Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.
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.
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.
Graphical abstractT. cruzi II strains accumulate more 8-oxoguanine in the kDNA after hydrogen peroxide-induced 18 oxidative stress than T. cruzi I strains. NT: untreated; T: treated.Research highlights▶ Distinct levels of DNA mismatch repair activity are found among T. cruzi strains. ▶ In T. cruzi and T. brucei, MSH2 has a mitochondrial function involved in the response to oxidative stress.
ObjectivesTo determine how adult juvenile idiopathic arthritis (JIA) patients fulfil classification criteria for adult rheumatic diseases, evaluate their outcomes and determine clinical predictors of inactive disease, functional status and damage.MethodsPatients with JIA registered on the Rheumatic Diseases Portuguese Register (Reuma.pt) older than 18 years and with more than 5 years of disease duration were included. Data regarding sociodemographic features, fulfilment of adult classification criteria, Health Assessment Questionnaire, Juvenile Arthritis Damage Index—articular (JADI-A) and Juvenile Arthritis Damage Index—extra-articular (JADI-E) damage index and disease activity were analysed.Results426 patients were included. Most of patients with systemic JIA fulfilled criteria for Adult Still's disease. 95.6% of the patients with rheumatoid factor (RF)-positive polyarthritis and 57.1% of the patients with RF-negative polyarthritis matched criteria for rheumatoid arthritis (RA). 38.9% of the patients with extended oligoarthritis were classified as RA while 34.8% of the patients with persistent oligoarthritis were classified as spondyloarthritis. Patients with enthesitis-related arthritis fulfilled criteria for spondyloarthritis in 94.7%. Patients with psoriatic arthritis maintained this classification. Patients with inactive disease had lower disease duration, lower diagnosis delay and corticosteroids exposure. Longer disease duration was associated with higher HAQ, JADI-A and JADI-E. Higher JADI-A was also associated with biological treatment and retirement due to JIA disability and higher JADI-E with corticosteroids exposure. Younger age at disease onset was predictive of higher HAQ, JADI-A and JADI-E and decreased the chance of inactive disease.ConclusionsMost of the included patients fulfilled classification criteria for adult rheumatic diseases, maintain active disease and have functional impairment. Younger age at disease onset was predictive of higher disability and decreased the chance of inactive disease.
The Pirarucu (Arapaima gigas) is one of the world’s largest freshwater fishes and member of the superorder Osteoglossomorpha (bonytongues), one of the oldest lineages of ray-finned fishes. This species is an obligate air-breather found in the basin of the Amazon River with an attractive potential for aquaculture. Its phylogenetic position among bony fishes makes the Pirarucu a relevant subject for evolutionary studies of early teleost diversification. Here, we present, for the first time, a draft genome version of the A. gigas genome, providing useful information for further functional and evolutionary studies. The A. gigas genome was assembled with 103-Gb raw reads sequenced in an Illumina platform. The final draft genome assembly was ∼661 Mb, with a contig N50 equal to 51.23 kb and scaffold N50 of 668 kb. Repeat sequences accounted for 21.69% of the whole genome, and a total of 24,655 protein-coding genes were predicted from the genome assembly, with an average of nine exons per gene. Phylogenomic analysis based on 24 fish species supported the postulation that Osteoglossomorpha and Elopomorpha (eels, tarpons, and bonefishes) are sister groups, both forming a sister lineage with respect to Clupeocephala (remaining teleosts). Divergence time estimations suggested that Osteoglossomorpha and Elopomorpha lineages emerged independently in a period of ∼30 Myr in the Jurassic. The draft genome of A. gigas provides a valuable genetic resource for further investigations of evolutionary studies and may also offer a valuable data for economic applications.
The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1−/− (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.
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