Protein detection by fluorescence of manually cleaned high-speed dental handpieces.

We analyzed the effectiveness of manual cleaning protocols performed on high-speed dental handpieces, using protein identification by fluorescence. Although one protocol was able to able to lower the amount of protein, >40% of the handpieces showed amounts of residual protein at unacceptable levels.

Genetic characterization of an X-STR decaplex system in the State of Mato Grosso, Brazil: distribution, forensic efficiency and population structure.

Studies with X-STR loci show population genetic substructure, which makes necessary the characterization of such markers in the different geographical and/or ethnic populations. Therefore, this study assessed the distribution and forensic efficiency of an X-STR decaplex system in the population of the State of Mato Grosso, as well as analysed the population structure of this State based on the aforementioned system. All X-STR markers were in Hardy-Weinberg equilibrium and linkage equilibrium, and the DXS6809 was the most informative marker. The power of discrimination value in females and males was 0.99999999995 and 0.9999994, respectively. Analysis of molecular variance indicated 1.10% (p < 0.00001) of heterogeneity among Europeans, Africans, Brazilians and other Latin Americans, and in relation to such groups, the population of the State of Mato Grosso showed lower genetic variation when compared with the Brazilian group (-0.10%, p = 0.67327). The genetic distance analysis showed lower values of F ST (0.0004 ≤ F ST ≤ 0.00331), with non-significant p value (p > 0.00024), between the populations of Mato Grosso and Mato Grosso do Sul, Paraná and the Southeast region of Brazil (except for one sample of Rio de Janeiro). F ST values with significant p values (p ≤ 0.00024) were obtained between the population of Mato Grosso and Iberian, African and some Latin American populations. The X-STR decaplex system proved to be extremely useful in the population of the State of Mato Grosso, and the data obtained does not show the need for a specific forensic database for this State in relation to the Brazilian populations compared in this study, except for population of Rio de Janeiro.

Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments

Abstract Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5 kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58 kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5 kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

Trypanocidal Activity of Flavokawin B, a Component of Polygonum ferrugineum Wedd.

The trypanocidal potential of the natural chalcone flavokawin B, which was isolated from the hexanic extract of Polygonum ferrugineum Wedd., is reported here. Although flavokawin B is widespread, this is the first report about its trypanocidal properties on both Trypanosoma cruzi (IC50 = 9.5 µM, IC50 = 34.7 µM benznidazol, Y strain) epimastigotes and Trypanosoma brucei (IC50 = 4.8 µM, IC50 = 6.4 µM pentamidine, 29-13 strain) procyclic forms, which was also corroborated on T. brucei strain 427 (IC50 = 6.2 µM). In order to learn more about its properties, unspecific cytotoxicity on Hep G2 cells was investigated as well as the trans-splicing inhibitory potential on T. brucei cells. The results shown here point to flavokawin B as a candidate in the search for new agents. It is also cheaper and less toxic than the available drugs to treat trypanosomiasis with a special focus on sleeping sickness disease.

Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments.

Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

Male-specific contributions to the Brazilian population of Espirito Santo.

Y chromosome markers have been widely studied due to their various applications in the fields of forensic and evolutionary genetics. In this study, 35 Y-SNPs and 17 Y-STRs were genotyped in 253 males from the State of Espirito Santo, Brazil. A total of 18 haplogroups and 243 haplotypes were detected; the haplogroup and haplotype diversities were 0.7794 and 0.9997, respectively. Genetic distance analysis using the Y-STR data showed no statistically significant differences between Espirito Santo and other admixed populations from Brazil. The classification of paternal lineages based on haplogroups showed a predominant European contribution (85.88%), followed by African (11.37%) and Amerindian (2.75%) contributions.

Mitochondrial DNA control region diversity in a population from Espirito Santo state, Brazil.

Mitochondrial DNA (mtDNA) analysis has proved to be useful for forensic identification, especially in cases which nuclear DNA markers fail, as in degraded samples or in cases where the biological material has few traces or no nuclear DNA. Moreover, it can be applied in population genetics, inferring the origin of a population. In this work, the entire mtDNA control region of 97 individuals from the state of Espirito Santo, Brazil, was analyzed. We have found 94 different haplotypes yielding a high haplotype diversity of 0.9994 ± 0.0016. The probability of a random match calculated was 1.09. Haplogroup distribution analysis confirmed a highly admixed Latin American population: African lineages (43.3 %), European lineages (32.0 %), Native American lineages (23.7 %) and Asian lineages (1.0 %). We have concluded that this type of tool can be used both in forensic genetics to the study of different human populations, such as highly admixed populations, and in the study of migration's history and colonization of different states and countries of the world.

Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini.

Multiple fragments of mitochondrial DNA genes (cytochrome b, cytochrome oxidase I, and 16S rDNA) were used to evaluate the phylogenetic relationships among Triatoma melanocephala, Triatoma tibiamaculata, Triatoma vitticeps, and other members of Triatoma brasiliensis subcomplex under a Bayesian framework and maximum parsimony criterion. With the addition of new sequences of T. tibiamaculata and T. vitticeps, Triatoma juazeirensis, Triatoma melanica and the newly sequenced T. melanocephala, the three first sylvatic species, T. melanocephala, T. tibiamaculata and T. vitticeps, were strongly recovered into a clade separate from the other with the remaining Triatoma species from South America, such as the members of T. brasiliensis subcomplex. Panstrongylus megistus was recovered as a sister to T. tibiamaculata, whereas T. vitticeps was a sister to T. melanocephala. This study revealed the non-monophyly of the T. brasiliensis subcomplex, and the polyphyly of Triatoma was reinforced by the placement of these three sylvatic species with Dipetalogaster, Meccus, Mepraia, and Panstrongylus. The results herein shown highlight the need of generic revision in Triatomini.

In vitro trypanocidal activity of solamargine and extracts from Solanum palinacanthum and Solanum lycocarpum of Brazilian cerrado.

The present investigation was to evaluate the potential trypanocidal activity of crude ethanolic extract of the fruits of Solanum palinacanthum, Solanum lycocarpum and the glycoalcaloid, solamargine. S. palinacanthum and S. lycocarpum fruit powders were submitted to exhaustively extraction with 96% ethanol and solamargine were isolated from the extract of S. palinacanthum. Both extracts and solamargine were analysed for trypanocidal activity by using MTT colorimetric assay. Extracts of S. palinacanthum showed to be more active (IC50 = 175.9 µg.ml-1) than S. lycocarpum (IC50 = 194.7 µg.ml-1). Solamargine presented a strong activity (IC50 = 15.3 µg.ml-1), which can explain the better activity of the both extracts. Benznidazol (IC50 = 9.0 µg.ml-1) is the only drug used to treat Chagas' disease. These findings demonstrate for the first time that ethanol extracts obtained from both fruits of S. palinacanthum and S. lycocarpum and also solamargine have a potential anti-trypanosomal activity.

Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic triatoma in Central-Western Brazil, based on mitochondrial DNA sequences.

The phylogenetic relationships among species of Triatoma matogrossensis subcomplex (T. baratai, T. guazu, T. matogrossensis, T. sordida, T. vandae, and T. williami) was addressed by using fragments of cytochrome oxidase I (COI), 16S rDNA (16S), and cytochrome b (Cytb) through Bayesian and parsimony analyses. We did not recover a monophyletic T. matogrossensis subcomplex, and their members were found clustered in three strongly supported clades, as follows: i) T. jurbergi + T. matogrossensis + T. vandae + T. garciabesi + T. sordida; ii) with T. guasayana as the sister group of clade (i); and iii) T. williami + T. guazu, however not closely related to the clade formed by the previously mentioned species. The other two endemic species from Central-Western Brazil, T. baratai and T. costalimai, were not recovered with strong clade support as related to other members of this subcomplex. Results call for a further revision in the classification of the subcomplexes within the genus Triatoma.

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae).

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC(50)) values of less than 0.88 nM. Both compounds showed IC(50) lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease.

Antiprotozoal sesquiterpene pyridine alkaloids from Maytenus ilicifolia.

