Validation of a reaction volume reduction protocol for analysis of Y chromosome haplotypes targeting DNA databases.

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA® cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex® Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Secondary contact and asymmetrical gene flow in a cosmopolitan marine fish across the Benguela upwelling zone.

The combination of oceanographic barriers and habitat heterogeneity are known to reduce connectivity and leave specific genetic signatures in the demographic history of marine species. However, barriers to gene flow in the marine environment are almost never impermeable which inevitably allows secondary contact to occur. In this study, eight sampling sites (five along the South African coastline, one each in Angola, Senegal and Portugal) were chosen to examine the population genetic structure and phylogeographic history of the cosmopolitan bluefish (Pomatomus saltatrix), distributed across a large South-east Atlantic upwelling zone. Molecular analyses were applied to mtDNA cytochrome b, intron AM2B1 and 15 microsatellite loci. We detected uncharacteristically high genetic differentiation (FST 0.15-0.20; P<0.001) between the fish sampled from South Africa and the other sites, strongly influenced by five outlier microsatellite loci located in conserved intergenic regions. In addition, differentiation among the remaining East Atlantic sites was detected, although mtDNA indicated past isolation with subsequent secondary contact between these East Atlantic populations. We further identified secondary contact, with unidirectional gene flow from South Africa to Angola. The directional contact is likely explained by a combination of the northward flowing offshore current and endogenous incompatibilities restricting integration of certain regions of the genome and limiting gene flow to the south. The results confirm that the dynamic system associated with the Benguela current upwelling zone influences species distributions and population processes in the South-east Atlantic.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR® Identifiler® Plus PCR and Argus-X-12® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Detecting multiple DNA human profile from a mosquito blood meal.

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

Trace samples of human blood in mosquitoes as a forensic investigation tool.

Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/µL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

Distribution of forensic marker allelic frequencies in Pernambuco, Northestern Brazil.

Pernambuco is one of the 27 federal units of Brazil, ranking seventh in the number of inhabitants. We examined the allele frequencies of 13 short tandem repeat loci (CFS1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, vWA, and TPOX), the minimum recommended by the Federal Bureau of Investigation and commonly used in forensic genetics laboratories in Brazil, in a sample of 609 unrelated individuals from all geographic regions of Pernambuco. The allele frequencies ranged from 5 to 47.2%. No significant differences for any loci analyzed were observed compared with other publications in other various regions of Brazil. Most of the markers observed were in Hardy-Weinberg equilibrium. The occurrence of the allele 47.2 (locus FGA) and alleles 35.1 and 39 (locus D21S11), also described in a single study of the Brazilian population, was observed. The other forensic parameters analyzed (matching probability, power of discrimination, polymorphic information content, paternity exclusion, complement factor I, observed heterozygosity, expected heterozygosity) indicated that the studied markers are very informative for human forensic identification purposes in the Pernambuco population.

Evaluating the resolution power of new microsatellites for species identification and stock delimitation in the Cape hakes Merluccius paradoxus and Merluccius capensis (Teleostei: Merlucciidae).

The utility of 15 new and 17 previously published microsatellite markers was evaluated for species identification and stock delimitation in the deep-water hake Merluccius paradoxus and the shallow-water hake Merluccius capensis. A total of 14 microsatellites were polymorphic in M. paradoxus and 10 in M. capensis. Two markers could individually discriminate the species using Bayesian clustering methods and a statistical power analysis showed that the set of markers for each species is likely to detect subtle genetic differentiation (FST < 0·006) that will be valuable to delimit and characterize genetic stocks.

Radon in indoor concentrations and indoor concentrations of metal dust particles in museums and other public buildings.

The aim of this study was to evaluate the public and occupational exposure to radon and metal-bearing particles in museums and public buildings located in the city of Rio de Janeiro, Brazil. For this study, four buildings were selected: two historic buildings, which currently house an art gallery and an art museum; and two modern buildings, a chapel and a club. Integrated radon concentration measurements were performed using passive radon detectors with solid state nuclear track detector-type Lexan used as nuclear track detector. Air samplers with a cyclone were used to collect the airborne particle samples that were analyzed by the particle-induced X-ray emission technique. The average unattached-radon concentrations in indoor air in the buildings were above 40 Bq/m(3), with the exception of Building D as measured in 2009. The average radon concentrations in indoor air in the four buildings in 2009 were below the recommended reference level by World Health Organization (100 Bq/m(3)); however, in 2011, the average concentrations of radon in Buildings A and C were above this level, though lower than 300 Bq/m(3). The average concentrations of unattached radon were lower than 148 Bq/m(3) (4pCi/L), the USEPA level recommended to take action to reduce the concentrations of radon in indoor air. The unattached-radon average concentrations were also lower than the value recommended by the European Union for new houses. As the unattached-radon concentrations were below the international level recommended to take action to reduce the radon concentration in air, it was concluded that during the period of sampling, there was low risk to human health due to the inhalation of unattached radon in these four buildings.

