Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

Quantitative phenetics and taxonomy of some phlebotomine taxa

Elucidating the evolution of Phlebotominae is important not only to revise their taxonomy, but also to help understand the origin of the genus Leishmania and its relationship with humans. Our study is a phenetic portrayal of this history based on the genetic relationships among some New Word and Old Word taxa. We used both multilocus enzyme electrophoresis and morphometry on 24 male specimens of the Old Word genus Phlebotomus (with three of its subgenera: Phlebotomus, Spelaeophlebotomus and Australophlebotomus), and on 67 male specimens of the three New World genera, Warileya, Brumptomyia and Lutzomyia, (with three subgenera of Lutzomyia: Lutzomyia, Oligodontomyia and Psychodopygus). Phenetic trees derived from both techniques were similar, but disclosed relationships that disagree with the present classification of sand flies. The need for a true evolutionary approach is stressed.

Brazilian Flavivirus phylogeny based on NS5

In this work, a comprehensive phylogenetic study based on 600 base pair nucleotide and on putative 200 amino acid sequences of NS5 was carried out in order to establish genetic relationships among 15 strains of 10 Brazilian flaviviruses: Bussuquara, Cacipacore, dengue type 1, 2 and 4, Iguape, Ilheus, Rocio, Saint Louis encephalitis (SLE), and yellow fever. Phylogenetic trees were created by neighbor-joining and maximum parsimony methods. These trees showed Brazilian flaviviruses grouped into three main branches: yellow fever branch, dengue branch subdivided in types 1, 2 and 4 branches, and Japanese encephalitis virus (JEV) complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara. Viruses transmitted by Aedes mosquitoes, such as dengue and urban yellow fever, that are also the only Flavivirus causing hemorrhagic fevers in Brazil, were grouped in the same cluster. Encephalitis associated viruses, transmitted by Culex mosquitoes such as JEV complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara were also grouped in the same clade.

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups: a review

The socalled enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheageniccategories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in São Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheageniccategory O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

First detection of a human astrovirus type 8 in a child with diarrhea in Belém, Brazil: comparison with other strains worldwide and identification of possible three lineages

This study describes the genetic relationships of the first human astrovirus type-8 (HAstV-8) detected in Belém-Brazil, during a public hospital-based study. This strain was compared with other HAstV-8 strains identified elsewhere which have sequences available at GeneBank. The regions ORF1a (primers Mon348/Mon340) and ORF2 (primers Mon269/Mon270) were analyzed by nucleotide sequencing and a high similarity rate was observed among the Belém strain and other HAstV-8 strains. In ORF1a, homology values of 93-100% were detected, and in ORF2 96-99%. Considering the sequence variation (7%) observed in ORF2 region, it was suggested that HAstV-8 strains could be divided in three different lineages.

Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA) report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.

Extreme homogeneity among Brazilian wheat genotypes determined by RAPD markers

Random amplified polymorphic DNA markers (RAPD) were used to estimate the variability of 14 genotypes of Brazilian wheat (Triticum aestivum L.), using a set of 50 random 10mer primers. A total of 256 reproducibly scorable DNA amplification products were obtained from 48 of the primers, 83% of which were polymorphic. Genetic distances among genotypes were calculated and a dendrogram and a principal coordinates analysis showing the genetic relationships among them were obtained. Despite the low variability found (average genetic distance of 27%), two groups of genotypes could be identified, which probably reflect how they were formed. Studies such as this one may be important in the planning and development of future improvement programs for this plant species.

Assessment of silver-stained AFLP markers for studying DNA polymorphism in proso millet (Panicum miliaceum L.)

