Phylogeographic review of Y chromosome haplogroups in Europe.

The Y chromosome has been widely explored for the study of human migrations. Due to its paternal inheritance, the Y chromosome polymorphisms are helpful tools for understanding the geographical distribution of populations all over the world and for inferring their origin, which is really useful in forensics. The remarkable historical context of Europe, with numerous migrations and invasions, has turned this continent into a melting pot. For this reason, it is interesting to study the Y chromosome variability and how it has contributed to improving our knowledge of the distribution and development of European male genetic pool as it is today. The analysis of Y lineages in Europe shows the predominance of four haplogroups, R1b-M269, I1-M253, I2-M438 and R1a-M420. However, other haplogroups have been identified which, although less frequent, provide significant evidence about the paternal origin of the populations. In addition, the study of the Y chromosome in Europe is a valuable tool for revealing the genetic trace of the different European colonizations, mainly in several American countries, where the European ancestry is mostly detected by the presence of the R1b-M269 haplogroup. Therefore, the objective of this review is to compile the studies of the Y chromosome haplogroups in current European populations, in order to provide an outline of these haplogroups which facilitate their use in forensic studies.

Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene.

Meat adulteration by substitution with lower value products and/or mislabeling involves economic, health, quality and socio-religious issues. Therefore, identification and traceability of meat species has become an important subject to detect possible fraudulent practices. In the present study the development of a high resolution melt (HRM) screening method for the identification of eight common meat species is reported. Samples from Bos taurus, Ovis aries, Sus scrofa domestica, Equus caballus, Oryctolagus cuniculus, Gallus gallus domesticus, Meleagris gallopavo and Coturnix coturnix were analyzed through the amplification of a 148 bp fragment from the cyt b gene with a universal primer pair in HRM analyses. Melting profiles from each species, as well as from several DNA mixtures of these species and blind samples, allowed a successful species differentiation. The results demonstrated that the HRM method here proposed is a fast, reliable, and low-cost screening technique.

Identification of new SNPs in native South American populations by resequencing the Y chromosome.

The Y-chromosomal genetic landscape of South America is relatively homogenous. The majority of native Amerindian people are assigned to haplogroup Q and only a small percentage belongs to haplogroup C. With the aim of further differentiating the major Q lineages and thus obtaining new insights into the population history of South America, two individuals, both belonging to the sub-haplogroup Q-M3, were analyzed with next-generation sequencing. Several new candidate SNPs were evaluated and four were confirmed to be new, haplogroup Q-specific, and variable. One of the new SNPs, named MG2, identifies a new sub-haplogroup downstream of Q-M3; the other three (MG11, MG13, MG15) are upstream of Q-M3 but downstream of M242, and describe branches at the same phylogenetic positions as previously known SNPs in the samples tested. These four SNPs were typed in 100 individuals belonging to haplogroup Q.

Association between ancient bone preservation and dna yield: a multidisciplinary approach.

Ancient molecular typing depends on DNA survival in archaeological bones. Finding valuable tools to predict DNA presence in ancient samples, which can be measured prior to undertaking a genetic study, has become an important issue as a consequence of the peculiarities of archaeological samples. Since the survival of DNA is explained by complex interrelations of multiple variables, the aim of the present study was to analyze morphological, structural, chemical, and biological aspects of a set of medieval human bones, to provide an accurate reflection of the state of preservation of the bony components and to relate it with DNA presence. Archaeological bones that yielded amplifiable DNA presented high collagen content (generally more than 12%), low racemization values of aspartic acid (lesser than 0.08), leucine and glutamic acid, low infrared splitting factor, small size of crystallite, and more compact appearance of bone in the scanning electron micrographs. Whether these patterns are characteristic of ancient bones or specific of each burial site or specimen requires further investigation.

Evaluation of the illegal use of clenbuterol in Portuguese cattle farms from drinking water, urine, hair and feed samples.

The recent discovery of clenbuterol contamination in Portuguese food led to the specific inspection of 16 cattle farms for beta-agonists, involving the analysis of a total of 486 samples (78 feed, 106 drinking water, 168 urine and 134 hair). The samples were screened for the beta-agonists: bromobuterol, cimaterol, clenbuterol, clenpenterol, clenproperol, hydroxymethylclenbuterol, mapenterol, salbutamol and terbutaline. Only clenbuterol was found in all analyzed matrices and the most likely method of illegal administration to animals was through drinking water. Of all samples analysed, 14.15% of drinking water were found positive in the range 0.03-3.80 mg l(-1) clenbuterol. Inclusion of hair samples in the Portuguese plan for clenbuterol residue control in live animals is discussed.

