Assessment of calcinosis in Portuguese patients with systemic sclerosis - a multicenter study.

INTRODUCTION/OBJECTIVES: The study aims to define the clinical and subclinical calcinosis prevalence, the sensitivity of radiographed site and clinical method for its diagnosis, and the phenotype of Portuguese systemic sclerosis (SSc) patients with calcinosis. METHOD: A cross-sectional multicenter study was conducted with SSc patients fulfilling Leroy/Medsger 2001 or ACR/EULAR 2013 classification criteria, registered in the Reuma.pt. Calcinosis was assessed through clinical examination and radiographs of hands, elbows, knees, and feet. Independent parametric or non-parametric tests, multivariate logistic regression, and sensitivity calculation of radiographed site and clinical method for calcinosis detection were performed. RESULTS: We included 226 patients. Clinical calcinosis was described in 63 (28.1%) and radiological calcinosis in 91 (40.3%) patients, of which 37 (40.7%) were subclinical. The most sensitive location to detect calcinosis was the hand (74.7%). Sensitivity of the clinical method was 58.2%. Calcinosis patients were more often female (p = 0.008) and older (p < 0.001) and had more frequently longer disease duration (p < 0.001), limited SSc (p = 0.017), telangiectasia (p = 0.039), digital ulcers (p = 0.001), esophageal (p < 0.001) and intestinal (p = 0.003) involvements, osteoporosis (p = 0.028), and late capillaroscopic pattern (p < 0.001). In multivariate analysis, digital ulcers (OR 2.63, 95% CI 1.02-6.78, p = 0.045) predicted overall calcinosis, esophageal involvement (OR 3.52, 95% CI 1.28-9.67, p = 0.015) and osteoporosis (OR 4.1, 95% CI 1.2-14.2, p = 0.027) predicted hand calcinosis, and late capillaroscopic pattern (OR 7.6, 95% CI 1.7-34.9, p = 0.009) predicted knee calcinosis. Anti-nuclear antibody positivity was associated with less knee calcinosis (OR 0.021, 95% CI 0.001-0477, p = 0.015). CONCLUSIONS: Subclinical calcinosis high prevalence suggests that calcinosis is underdiagnosed and radiographic screening might be relevant. Multifactorial pathogenesis may explain calcinosis predictors' variability. Key Points • Prevalence of subclinical calcinosis in SSc patients is substantial. • Hand radiographs are more sensitive to detect calcinosis than other locations or clinical method. • Digital ulcers were associated with overall calcinosis, esophageal involvement and osteoporosis were associated with hand calcinosis, and late sclerodermic pattern in nailfold capillaroscopy was associated with knee calcinosis. • Anti-nuclear antibody positivity may be a protective factor for knee calcinosis.

Familial history and prevalence of BRCA1, BRCA2 and TP53 pathogenic variants in HBOC Brazilian patients from a public healthcare service.

Several studies have demonstrated the cost-effectiveness of genetic testing for surveillance and treatment of carriers of germline pathogenic variants associated with hereditary breast/ovarian cancer syndrome (HBOC). In Brazil, seventy percent of the population is assisted by the public Unified Health System (SUS), where genetic testing is still unavailable. And few studies were performed regarding the prevalence of HBOC pathogenic variants in this context. Here, we estimated the prevalence of germline pathogenic variants in BRCA1, BRCA2 and TP53 genes in Brazilian patients suspected of HBOC and referred to public healthcare service. Predictive power of risk prediction models for detecting mutation carriers was also evaluated. We found that 41 out of 257 tested patients (15.9%) were carriers of pathogenic variants in the analyzed genes. Most frequent pathogenic variant was the founder Brazilian mutation TP53 c.1010G > A (p.Arg337His), adding to the accumulated evidence that supports inclusion of TP53 in routine testing of Brazilian HBOC patients. Surprisingly, BRCA1 c.5266dupC (p.Gln1756fs), a frequently reported pathogenic variant in Brazilian HBOC patients, was not observed. Regarding the use of predictive models, we found that familial history of cancer might be used to improve selection or prioritization of patients for genetic testing, especially in a context of limited resources.

Corrigendum: Somatic DNA damage response and homologous repair gene alterations and its association with tumor variant burden in breast cancer patients with occupational exposure to pesticides.

[This corrects the article DOI: 10.3389/fonc.2022.904813.].

Somatic DNA Damage Response and Homologous Repair Gene Alterations and Its Association With Tumor Variant Burden in Breast Cancer Patients With Occupational Exposure to Pesticides.