Proso millet (Panicum miliaceum L.) is a serious weed in North America. A high number of wild proso millet biotypes are known but the genetic basis of its phenotypic variation is poorly understood. In the present study, a non-radioactive silver staining method for PCR-Amplified Fragment Length Polymorphism (AFLP) was evaluated for studying genetic polymorphism in American proso millet biotypes. Twelve biotypes and eight primer combinations with two/three and three/three selective nucleotides were used. Pair of primers with two/three selective nucleotides produced the highest number of amplified DNA fragments, while pair of primers with three/three selective nucleotides were more effective for revealing more polymorphic DNA fragments. The two better primer combinations were EcoR-AAC/Mse-CTT and EcoR-ACT/Mse-CAA with seven and eleven polymorphic DNA fragments, respectively. In a total of 450 amplified fragments, at least 339 appeared well separated in a silver stained acrylamide gel and 39 polymorphic DNA bands were scored. The level of polymorphic DNA (11.5%) using only eight pairs of primers were effective for grouping proso millet biotypes in two clusters but insufficient for separating hybrid biotypes from wild and crop. Nevertheless, the present result indicates that silver stained AFLP markers could be a cheap and important tool for studying genetic relationships in proso millet.

Genetic diversity of Brazilian Pantaneiro horse and relationships among horse breeds

The objective of this work was to evaluate the genetic diversity of Brazilian Pantaneiro horse by microsatellite markers, investigate the effect of genetic bottlenecks and estimate genetic differentiation among four horse breeds. Genetic variation was estimated through allele frequencies and mean breed heterozygosity. Nei's genetic distances among the breeds Pantaneiro, Thoroughbred, Arabian, Spanish Pure Breed (Andalusian), and Uruguay Creole were calculated, and it was used to construct an UPGMA dendrogram. Clustering at different K values was calculated to infer population structure and assign individuals to populations. Nei's distances showed a minimum distance between Pantaneiro horse and Spanish Pure Breed (0.228), and similar distances from Spanish Pure Breed to Thoroughbred and to Arabian (0.355 and 0.332). It was observed a great level of diversity, clear distance from Pantaneiro horse to other breeds, and genetic uniformity within breed. It was verified a certain level of substructure of Pantaneiro horse showing no influences from the other studied breeds.

Smoking, consumption of alcohol and sedentary life style in population grouping and their relationships with lipemic disorders

The study, part of the project "Atherosclerotic cardiovascular diseases, lipemic disorders, hypertension, obesity and diabetis mellitus in a population of the metropolitan area of the southeastern region of Brazil", had the following objectives: a) the characterization and distribution among typical human socio-economic groupings, of the prevalence of some particular habits which constitute aspects of life-style-the use of tobacco, the use of alcohol and sedentary activity; b) the establishment of the interrelation between the above-mentioned habits and some lipemic disorders. The prevalence of the habits cited behaved in the following manner: the use of tobacco predominated among men, distributed uniformly throughout the social strata; among the women the average percentage of smokers was 18,9%, a significant difference occurring among the highest socio-economic class, where the average was of 40.2%. The sedentary style of life presented high prevalence, among both men and women with exception of the women of the highest socio-economic level and of the skilled working class. The use of alcohol, as one would expect, is a habit basically practised by the men, without any statistically significant differences between classes. For the purpose of establishing associations between these risk fictors and lipemic conditions four situations were chosen, of the following characteristics: 1- total cholesterol > or = 220 mg/dl and triglycerides > or = 150 mg/dl; 2- HDL cholesterol <35 mg/dl for men and <45 mg/dl for women and triglycerides levels > or = 150 mg/dl; 3- HDL cholesterol <35 mg/dl for men and <45 mg/dl for women and triglycerides levels <150 mg/dl; 4- total cholesterol 220 mg/dl with triglycerides levels <150 mg/dl. Six models of multiple (backward) regression were established, with seven independent variables- age, sex, use of tobacco, consumption of alcohol, light physical activity, hypertension and obesity. Significant associations (P<0,05) were revealed with hypercholesterolemia, accompanied by triglyceride levels > or = 150 mg/dl, and the following independent variables: age, use of tobacco and the interactions between obesity and smoking, age and sedentary lifestyle, sex and obesity (R2=22%); the standardized B coefficient showed that the variables with the greatest weight in the forecasting of the variation in the levels of cholesterol were smoking and the interaction between obesity and smoking. The hypercholesterolemia accompanied by triglycerides levels <150 mg/dl showed a positive association between total cholesterol and sex and the interactions obesity/smoking and sex/obesity. As regards HDL cholesterol accompanied by triglyceride/ levels > or = 150 mg/dl was inversely associated with obesity and the interaction smoking/ age and directly with age (R=31%). The standardized B coeffients, indicated that the variables obesity and the interactions smoking/age possessed a weight three times greater than age alone in accounting for the variation in the serum levels of HDL cholesterol. When accompanied by triglycerides <150 mg/dl there was no association between and the independent variables and the set of them presented R equal to 22%. The sum of top, in the population stutied in this project, the component habits of life-style (smoking, alcohol consumption and sedentary activity) which constitute risk factors which determine morbidity from atherosclerotic cardiovascular diseases are be found distributed through all the typical social groupings of this particular form of social organization. On the other hand, the seven independent variables used in the multiple regression models for the explanation of the lipemic conditions considered presented multiple determination coefficients which varied, approximately, between 20% and 30%. Thus it is important that in the genetic epidemiology the study of the morbidities in question be emphasized.

Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs)

Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers) were used to amplify the extracted DNA by the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Genetic Data Showing Evolutionary Links between Leishmania and Endotrypanum

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Coevolutionary networks: a novel approach to understanding the relationships of humans with the infectious agents

Human organism is interpenetrated by the world of microorganisms, from the conception until the death. This interpenetration involves different levels of interactions between the partners including trophic exchanges, bi-directional cell signaling and gene activation, besides genetic and epigenetic phenomena, and tends towards mutual adaptation and coevolution. Since these processes are critical for the survival of individuals and species, they rely on the existence of a complex organization of adaptive systems aiming at two apparently conflicting purposes: the maintenance of the internal coherence of each partner, and a mutually advantageous coexistence and progressive adaptation between them. Humans possess three adaptive systems: the nervous, the endocrine and the immune system, each internally organized into subsystems functionally connected by intraconnections, to maintain the internal coherence of the system. The three adaptive systems aim at the maintenance of the internal coherence of the organism and are functionally linked by interconnections, in such way that what happens to one is immediately sensed by the others. The different communities of infectious agents that live within the organism are also organized into functional networks. The members of each community are linked by intraconnections, represented by the mutual trophic, metabolic and other influences, while the different infectious communities affect each other through interconnections. Furthermore, by means of its adaptive systems, the organism influences and is influenced by the microbial communities through the existence of transconnections. It is proposed that these highly complex and dynamic networks, involving gene exchange and epigenetic phenomena, represent major coevolutionary forces for humans and microorganisms.

Genetic epidemiology of fecal egg excretion during Schistosoma mansoni infection in an endemic area in Minas Gerais, Brazil

There is considerable variation in the level of fecal egg excretion during Schistosoma mansoni infections. Within a single endemic area, the distribution of egg counts is typically overdispersed, with the majority of eggs excreted coming from a minority of residents. The purpose of this study was to quantify the influence of genetic factors on patterns of fecal egg excretion in a rural study sample in Brazil. Individual fecal egg excretions, expressed in eggs per gram of feces, were determined by the Kato-Katz method on stool samples collected on three different days. Detailed genealogic information was gathered at the time of sampling, which allowed assignment of 461 individuals to 14 pedigrees containing between 3 and 422 individuals. Using a maximum likelihood variance decomposition approach, we performed quantitative genetic analyses to determine if genetic factors could partially account for the observed pattern of fecal egg excretion. The quantitative genetic analysis indicated that between 21-37% of the variation in S. mansoni egg counts was attributable to additive genetic factors and that shared environment, as assessed by common household, accounted for a further 12-21% of the observed variation. A maximum likelihood heritability (h²) estimate of 0.44 ± 0.14 (mean ± SE) was found for the 9,604 second- and higher-degree pairwise relationships in the study sample, which is consistent with the upper limit (37%) of the genetic factor determined in the variance decomposition analysis. These analyses point to the significant influence of additive host genes on the pattern of S. mansoni fecal egg excretion in this endemic area.