A preliminary study on the incidence of heteroplasmy in mitochondrial DNA from vitreous humour.

Vitreous humour is routinely sampled in Forensic Medicine as several post-mortem analyses can be performed. However, it is not used for DNA analyses probably due to its scarce cellularity. In these samples, in which the study of nuclear DNA is difficult, the analysis of mtDNA is an alternative approach. The aim of this study was to investigate the utility of vitreous humour for forensic identification purposes. Samples were collected during vitrectomy from retinopathy patients, in collection bags with saline solution. Blood samples were also obtained in order to contrast results. Before DNA organic extraction, several centrifugation steps were needed to concentrate the vitreous humour samples. Unlike blood, direct amplification of 400-bp fragments of the hipervariable regions I and II (HVI and HVII) was not successful, possibly due to damage to the DNA strand caused by the surgery conditions (UV radiation, oxidative stress). Therefore, amplification of two overlapping fragments for each control region was performed in vitreous humour. In order to eliminate undesired products, all samples were purified by an enzymatic method. Thereafter, mtDNA fragments were sequenced using dye terminators in a MegaBACE 500 capillary sequencer. Sequences of HVI and HVII of approximately 400 bp were obtained from all samples. The sequences obtained from each patient matched almost perfectly those from blood. In summary, herein we describe for the first time a methodology suitable for the mtDNA analysis of vitreous humour samples.

Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree.

Urban and rural population exposure to ochratoxin A (OTA) in central zone of Portugal was investigated in three places: Coimbra, Verride and Ereira. The analytical method proposed for the determination of ochratoxin A involved extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column (IAC), high performance liquid chromatography (HPLC) with spectrofluorimetric detection (FD) for separation and identification of ochratoxin A, and confirmation with HPLC-FD after OTA methylation in serum. The limit of quantification of the proposed method was 0.1 microg/L for serum and 0.05 microg/L for blood. OTA recoveries in serum ranged from 70.3% to 115.3% for levels at 0.25 microg/L and 0.5 microg/L, respectively, with a within-day RSD between 8.0% and 16.2%. Ochratoxin A serum levels were evaluated in an hundred and four donors from Coimbra city, Verride, and Ereira. The study revealed a frequency of detection of 100%. The ratio of ochratoxin A level in serum to whole blood was 2.0+/-0.7. The overall concentrations range from 0.25 to 2.49 microg/L, 0.14 to 1.91 microg/L, and 0.19 to 0.96 microg/L, for samples of Verride, Ereira, and Coimbra, respectively. The mean concentration and standard deviation were 0.78+/-0.53 microg/L, 0.44+/-0.31 microg/L, and 0.42+/-0.18 microg/L for the same samples. A significant difference was found in Verride population (P-value=0.000). Levels of OTA are clearly higher in males from rural areas than in females. For all samples, a significant difference was found in Verride male population (P-value=0.014).

A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 microg kg(-1). Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 microg kg(-1), respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg(-1) body weight per day for swine samples and 18 pg kg(-1) body weight per day for turkey samples.

[Bacterial contamination of breast milk collected through manual expression and stored at room temperature] / Contaminação bacteriana do leite humano coletado por expressão manual e estocado à temperatura ambiente.

OBJECTIVES: To the determine the bacterial contamination profile of unheated expressed breast milk, collected without rigid hygienic precautions and stored at room temperature for nine hours. The purpose was to give poor lactating mothers the alternative of storing their own milk out of refrigerator. A research on cultural, social and economical aspects as well as on donators knowledge about breastfeeding was considered necessary. METHODS: 35 donators were interviewed and an experimental investigation was performed with 33 samples of breast milk stored at room temperature (17 masculine C to 30.5 masculine C) and bacteriologically analyzed at zero, three, six and nine hours after collection. The same breast milk was stored at refrigerator (2 masculine C to 6 masculine C) as a control procedure. Total count of bacterial contents and identification of Staphylococcus aureus and Escherichia coli were evaluated.RESULTS: The enterviews revealed the low socio-economical and cultural level of lactating mothers and their little experience in expressing, collecting and using their own milk. Bacteriological data analysis showed mesophyllous average of 7.1x10(3)UFC/mL, acceptable outline of bacterial contamination, despite the use of a simplified hygiene technique. After nine hours, samples stored at room temperature showed final average of bacterial contents similar to the first ones (7.3x10(3)UFC/mL) and without relevant statistic differences from the ones kept under refrigeration (p=0.05) for studied bacterias.CONCLUSION: This study shows that it is possible to use unprocessed breast milk for babýs consumption if it is stored at room temperatures until nine hours after it has been collected. However, mothers have to be told about the possibility of storing breast milk for babies later consumption.