Homologous recombination is a crucial pathway that is specialized in repairing double-strand breaks; thus, alterations in genes of this pathway may lead to loss of genomic stability and cell growth suppression. Pesticide exposure potentially increases cancer risk through several mechanisms, such as the genotoxicity caused by chronic exposure, leading to gene alteration. To analyze this hypothesis, we investigated if breast cancer patients exposed to pesticides present a different mutational pattern in genes related to homologous recombination (BRCA1, BRCA2, PALB2, and RAD51D) and damage-response (TP53) concerning unexposed patients. We performed multiplex PCR-based assays and next-generation sequencing (NGS) of all coding regions and flanking splicing sites of BRCA1, BRCA2, PALB2, TP53, and RAD51D in 158 unpaired tumor samples from breast cancer patients on MiSeq (Illumina) platform. We found that exposed patients had tumors with more pathogenic and likely pathogenic variants than unexposed patients (p = 0.017). In general, tumors that harbored a pathogenic or likely pathogenic variant had a higher mutational burden (p < 0.001). We also observed that breast cancer patients exposed to pesticides had a higher mutational burden when diagnosed before 50 years old (p = 0.00978) and/or when carrying BRCA1 (p = 0.0138), BRCA2 (p = 0.0366), and/or PALB2 (p = 0.00058) variants, a result not found in the unexposed group. Our results show that pesticide exposure impacts the tumor mutational landscape and could be associated with the carcinogenesis process, therapy response, and disease progression. Further studies should increase the observation period in exposed patients to better evaluate the impact of these findings.

Naso-oropharyngeal microbiome from breast cancer patients diagnosed with COVID-19.

Due to immunosuppressive cancer therapies, cancer patients diagnosed with COVID-19 have a higher chance of developing severe symptoms and present a higher mortality rate in comparison to the general population. Here we show a comparative analysis of the microbiome from naso-oropharyngeal samples of breast cancer patients with respect to SARS-CoV-2 status and identified bacteria associated with symptom severity. Total DNA of naso-oropharyngeal swabs from 74 women with or without breast cancer, positive or negative for SARS-CoV-2 were PCR-amplified for 16S-rDNA V3 and V4 regions and submitted to massive parallel sequencing. Sequencing data were analyzed with QIIME2 and taxonomic identification was performed using the q2-feature-classifier QIIME2 plugin, the Greengenes Database, and amplicon sequence variants (ASV) analysis. A total of 486 different bacteria were identified. No difference was found in taxa diversity between sample groups. Cluster analysis did not group the samples concerning SARS-CoV-2 status, breast cancer diagnosis, or symptom severity. Three taxa (Pseudomonas, Moraxella, and Klebsiella,) showed to be overrepresented in women with breast cancer and positive for SARS-CoV-2 when compared to the other women groups, and five bacterial groups were associated with COVID-19 severity among breast cancer patients: Staphylococcus, Staphylococcus epidermidis, Scardovia, Parasegitibacter luogiensis, and Thermomonas. The presence of Staphylococcus in COVID-19 breast cancer patients may possibly be a consequence of nosocomial infection.

A quick UPLC-MS/MS method for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in human plasma.

Abiraterone acetate efficacy against prostate cancer is dependent on the circulating levels of abiraterone and its active metabolites, which present significant pharmacokinetic variability among patients. Thus, therapeutic drug monitoring can be performed to improve treatment outcomes. To support such studies, there are only a limited number of bioanalytical methods in current literature. This work presents a fast method to quantify abiraterone and D4A in plasma in 4 min by UPLC-MS/MS. Bioanalytical method validation was performed according to the recommendations of the US Food and Drug Administration. The method was linear within the range of 1-400 ng/ml for abiraterone and 0.2-20 ng/ml for D4A (r2 > 0.99). Based on the analysis of quality control samples at the lower limit of quantification, low, medium and high concentrations, the method was precise (CVabiraterone ≤ 9.72%; CVD4A ≤ 14.64%) and accurate (CVabiraterone 95.51-107.59%; CVD4A 98.04-99.89%). Application of the method to the quantification of abiraterone and D4A in 10 clinical samples revealed important variability in the conversion ratio of abiraterone to D4A (CV 90.85%). Considering the current literature, this is the fastest method to quantify abiraterone and D4A in plasma, allowing for optimization of the analytical routine.

miRNome profiling of LSC-enriched CD34+CD38-CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool.

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38-CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38-CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1 + ) vs. CD26- (BCR-ABL1 -) CD34+CD38- fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38- fractions that distinguishes between CD26+ (BCR-ABL1 + ) and their CD26- (BCR-ABL1 - ) counterparts, providing valuable data for future studies.

