Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi.

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.

A Protocol for Preconceptional Screening of Consanguineous Couples Using Whole Exome Sequencing.

Genetic studies performed in consanguineous couples suggest that the reproductive risk that distinguish them from other couples in the general population is related to autosomal recessive (AR) diseases. This risk is scattered among the thousands of known and potential AR diseases. Thus, for effective preconceptional screening of consanguineous couples it is necessary a test that encompasses the largest number of genes possible. For that reason, we decided to create a protocol based on whole exome sequencing (WES). We sequenced completely the exomes of 39 consanguineous couples at high coverage (∼100×). Applying bioinformatics filters, we could detect genetic variants that were simultaneously present in both members of the couple in all genes listed in the Clinical Genomics Database as causally related to AR diseases. Shared variants were then assessed for pathogenicity. For non-truncating variants (missense and in-frame indels) we considered as pathogenic or likely pathogenic only the variants included as such in the ClinVar database. Shared truncating variants (frameshift, non-sense, and canonical splice variants) were considered likely pathogenic when loss-of-function was a known mechanism of disease. The 39 consanguineous cases included two couples with a coefficient of genetic relationship (CGR) of 0.25, 26 couples with a CGR of 0.125, three couples with a CGR of 0.0625 and eight couples with a CGR of 0.03125. In 21 of the 39 couples (53.8%) we ascertained sharing of heterozygosity for at least one variant considered pathogenic or likely pathogenic for an AR disease. In eight couples we found sharing of heterozygosity for at least two pathogenic variants. Once the specific pathogenic variant was identified, it became possible for the couple to undergo prenatal diagnosis or, if desired, preimplantation genetic diagnosis (PGD) involving in vitro fertilization and embryo screening. In conclusion, our results demonstrate that preconceptional screening by WES is a useful new procedure that should be incorporated in the genetic counseling of all consanguineous couples.

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.

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.

Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance.

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci--six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.

Pre- and post-Columbian gene and cultural continuity: the case of the Gaucho from southern Brazil.

OBJECTIVE: To investigate the evolutionary and demographic history of the Gaucho, a distinct population of southern Brazil, relating it to their culture, to assess possible parallel continuity. METHODS: Six binary polymorphisms, an Alu insertion polymorphism (YAP) and 12 short tandem repeat loci in the non-recombining region of the Y-chromosome, as well as the sequence of the first hypervariable segment (HVS-I) of the mitochondrial DNA (mtDNA) control region were studied in 150 unrelated males born in the Pampa region of Rio Grande do Sul. RESULTS: Comparison of the results with the other Brazilian and Uruguayan populations, as well as with their putative ancestors, indicated a stronger male Spanish influence than that observed elsewhere in Brazil, a former Portuguese colony. Extensive mtDNA analyses of their Amerindian component gave clear indications of the presence there of material from extinct (Charrua), as well as extant (Guarani) tribes. CONCLUSIONS: The genetic analyses contributed in a significant way to reveal that the known cultural continuity between pre- and post-Columbian Pampa populations was also accompanied by an extraordinary genetic continuity.

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.

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.

Up-regulation of the error-prone DNA polymerase {kappa} promotes pleiotropic genetic alterations and tumorigenesis.

It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberrations, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase kappa, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase kappa favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype.

Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response.

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.

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.

A novel polymorphic Alu insertion embedded in a LINE 1 retrotransposon in the human X chromosome (DXS225): identification and worldwide population study.

We describe a novel polymorphic Alu insertion (DXS225) on the human X chromosome (Xq21.3) embedded into an L1 retrotransposon. The DXS225 polymorphism was genotyped in 684 males from the CEPH Human Genome Diversity Panel. This insertion was found in all regions of the globe, suggesting that it took place before modern humans spread from Africa ca. 100,000 years ago. However, only one Amerindian population (Karitiana) showed this insertion allele, which may have been introduced by European admixture. Thus, it appears likely that the Alu insertion was absent from pre-Columbian America. Analysis of molecular variance worldwide demonstrated that 92.2% of the genetic variance was concentrated within populations. DXS225 is flanked by two microsatellites (DXS8114 and DXS1002), which are 86 kb apart and are in very strong linkage disequilibrium. The combination of a unique event polymorphism on the X chromosome in linkage disequilibrium with two rapidly evolving microsatellites should provide a useful tool for studies of human evolution.

Characterization and comparative functional analysis in yeast of a Schistosoma mansoni Rho1 GTPase gene.

Low-molecular weight GTP-binding proteins (LMWGPs) of the Ras superfamily are believed to play a role in Schistosoma mansoni female development and egg production. Here we describe the characterization of a novel S. mansoni gene (SMRHO1), highly homologous to Rho-type LMWGPs from several other organisms and encoding a polypeptide with 193 amino acids and an estimated molecular mass of 21.8 kDa. SMRHO1 complemented a Saccharomyces cerevisiae rho1 null mutant strain even in restrictive temperature and calcium concentration, in contrast with the human RHOA GTPase that was not able to provide complementation in such conditions. Comparison of the amino acid sequence of the alpha3-helix loop7 regions of the two proteins allowed the identification of the proline 96 and threonine 100 amino acid residues of human RHOA as the most probable determinants of the complementation differences. We generated SMRHO1 mutants (smrho1(E97P), smrho1(L101T) and smrho1(E97P,) L101T) by site directed mutagenesis and reproduced the conditional lethality phenotype at high temperature, providing strong evidence that the related amino acid positions (Gln(101) and Ile(105)) in the Rho1 GTPase are indeed important for regulation of the cell wall synthesis performed by this protein in yeast. The observation that specific amino acid positions seem to be important for the different functions performed by the Rho GTPases leads to the idea that SMRHO1 might be a useful target in the development of new anti-schistosomiasis drugs, although it does share high sequence homology with the human RhoA GTPase.