Environmental and ichthyofaunistic characteristics of Amazonian streams with and without fish farm.

The environmental impacts caused by fish farming can lead to changes in aquatic ecosystems, especially in fish communities. In this study, we investigated possible changes in water quality, in the number of species and individuals of the same species caused by the construction of fish nurseries in dammed Amazonian streams. For this purpose, four streams located in the municipalities of Novo Airão and Presidente Figueiredo were selected. Samples were collected in streams without fish farming and in streams with stretches used for fish farming, where collections took place both downstream and upstream. The fish were captured, identified, quantified and the water was collected for physical and chemical analyses, in addition to the environmental characterization that was carried out. Comparisons were made using fish richness and abundance indices between the sampled points and correspondence analysis of the species identified at the collection sites, using the Mantel test and principal component analysis (PCA) for the environmental characteristics of the water and variables physical and chemical, and permutational multivariate analysis of variance (PERMANOVA) to verify the possible effects of species composition on the sampled conditions and on the analyzed environmental variables. In total, 2,302 fish belonging to 53 species, 15 families and six orders were found. We conclude that the Amazonian streams dammed for fish farming, under the analyzed conditions, can be characterized as environments with different and specific levels of richness and abundance, however, the data set analyzed in this study did not show that these characteristics are directly related to productive activity. We recommend that further studies be carried out following the current research.

Trophic relationships among three species of ornamental fish from the region of Lake Amanã, Amazon.

The present study objectified to evaluate the trophic relationships and the possible aggressive mimicry involving Carnegiella strigata, Carnegiella marthae and Gnathocharax steindachneri, in lowland forest streams. Samplings occurred in November 2002, March and August 2003 in Amanã Sustainable Development Reserve (ASDR). The total of 943 fish was caught, being 79.43% of C. marthae, 3.18% of C. strigata and 17.39% of G. steindachneri. Stomach contents, degree of stomach repletion, relative volume and frequency of occurrence of food items were evaluated. Thirteen food items were identified, and the presence of terrestrial and aquatic insects indicates the high dependence of these species and the flooded forest. Low feeding overlap was observed between Carnegiella strigata and C. marthae compared to Gnathocharax steindachneri. However, the overlap between the two Carnegiella species was relatively high, which could explain the low frequency of syntopic occurrence among these species, suggesting a possible case of competitive exclusion.

Idiopathic gingival fibromatosis: description of two cases.

Gingival fibromatosis is an enlargement localized or generalized of the gingival tissue characterized by an expansion and accumulation of the connective tissue, predominantly type I collagen, with occasional presence of increased number of cells, supposed fibroblastic proliferation. Gingival fibromatosis can be induced as a side effect of systemic drugs, such as phenytoin, cyclosporin, and nifedipine, or due to hereditary factors. However, in some cases, the gingival overgrowth is idiopathic. This paper reports two cases of idiopathic gingival fibromatosis and discusses the diagnosis, histopathological features, treatment and immunohistochemical evaluation of myofibroblasts of this condition. The tissues removed were fixed in formalin, and sections used for hematoxylin and eosin and Masson tricromic stain. To determine the presence of myofibroblasts, we performed immunohistochemistry against a-SMA protein. Histological examination revealed epithelial hyperplasia with long rete pegs and increase in the dense fibrous connective tissue. The Masson tricromic stain revealed wide bundles of collagen strongly stained. It was showed negative labeling to a-SMA. These results strongly suggest that myofibroblasts are not involved in gingival overgrowth in the cases of IGF reported. Future studies will be necessary to determine the pathogenesis of idiopathic gingival fibromatosis.

Seasonal variation of peptidase activities in the reproductive tract of Crotalus durissus terrificus.

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-I activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus.

Strategy to assess the overall cytokine profile of circulating leukocytes and its association with distinct clinical forms of human Chagas disease.

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate-IND and cardiac-CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine-positive leukocytes of our study sample to establish, for each cytokine-positive cell population, the cut-off edge that would segregate 'low' and 'high' cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4+ T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4+ T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL-10-producing CD4+ T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.