Genes Controlled by DNA Methylation Are Involved in Wilms Tumor Progression.

To identify underlying mechanisms involved with metastasis formation in Wilms tumors (WTs), we performed comprehensive DNA methylation and gene expression analyses of matched normal kidney (NK), WT blastemal component, and metastatic tissues (MT) from patients treated under SIOP 2001 protocol. A linear Bayesian framework model identified 497 differentially methylated positions (DMPs) between groups that discriminated NK from WT, but MT samples were divided in two groups. Accordingly, methylation variance grouped NK and three MT samples tightly together and all WT with four MT samples that showed high variability. WT were hypomethylated compared to NK, and MT had a hypermethylated pattern compared to both groups. The methylation patterns were in agreement with methylases and demethylases expression. Methylation data pointed to the existence of two groups of metastases. While hierarchical clustering analysis based on the expression of all 2569 differentially expressed genes (DEGs) discriminated WT and MT from all NK samples, the hierarchical clustering based on the expression of 44 genes with a differentially methylated region (DMR) located in their promoter region revealed two groups: one containing all NKs and three MTs and one containing all WT and four MTs. Methylation changes might be controlling expression of genes associated with WT progression. The 44 genes are candidates to be further explored as a signature for metastasis formation in WT.

Mutations, Differential Gene Expression, and Chimeric Transcripts in Esophageal Squamous Cell Carcinoma Show High Heterogeneity.

Esophageal squamous cell carcinoma (ESCC) is a frequent and lethal neoplasia. As recent advances in targeted therapy have not improved ESCC prognosis, characterization of molecular alterations associated to this tumor is of foremost relevance. In this study, we analyze, for the first time, the complete genomic profile of ESCC by RNA-seq. TP53 was the most frequently mutated gene in the investigation and validation sets (78.6% and 67.4%, respectively). Differential expression analysis between tumor and nontumor adjacent mucosa showed 6698 differentially expressed genes, most of which were overexpressed (74%). Enrichment analysis identified overrepresentation of Wnt pathway, with overexpressed activators and underexpressed inactivators, suggesting activation of canonical and noncanonical Wnt signaling pathways. Higher WNT7B expression was associated with poor prognosis. Twenty-one gene fusions were identified in 50% of tumors, none of which involving the same genes in different patients; 71% of fusions involved syntenic genes. Comparisons with TCGA data showed co-amplification of seven gene pairs involved in fusions in the present study (~33%), suggesting that these rearrangements might have been driven by chromoanagenesis. In conclusion, genomic alterations in ESCC are highly heterogeneous, impacting negatively in target therapy development.

Angiogenesis and evading immune destruction are the main related transcriptomic characteristics to the invasive process of oral tongue cancer.

Metastasis of head and neck tumors is responsible for a high mortality rate. Understanding its biochemistry may allow insights into tumorigenesis. To that end we carried out RNA-Seq analyses of 5 SCC9 derived oral cancer cell lines displaying increased invasive potential. Differentially expressed genes (DEGs) were annotated based on p-values and false discovery rate (q-values). All 292 KEGG pathways related to the human genome were compared in order to pinpoint the absolute and relative contributions to the invasive process considering the 8 hallmarks of cancer plus 2 new defined categories, as well as we made with our transcriptomic data. In terms of absolute contribution, the highest correlations were associated to the categories of evading immune destruction and energy metabolism and for relative contributions, angiogenesis and evading immune destruction. DEGs were distributed into each one of all possible modes of regulation, regarding up, down and continuum expression, along the 3 stages of metastatic progression. For p-values twenty-six genes were consistently present along the tumoral progression and 4 for q-values. Among the DEGs, we found 2 novel potentially informative metastatic markers: PIGG and SLC8B1. Furthermore, interactome analysis showed that MYH14, ANGPTL4, PPARD and ENPP1 are amenable to pharmacological interventions.

Oxidation of monoterpenes in Protium heptaphyllum oleoresins.