As part of a bioprospecting program aimed at the discovery of antiprotozoal agents from the Brazilian flora, two new sesquiterpene pyridine alkaloids, ilicifoliunines A (1) and B (2), along with the known alkaloids aquifoliunine E-I (3) and mayteine (4), were isolated from the root bark of Maytenus ilicifolia. The structures of 1 and 2 were established on the basis of spectroscopic data interpretation. Alkaloid 3 displayed potent in vitro antiprotozoal activity against Leishmania chagasi and Trypanosoma cruzi, with IC(50) values of 1.4 and 41.9 µM, respectively, as well as low cytotoxicity against murine peritoneal macrophages (IC(50) of 1.8 mM).

New insights into trypanosomatid U5 small nuclear ribonucleoproteins.

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.

Absolute configuration and selective trypanocidal activity of gaudichaudianic acid enantiomers.

Gaudichaudianic acid, a prenylated chromene isolated from Piper gaudichaudianum, has been described as a potent trypanocidal compound against the Y-strain of Trypanosoma cruzi. We herein describe its isolation as a racemic mixture followed by enantiomeric resolution using chiral HPLC and determination of the absolute configuration of the enantiomers as (+)-S and (-)-R by means of a combination of electronic and vibrational circular dichroism using density functional theory calculations. Investigation of the EtOAc extract of the roots, stems, and leaves from both adult specimens and seedlings of P. gaudichaudianum revealed that gaudichaudianic acid is biosynthesized as a racemic mixture from the seedling stage onward. Moreover, gaudichaudianic acid was found exclusively in the roots of seedlings, while it is present in all organs of the adult plant. Trypanocidal assays indicated that the (+)-enantiomer was more active than its antipode. Interestingly, mixtures of enantiomers showed a synergistic effect, with the racemic mixture being the most active.

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups.

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction, in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. Complete SNP profiles were obtained from 10 pg of total DNA. We conclude that it is possible to build and genotype more than forty mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations.

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs.

The analysis of heteroplasmy (presence of more than one type of mitochondrial DNA in an individual) is used as a tool in human identification studies, anthropology, and most currently in studies that relate heteroplasmy with longevity. The frequency of heteroplasmy and its correlation with age has been analyzed using different tissues such as blood, muscle, heart, bone and brain and in different regions of mitochondrial DNA, but this analysis had never been performed using hair samples. In this study, samples of hair were sequenced in order to ascertain whether the presence or not of heteroplasmy varied according to age, sex and origin of haplogroup individuals. The samples were grouped by age (3 groups), gender (male and female) and haplogroup of origin (European, African and Native American), and analyzed using the chi-square statistical test (χ(2)). Based in statistical results obtained, we conclude that there is no relationship between heteroplasmy and sex, age and haplogroup origin using hair samples.

Genetic profile characterization of 10 X-STRs in four populations of the southeastern region of Brazil.

Ten X-chromosomal short tandem repeats (DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08 and DXS7423) were analyzed in four populations of the southeastern region of Brazil (São Paulo, Rio de Janeiro, Vitória and Belo Horizonte). No deviations from the Hardy-Weinberg equilibrium were observed for any of the analyzed loci in the four populations. The average diversity per locus varied between 68% for DXS8378, DXS7133, and DXS7423 and 83%, for DXS6809, with Rio de Janeiro being the most diverse population. Overall power of discrimination values in females varied between 0.99999999990 and 0.99999999997 and between 0.9999991 and 0.9999995 in males. These high values show the potential of this system for forensic application and relationships' testing in the studied groups. Genetic comparisons (exact tests of population differentiation and pairwise genetic distances) revealed significant differences between Brazilian and other populations from Europe, Latin America and Africa, as well as among different Brazilian populations.

Heteroplasmy in hair: study of mitochondrial DNA third hypervariable region in hair and blood samples.

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA "alleles" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) inferred from two mitochondrial genes suggests its location within the Triatoma brasiliensis complex.

The phylogenetic position of Triatoma sherlocki within triatomines group was inferred by analyzing mtDNA fragments of Cyt B and 16S ribosomal RNA by using maximum parsimony and Bayesian analysis. Despite being differentiated from members of the T. brasiliensis complex on morphologic grounds, molecular phylogenetic analysis suggests T. sherlocki is a member of this complex; moreover, it was placed as a sister species of T. melanica. These suggestions were supported by robust credibility rates. Hence, we show evidence for the paraphyletic group of the "Triatoma brasiliensis complex," which should be composed of T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki.

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate.

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.