Idiotypic sensitization in utero of children born to mothers with schistosomiasis or Chagas' disease.

Cord blood mononuclear cells (CBMC) of neonates born of mothers with Chagas' disease or schistosomiasis exhibited strong proliferative responses against idiotypes expressed on antibodies with specificity for Trypanosoma cruzi or Schistosoma mansoni antigens, respectively. These immunoaffinity-purified preparations were stimulatory if they were prepared from pools of patients' sera or from the mother's own serum, taken early during her pregnancy. These CBMC did not respond to normal immunoglobulin, and CBMC of neonates born of uninfected mothers did not respond to antibodies against either T. cruzi or S. mansoni, or normal immunoglobulin preparations. We propose that in utero exposure of a fetus to some idiotypes expressed on placentally transferred antibodies induces anti-Id T lymphocyte sensitization, which we detect as a proliferative response by CBMC exposed to immunoaffinity-purified antibodies expressing the relevant idiotypes. This is the first experimental evidence that children born of mothers with chronic infections undergo natural in utero idiotypic manipulations and are born possessing cellular anti-Id reactivity.

Evolutionary relationships based on genetic and phenetic characters between Triatoma maculata, Triatoma pseudomaculata and morphologically related species (Reduviidae: Triatominae).

The maculata group currently comprises two species of Triatominae, Triatoma maculata and Triatoma pseudomaculata, which share morphologic and chromatic characteristics. In order to clarify the systematic status of these two vectors of Trypanosoma cruzi and to infer their evolutionary relationships, we performed an enzymatic, morphometric and cytogenetic comparison of them, also taking into account two sister species not included in the group (T. arthurneivai and T. wygodzinskyi). According to our results, T. maculata and T. pseudomaculata belong to distinct evolutionary lineages. Similarly, T. arthurneivai topotypes from Minas Gerais form an independent isolated group by morphometrics. Our results also support the specific status of the Triatoma population from São Paulo State (formerly referred to T. arthurneivai), and suggest the possibility that it is T. wygodzinskyi. Finally, we suggest that only the arboricolous T. pseudomaculata from northeast Brazil and the rupicolous sister species originated from São Paulo State should be classified together in the same group.

Trophic relationships among three species of ornamental fish from the region of Lake Amanã, Amazon / Relações tróficas entre três espécies de peixes ornamentais da região do lago de Amanã, Amazonas

The present study objectified to evaluate the trophic relationships and the possible aggressive mimicry involving Carnegiella strigata, Carnegiella marthae and Gnathocharax steindachneri, in lowland forest streams. Samplings occurred in November 2002, March and August 2003 in Amanã Sustainable Development Reserve (ASDR). The total of 943 fish was caught, being 79.43% of C. marthae, 3.18% of C. strigata and 17.39% of G. steindachneri. Stomach contents, degree of stomach repletion, relative volume and frequency of occurrence of food items were evaluated. Thirteen food items were identified, and the presence of terrestrial and aquatic insects indicates the high dependence of these species and the flooded forest. Low feeding overlap was observed between Carnegiella strigata and C. marthae compared to Gnathocharax steindachneri. However, the overlap between the two Carnegiella species was relatively high, which could explain the low frequency of syntopic occurrence among these species, suggesting a possible case of competitive exclusion.

Este estudo teve como objetivo analisar as relações tróficas e a possível existência de uma relação de mimetismo agressivo envolvendo Carnegiella strigata, Carnegiella marthae e Gnathocharax steindachneri, em igarapés de terra firme. As coletas ocorreram em novembro de 2002, março e agosto de 2003 e foram realizadas na Reserva de Desenvolvimento Sustentável de Amanã (RDSA). Foram capturados 943 peixes, onde C. marthae representou 79,43%, C. strigata 3,18% e G. steindachneri 17,39%. Foram analisados os conteúdos estomacais e verificados grau de repleção, volume relativo e frequência de ocorrência dos alimentos. Foram identificados 13 itens alimentares, onde insetos terrestres e aquáticos denotaram alto grau de dependência destas espécies em relação à floresta alagada. Ocorreu baixa sobreposição alimentar das espécies Carnegiella strigata e C. marthae em relação a Gnathocharax steindachneri. Entretanto, a sobreposição entre as duas espécies de Carnegiella foi relativamente alta, o que poderia explicar a baixa frequência de ocorrência sintópica entre essas espécies, sugerindo um possível caso de exclusão competitiva.