Protium heptaphyllum (Burseraceae) oleoresins are rich in volatile monoterpenes, exhibiting a chemical composition that can be strongly altered with time. The present work aimed to discuss the temporal change of the volatile composition of these oleoresins, and search for related supporting evidence. Samples of P. heptaphyllum oleoresin were collected separately for fresh (n = 10) and aged (n = 8) oleoresins, with the essential oils obtained by hydrodistillation analyzed by GC-FID and GC-MS. Fresh oleoresins were characterized by a high content of terpinolene (28.2-69.7%), whereas aged ones contained large amounts of p-cymene (18.7-43.0%) and p-cymen-8-ol (8.2-31.8%). Multivariate analyses were performed based on the yield and major essential oil components to clearly demonstrate the existence of two subsets (fresh and aged oleoresins). In addition, an analysis of the partial genome sequencing of the species was carried out, producing the largest amount of data for the genus Protium. Subsequently, were searched for nucleotide sequences responsible for the enzymes involved in the biosynthesis of monoterpenes. Two hypotheses were formulated to understand the oxidation process during aging of the oleoresins: (i) a natural chemical oxidation of terpenes and (ii) an oxidation catalyzed by enzymes produced by microorganisms associated with the plant. The results suggested that terpinolene was most likely oxidized to p-cymene, which, in turn, was oxidized into p-cymen-8-ol during natural aging of the exudate due to abiotic factors.

Tetrapod limb and sarcopterygian fin regeneration share a core genetic programme.

Salamanders are the only living tetrapods capable of fully regenerating limbs. The discovery of salamander lineage-specific genes (LSGs) expressed during limb regeneration suggests that this capacity is a salamander novelty. Conversely, recent paleontological evidence supports a deeper evolutionary origin, before the occurrence of salamanders in the fossil record. Here we show that lungfishes, the sister group of tetrapods, regenerate their fins through morphological steps equivalent to those seen in salamanders. Lungfish de novo transcriptome assembly and differential gene expression analysis reveal notable parallels between lungfish and salamander appendage regeneration, including strong downregulation of muscle proteins and upregulation of oncogenes, developmental genes and lungfish LSGs. MARCKS-like protein (MLP), recently discovered as a regeneration-initiating molecule in salamander, is likewise upregulated during early stages of lungfish fin regeneration. Taken together, our results lend strong support for the hypothesis that tetrapods inherited a bona fide limb regeneration programme concomitant with the fin-to-limb transition.

Identification of novel human papillomavirus lineages and sublineages in HIV/HPV-coinfected pregnant women by next-generation sequencing.

Infection by human papillomavirus (HPV) is a necessary condition for development of cervical cancer, and has also been associated with malignancies of other body anatomical sites. Specific HPV types have been associated with premalignant lesions and invasive carcinoma, but mounting evidence suggests that within-type lineages and sublineages also display distinct biological characteristics associated with persistent infections and evolution to cervical cancer. In the present study, we have assessed HPV multiple infection and variation from a cohort of highly susceptible, HIV(+) pregnant women using next-generation sequencing and an in-house pipeline for HPV full-length genome assembly. Seventy-two consensus sequences representing complete or near-complete (>97%) HPV genomes were assembled, spanning 28 different types. Genetic distance and phylogenetic analyses allowed us to propose the classification of novel HPV lineages and sublineages across nine HPV types, including two high-risk types. HPV diversity may be a hallmark of immunosuppressed patients upon HIV infection and AIDS progression.

Effectiveness and long-term retention of anti-tumour necrosis factor treatment in juvenile and adult patients with juvenile idiopathic arthritis: data from Reuma.pt.

OBJECTIVES: Assess the effectiveness and safety of biologic therapy as well as predictors of response at 1 year of therapy, retention rate in biologic treatment and predictors of drug discontinuation in JIA patients in the Portuguese register of rheumatic diseases. METHODS: We prospectively collected patient and disease characteristics from patients with JIA who started biological therapy. Adverse events were collected during the follow-up period. Predictors of response at 1 year and drug retention rates were assessed at 4 years of treatment for the first biologic agent. RESULTS: A total of 812 JIA patients [65% females, mean age at JIA onset 6.9 years (s.d. 4.7)], 227 received biologic therapy; 205 patients (90.3%) were treated with an anti-TNF as the first biologic. All the parameters used to evaluate disease activity, namely number of active joints, ESR and Childhood HAQ/HAQ, decreased significantly at 6 months and 1 year of treatment. The mean reduction in Juvenile Disease Activity Score 10 (JADAS10) after 1 year of treatment was 10.4 (s.d. 7.4). According to the definition of improvement using the JADAS10 score, 83.3% respond to biologic therapy after 1 year. Fourteen patients discontinued biologic therapies due to adverse events. Retention rates were 92.9% at 1 year, 85.5% at 2 years, 78.4% at 3 years and 68.1% at 4 years of treatment. Among all JIA subtypes, only concomitant therapy with corticosteroids was found to be univariately associated with withdrawal of biologic treatment (P = 0.016). CONCLUSION: Biologic therapies seem effective and safe in patients with JIA. In addition, the retention rates for the first biologic agent are high throughout 4 years.