Association of genetic variants with self-assessed color categories in Brazilians.

The Brazilian population was formed by extensive admixture of three different ancestral roots: Amerindians, Europeans and Africans. Our previous work has shown that at an individual level, ancestry, as estimated using molecular markers, was a poor predictor of color in Brazilians. We now investigate if SNPs known to be associated with human skin pigmentation can be used to predict color in Brazilians. For that, we studied the association of fifteen SNPs, previously known to be linked with skin color, in 243 unrelated Brazilian individuals self-identified as White, Browns or Blacks from Rio de Janeiro and 212 unrelated Brazilian individuals self-identified as White or Blacks from São Paulo. The significance of association of SNP genotypes with self-assessed color was evaluated using partial regression analysis. After controlling for ancestry estimates as covariates, only four SNPs remained significantly associated with skin pigmentation: rs1426654 and rs2555364 within SLC24A5, rs16891982 at SLC45A2 and rs1042602 at TYR. These loci are known to be involved in melanin synthesis or transport of melanosomes. We found that neither genotypes of these SNPs, nor their combination with biogeographical ancestry in principal component analysis, could predict self-assessed color in Brazilians at an individual level. However, significant correlations did emerge at group level, demonstrating that even though elements other than skin, eye and hair pigmentation do influence self-assessed color in Brazilians, the sociological act of self-classification is still substantially dependent of genotype at these four SNPs.

Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

Evidence of substantial recombination among Trypanosoma cruzi II strains from Minas Gerais.

Due to the scarcity of evidence of sexuality in Trypanosoma cruzi, the causative agent of Chagas disease, it has been general accepted that the parasite reproduction is essentially clonal with infrequent genetic recombination. This assumption is mainly supported by indirect evidence, such as Hardy-Weinberg imbalances, linkage disequilibrium and a strong correlation between independent sets of genetic markers of T. cruzi populations. However, because the analyzed populations are usually isolated from different geographic regions, the possibility of population substructuring as generating these genetic marker imbalances cannot be eliminated. To investigate this possibility, we firstly compared the allele frequencies and haplotype networks using seven different polymorphic loci (two from mitochondrial and five from different nuclear chromosomes) in two groups of TcII strains: one including isolates obtained from different regions in Latin America and the other including isolates obtained only from patients of the Minas Gerais State in Brazil. Our hypothesis was that if the population structure is essentially clonal, Hardy-Weinberg disequilibrium and a sharp association between the clusters generated by analyzing independent markers should be observed in both strain groups, independent of the geographic origin of the samples. The results demonstrated that the number of microsatellite loci in linkage disequilibrium decreased from 4 to 1 when only strains from Minas Gerais were analyzed. Moreover, we did not observed any correlation between the clusters when analyzing the nuclear and mitochondrial loci, suggesting independent inheritance of these markers among the Minas Gerais strains. Besides, using a second subset of five physically linked microsatellite loci and the Minas Gerais strains, we could also demonstrate evidence of homologous recombination roughly proportional to the relative distance among them. Taken together, our results do not support a clonal population structure for T. cruzi, particularly in TcII, which coexists in the same geographical area, suggesting that genetic exchanges among these strains may occur more frequently than initially expected.

Unveiling benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi.

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.

Rapid and inexpensive screening of genomic copy number variations using a novel quantitative fluorescent PCR method.

Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.

LSSP-PCR of Trypanosoma cruzi: how the single primer sequence affects the kDNA signature.

BACKGROUND: Low-stringency single specific primer PCR (LSSP-PCR) is a highly sensitive and discriminating technique that has been extensively used to genetically characterize Trypanosoma cruzi populations in the presence of large amounts of host DNA. To ensure high sensitivity, in most T. cruzi studies, the variable regions of the naturally amplified kinetoplast DNA (kDNA) minicircles were targeted, and this method translated the intraspecific polymorphisms of these molecules into specific and reproducible kDNA signatures. Although the LSSP-PCR technique is reproducible under strict assay conditions, the complex banding pattern generated can be significantly altered by even a single-base change in the target DNA. Our survey of the literature identified eight different primers with similar, if not identical, names that have been used for kDNA amplification and LSSP-PCR of T. cruzi. Although different primer sequences were used in these studies, many of the authors cited the same reference report to justify their primer choice. We wondered whether these changes in the primer sequence could affect also the parasite LSSP-PCR profiles. FINDINGS: To answer this question we compared the kDNA signatures obtained from three different and extensively studied T. cruzi populations with the eight primers found in the literature. Our results clearly demonstrate that even minimal modifications in the oligonucleotide sequences, especially in the 3' or 5' end, can significantly change the kDNA signature of a T. cruzi strain. CONCLUSIONS: These results highlight the necessity of careful preservation of primer nomenclature and sequence when reproducing an LSSP-PCR work to avoid confusion and allow comparison of results among different laboratories.