Trophic relationships among three species of ornamental fish from the region of Lake Amanã, Amazon

Abstract The present study objectified to evaluate the trophic relationships and the possible aggressive mimicry involving Carnegiella strigata, Carnegiella marthae and Gnathocharax steindachneri, in lowland forest streams. Samplings occurred in November 2002, March and August 2003 in Amanã Sustainable Development Reserve (ASDR). The total of 943 fish was caught, being 79.43% of C. marthae, 3.18% of C. strigata and 17.39% of G. steindachneri. Stomach contents, degree of stomach repletion, relative volume and frequency of occurrence of food items were evaluated. Thirteen food items were identified, and the presence of terrestrial and aquatic insects indicates the high dependence of these species and the flooded forest. Low feeding overlap was observed between Carnegiella strigata and C. marthae compared to Gnathocharax steindachneri. However, the overlap between the two Carnegiella species was relatively high, which could explain the low frequency of syntopic occurrence among these species, suggesting a possible case of competitive exclusion.

Resumo Este estudo teve como objetivo analisar as relações tróficas e a possível existência de uma relação de mimetismo agressivo envolvendo Carnegiella strigata, Carnegiella marthae e Gnathocharax steindachneri, em igarapés de terra firme. As coletas ocorreram em novembro de 2002, março e agosto de 2003 e foram realizadas na Reserva de Desenvolvimento Sustentável de Amanã (RDSA). Foram capturados 943 peixes, onde C. marthae representou 79,43%, C. strigata 3,18% e G. steindachneri 17,39%. Foram analisados os conteúdos estomacais e verificados grau de repleção, volume relativo e frequência de ocorrência dos alimentos. Foram identificados 13 itens alimentares, onde insetos terrestres e aquáticos denotaram alto grau de dependência destas espécies em relação à floresta alagada. Ocorreu baixa sobreposição alimentar das espécies Carnegiella strigata e C. marthae em relação a Gnathocharax steindachneri. Entretanto, a sobreposição entre as duas espécies de Carnegiella foi relativamente alta, o que poderia explicar a baixa frequência de ocorrência sintópica entre essas espécies, sugerindo um possível caso de exclusão competitiva.

Trophic relationships among three species of ornamental fish from the region of Lake Amanã, Amazon / Relações tróficas entre três espécies de peixes ornamentais da região do lago de Amanã, Amazonas

The present study objectified to evaluate the trophic relationships and the possible aggressive mimicry involving Carnegiella strigata, Carnegiella marthae and Gnathocharax steindachneri, in lowland forest streams. Samplings occurred in November 2002, March and August 2003 in Amanã Sustainable Development Reserve (ASDR). The total of 943 fish was caught, being 79.43% of C. marthae, 3.18% of C. strigata and 17.39% of G. steindachneri. Stomach contents, degree of stomach repletion, relative volume and frequency of occurrence of food items were evaluated. Thirteen food items were identified, and the presence of terrestrial and aquatic insects indicates the high dependence of these species and the flooded forest. Low feeding overlap was observed between Carnegiella strigata and C. marthae compared to Gnathocharax steindachneri. However, the overlap between the two Carnegiella species was relatively high, which could explain the low frequency of syntopic occurrence among these species, suggesting a possible case of competitive exclusion.

Este estudo teve como objetivo analisar as relações tróficas e a possível existência de uma relação de mimetismo agressivo envolvendo Carnegiella strigata, Carnegiella marthae e Gnathocharax steindachneri, em igarapés de terra firme. As coletas ocorreram em novembro de 2002, março e agosto de 2003 e foram realizadas na Reserva de Desenvolvimento Sustentável de Amanã (RDSA). Foram capturados 943 peixes, onde C. marthae representou 79,43%, C. strigata 3,18% e G. steindachneri 17,39%. Foram analisados os conteúdos estomacais e verificados grau de repleção, volume relativo e frequência de ocorrência dos alimentos. Foram identificados 13 itens alimentares, onde insetos terrestres e aquáticos denotaram alto grau de dependência destas espécies em relação à floresta alagada. Ocorreu baixa sobreposição alimentar das espécies Carnegiella strigata e C. marthae em relação a Gnathocharax steindachneri. Entretanto, a sobreposição entre as duas espécies de Carnegiella foi relativamente alta, o que poderia explicar a baixa frequência de ocorrência sintópica entre essas espécies, sugerindo um possível caso de exclusão competitiva.