The complete mitochondrial genome of Corydoras nattereri (Callichthyidae: Corydoradinae)

The complete mitogenome of Corydoras nattereri , a species of mailed catfishes from southeastern Brazil, was reconstructed using next-generation sequencing techniques. The mitogenome was assembled using mitochondrial transcripts from the liver transcriptomes of three individuals, and produced a circular DNA sequence of 16,557 nucleotides encoding 22 tRNA genes, two rRNA genes, 13 protein-coding genes and two noncoding control regions (D-loop, OrigL). Phylogeographic analysis of closely related sequences of Cytochrome Oxydase C subunit I (COI) demonstrates high diversity among morphologically similar populations of C. nattereri . Corydoras nattereri is nested within a complex of populations currently assigned to C. paleatus and C. ehrhardti . Analysis of mitogenome structure demonstrated that an insertion of 21 nucleotides between the ATPase subunit-6 and COIII genes may represent a phylogenetically informative character associated with the evolution of the Corydoradinae.

O mitogenoma completo de Corydoras nattereri , uma espécie de bagres encouraçados do sudeste do Brasil, foi reconstruído através de técnicas de sequencimento de DNA de próxima geração. O mitogenoma foi produzido a partir de produtos de transcrição mitocondrial dos transcriptomas hepáticos de três indivíduos, resultando numa sequência de DNA circular de 16.557 nucleotídeos abrangendo 22 genes de tRNA, dois genes de rRNA, 13 genes codificadores de proteínas e duas regiões de controle não codificadoras (D-loop, OrigL). A análise filogenética de sequências proximamente relacionadas da subunidade I do gene Citocrome Oxidase C (COI) demonstrou a existência de elevada diversidade entre populações morfologicamente similares de C. nattereri . Corydoras nattereri está inserida num complexo de populações atualmente identificadas como C. paleatus e C. ehrhardti . A análise da estrutura do mitogenoma demonstra que a inserção de uma sequência de 21 nucleotídeos entre os genes da subunidade 6 da ATPase e do COIII representa um caráter filogeneticamente informativo associado à evolução de Corydoradinae.

Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.

2011 Portuguese recommendations for the use of biological therapies in patients with psoriatic arthritis.

OBJECTIVE: To develop recommendations for the treatment of psoriatic arthritis (PsA) with biological therapies, endorsed by the Portuguese Society of Rheumatology. METHODS: These treatment recommendations were formulated by Portuguese rheumatologists based on literature evidence and consensus opinion. A draft of the recommendations was first circulated to all Portuguese rheumatologists and their suggestions were incorporated in the draft. At a national meeting the recommendations were discussed and all attending rheumatologists voted on the level of agreement for each recommendation. A second draft was again circulated before publication. RESULTS: A consensus was achieved regarding the initiation, assessment of response and switching biological therapies in patients with PsA. Specific recommendations were developed for several disease domains: peripheral arthritis, axial disease, enthesitis and dactylitis. CONCLUSION: These recommendations may be used for guidance in deciding which patients with PsA should be treated with biological therapies. They cover a rapidly evolving area of therapeutic intervention. As more evidence becomes available and more biological therapies are licensed, these recommendations will have to be updated.

Trypanosoma cruzi MSH2: Functional analyses on different parasite strains provide evidences for a role on the oxidative stress response.

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei.

Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation.

The Rad51 gene encodes a highly conserved enzyme involved in DNA double-strand break (DSB) repair and recombination processes. We cloned and characterized the Rad51 gene from Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. This gene is expressed in all three forms of the parasite life cycle, with mRNA levels that are two-fold more abundant in the intracellular amastigote form. The recombinase activity of the TcRad51 gene product was verified by an increase in recombination events observed in transfected mammalian cells expressing TcRad51 and containing two inactive copies of the neomycin-resistant gene. As a component of the DSB repair machinery, we investigated the role of TcRad51 in the resistance to ionizing radiation and zeocin treatment presented by T. cruzi. When exposed to gamma irradiation, different strains of the parasite survive to dosages as high as 1 kGy. A role for TcRad51 in this process was evidenced by the increased expression of its mRNA after irradiation. Furthermore, transfected parasites over-expressing TcRad51 have a faster kinetics of recovery of the normal pattern of chromosomal bands after irradiation as well as a higher resistance to zeocin treatment than do wild-type cultures.