or an equivalent mixture of both parasites during the initial phase of infection. This study demonstrates the clear differences in modulation of host gene expression by both parasites. Col1.7G2 strongly activated Th1-polarized immune signature genes, whereas JG caused only minor activation of the host immune response. Moreover, JG strongly reduced the expression of genes encoding ribosomal proteins and mitochondrial proteins related to the electron transport chain. Interestingly, the evaluation of gene expression in mice inoculated with a mixture of the parasites produced expression profiles with both up- and downregulated genes, indicating the coexistence of both parasite strains in the heart during the acute phase. This study suggests that different strains of T. cruzi may be distinguished by their efficiency in activating the immune system, modulating host energy metabolism and reactive oxygen species production and decreasing protein synthesis during early infection, which may be crucial for parasite persistence in specific organs.

The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.

Trypanosoma cruzi RNA-binding protein ALBA30 aggregates into cytoplasmic foci under nutritional stress.

Trypanosoma cruzi, the causative agent of Chagas disease, has a complex life cycle that requires the adaptation to different environments. In the absence of traditional mechanisms for regulation of gene expression, this parasite relies on posttranscriptional control events, which allow the progression of its life cycle in different hosts and stress conditions. In this context, different stress conditions trigger the aggregation of RNA-binding proteins and their target mRNAs into cytoplasmic foci known as RNA granules, which act as RNA-sorting centers. In this study, we have characterized the T. cruzi RNA-binding protein ALBA30 during nutritional stress conditions. Using a recombinant form of TcALBA30 to facilitate its detection (rTcALBA30), we showed that this protein resides in the cytoplasm in normal growth conditions but is recruited into cytoplasmic foci after starvation. Moreover, evaluation of rTcALBA30 in parasites that reached the stationary phase of growth also showed the recruitment of this protein into cytoplasmic foci. Our results indicate that, similar to TbALBA3, TcALBA30 aggregates into stress granules in parasites submitted to nutritional stress.

Metabolomic analysis of Cyrtopodium glutiniferum extract by UHPLC-MS/MS and in vitro antiproliferative and genotoxicity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of orchids have been traditionally used as human phytotherapeutics. Cyrtopodium flavum, for example, due to the analgesic and anti-inflammatory properties, beside the capacity of heal skin lesions has been focus of research. Also Cyrtopodium glutiniferum, an orchid found in the Brazilian southeastern rainforest, is known to synthesize anti-inflammatory glucomannans in the pseudobulbs, as other potentially therapeutic compounds. AIM OF THE STUDY: We have reported the first metabolomic analysis focused on the phenols expression of the neotropical orchid Cyrtopodium glutiniferum Raddi, besides free radical scavenging, anti-inflammatory and antiproliferative activities, and the genotoxicity properties of the aqueous extract. MATERIAL AND METHODS: The metabolomics of C. glutiniferum aqueous extract was performed through UHPLC-MSn acquisition. We have detected the scavenging potential of the extract using DPPH assay. The genotoxic potential was performed by Ames Test (0-5000 µg mL-1) and micronucleous assay (0-5000 µg mL-1) in RAW264.7 cells. The cytotoxic potential of the extract against RAW264.7 was tested by WST-1 assay (0-500 µg mL-1). And after all, the RAW264.7 cells were treated with non-cytotoxic concentrations of C. glutiniferum (0-50 µg mL-1) to evaluate the antiproliferative and anti-inflammatory potential, besides the mitochondrial activity. RESULTS: From the 55 molecules identified, 45.5% belonged to the phenolic compounds database from Phenol Explorer, 29% to an in-house Orchidaceae molecules database, and 25.5% to both. Among the identified phenolic compounds, 18 subclasses were discriminated, being phenanthrenes the most abundant. Doses-dependent of C. glutiniferum extracts were able to induce DPPH free radicals scavenging and also to increase TA100 His+ revertants, in metabolic environment, showing mutagenicity just in the highest concentration, of 5 mg/plate. On Eukaryotic cell models, the extract also has induced dose-response and time-response cytotoxicity against RAW264.7 macrophages, mainly after 48 h and 72 h, even though the extract has not been able to induce the increase of micronucleated cells and mitotic index alteration on Micronucleus assay. The activation and proliferation of macrophages cultures were downregulated after 24 h and 48 h by the non-cytotoxic concentrations of the extract in a dose-dependent manner. CONCLUSIONS: The Cyrtopodium glutiniferum metabolomics, anti-inflammatory and anti-proliferative properties observed in this study suggest a therapeutic efficacy of the orchid extract applied in folk medicine.

Artemisia annua L. and photoresponse: from artemisinin accumulation, volatile profile and anatomical modifications to gene expression.

KEY MESSAGE: Blue and yellow light affected metabolism and the morphology. Blue and red promote the DOXP/MEP pathway. ADS gene expression was increased in plants cultivated under blue, promoting artemisinin content. Artemisinin-based combination therapies are the most effective treatment for highly lethal malaria. Artemisinin is produced in small quantities in the glandular trichomes of Artemisia annua L. Our aim was to evaluate the effect of light quality in A. annua cultivated in vitro under different light qualities, considering anatomical and morphological changes, the volatile composition, artemisinin content and the expression of two key enzymes for artemisinin biosynthesis. Yellow light is related to the increase in the number of glandular trichomes and this seemed to positively affect the molecular diversity in A. annua. Yellow light-stimulated glandular trichome frequency without triggered area enhancement, whereas blue light stimulated both parameters. Blue light enhanced the thickness of the leaf epidermis. The B-promoting effect was due to increased cell size and not to increased cell numbers. Green and yellow light positively influenced the volatile diversity in the plantlets. Nevertheless, blue and red light seemed to promote the DOXP/MEP pathway, while red light stimulates MVA pathway. Amorpha-4,11-diene synthase gene expression was significantly increased in plants cultivated under blue light, and not red light, promoting artemisinin content. Our results showed that light quality, more specifically blue and yellow light, positively affected secondary metabolism and the morphology of plantlets. It seemed that steps prior to the last one in the artemisinin biosynthesis pathway could be strongly influenced by blue light. Our work provides an alternative method to increase the amount of artemisinin production in A. annua without the use of transgenic plants, by the employment of blue light.

Vanilla bahiana, a contribution from the Atlantic Forest biodiversity for the production of vanilla: A proteomic approach through high-definition nanoLC/MS.

Only a few cultivated species of Vanilla are used to produce vanilla, despite the high demand, predatory exploitation, and low genetic variability that threaten the production of natural vanilla. Vanilla bahiana pods from the Atlantic Forest may be an alternative source of natural vanilla. This study applied bottom-up and shotgun proteomics analysis to identify proteins related to flowering, fruiting, and vanilla-flavor production. Extraction solutions, including Tris-HCl buffer, ß-mercaptoethanol and SDS, were assayed. SDS proved to be feasible for extraction of Vanilla fruit proteins and could be an alternative to the phenol method of protein extraction. Progenesis QI for Proteomics (QIP) software loaded with an Orchidaceae database identified 2326 proteins in our samples. Among these, 75 were highlighted as useful for the synthesis of compounds related to vanilla flavor, such as vanillin synthase, which was successfully extracted with 1% SDS, which also improved the variety of the extracted proteins. The proteins identified in V. bahiana pods indicate the enzymatic potential of this species, as further validated by quantifying the vanilla in the samples.

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite.

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi.

Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 - a recombinase involved in homologous recombination - in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi's intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi's spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi's mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.