Inference of tobacco and alcohol consumption habits from DNA methylation analysis of blood.

DNA methylation has become a biomarker of great interest in the forensic and clinical fields. In criminal investigations, the study of this epigenetic marker has allowed the development of DNA intelligence tools providing information that can be useful for investigators, such as age prediction. Following a similar trend, when the origin of a sample in a criminal scenario is unknown, the inference of an individual's lifestyle such as tobacco use and alcohol consumption could provide relevant information to help in the identification of DNA donors at the crime scene. At the same time, in the clinical domain, prediction of these trends of consumption could allow the identification of people at risk or better identification of the causes of different pathologies. In the present study, DNA methylation data from the UK AIRWAVE study was used to build two binomial logistic models for the inference of smoking and drinking status. A total of 348 individuals (116 non-smokers, 116 former smokers and 116 smokers) plus a total of 237 individuals (79 non-drinkers, 79 moderate drinkers and 79 drinkers) were used for development of tobacco and alcohol consumption prediction models, respectively. The tobacco prediction model was composed of two CpGs (cg05575921 in AHRR and cg01940273) and the alcohol prediction model three CpGs (cg06690548 in SLC7A11, cg0886875 and cg21294714 in MIR4435-2HG), providing correct classifications of 86.49% and 74.26%, respectively. Validation of the models was performed using leave-one-out cross-validation. Additionally, two independent testing sets were also assessed for tobacco and alcohol consumption. Considering that the consumption of these substances could underlie accelerated epigenetic ageing patterns, the effect of these lifestyles on the prediction of age was evaluated. To do that, a quantile regression model based on previous studies was generated, and the potential effect of tobacco and alcohol consumption with the epigenetic age was assessed. The Wilcoxon test was used to evaluate the residuals generated by the model and no significant differences were observed between the categories analyzed.

Adapting an established Ampliseq microhaplotype panel to nanopore sequencing through direct PCR.

We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5-10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2-3 orders of magnitude higher when testing with the MinION-based haplotype data.

Development and evaluations of the ancestry informative markers of the VISAGE Enhanced Tool for Appearance and Ancestry.

The VISAGE Enhanced Tool for Appearance and Ancestry (ET) has been designed to combine markers for the prediction of bio-geographical ancestry plus a range of externally visible characteristics into a single massively parallel sequencing (MPS) assay. We describe the development of the ancestry panel markers used in ET, and the enhanced analyses they provide compared to previous MPS-based forensic ancestry assays. As well as established autosomal single nucleotide polymorphisms (SNPs) that differentiate sub-Saharan African, European, East Asian, South Asian, Native American, and Oceanian populations, ET includes autosomal SNPs able to efficiently differentiate populations from Middle East regions. The ability of the ET autosomal ancestry SNPs to distinguish Middle East populations from other continentally defined population groups is such that characteristic patterns for this region can be discerned in genetic cluster analysis using STRUCTURE. Joint cluster membership estimates showing individual co-ancestry that signals North African or East African origins were detected, or cluster patterns were seen that indicate origins from central and Eastern regions of the Middle East. In addition to an augmented panel of autosomal SNPs, ET includes panels of 85 Y-SNPs, 16 X-SNPs and 21 autosomal Microhaplotypes. The Y- and X-SNPs provide a distinct method for obtaining extra detail about co-ancestry patterns identified in males with admixed backgrounds. This study used the 1000 Genomes admixed African and admixed American sample sets to fully explore these enhancements to the analysis of individual co-ancestry. Samples from urban and rural Brazil with contrasting distributions of African, European, and Native American co-ancestry were also studied to gauge the efficiency of combining Y- and X-SNP data for this purpose. The small panel of Microhaplotypes incorporated in ET were selected because they showed the highest levels of haplotype diversity amongst the seven population groups we sought to differentiate. Microhaplotype data was not formally combined with single-site SNP genotypes to analyse ancestry. However, the haplotype sequence reads obtained with ET from these loci creates an effective system for de-convoluting two-contributor mixed DNA. We made simple mixture experiments to demonstrate that when the contributors have different ancestries and the mixture ratios are imbalanced (i.e., not 1:1 mixtures) the ET Microhaplotype panel is an informative system to infer ancestry when this differs between the contributors.

Eurasiaplex-2: Shifting the focus to SNPs with high population specificity increases the power of forensic ancestry marker sets.

To compile a new South Asian-informative panel of forensic ancestry SNPs, we changed the strategy for selecting the most powerful markers for this purpose by targeting polymorphisms with near absolute specificity - when the South Asian-informative allele identified is absent from all other populations or present at frequencies below 0.001 (one in a thousand). More than 120 candidate SNPs were identified from 1000 Genomes datasets satisfying an allele frequency screen of ≥ 0.1 (10 % or more) allele frequency in South Asians, and ≤ 0.001 (0.1 % or less) in African, East Asian, and European populations. From the candidate pool of markers, a final panel of 36 SNPs, widely distributed across most autosomes, were selected that had allele frequencies in the five 1000 Genomes South Asian populations ranging from 0.4 to 0.15. Slightly lower average allele frequencies, but consistent patterns of informativeness were observed in gnomAD South Asian datasets used to validate the 1000 Genomes variant annotations. We named the panel of 36 South Asian-specific SNPs Eurasiaplex-2, and the informativeness of the panel was evaluated by compiling worldwide population data from 4097 samples in four genome variation databases that largely complement the global sampling of 1000 Genomes. Consistent patterns of allele frequency distribution, which were specific to South Asia, were observed in all populations in, or closely sited to, the Indian sub-continent. Pakistani populations from the HGDP-CEPH panel had markedly lower allele frequencies, highlighting the need to develop a statistical system to evaluate the ancestry inference value of counting the number of population-specific alleles present in an individual.

Epigenetic age estimation in saliva and in buccal cells.

Age estimation based on epigenetic markers is a DNA intelligence tool with the potential to provide relevant information for criminal investigations, as well as to improve the inference of age-dependent physical characteristics such as male pattern baldness or hair color. Age prediction models have been developed based on different tissues, including saliva and buccal cells, which show different methylation patterns as they are composed of different cell populations. On many occasions in a criminal investigation, the origin of a sample or the proportion of tissues is not known with certainty, for example the provenance of cigarette butts, so use of combined models can provide lower prediction errors. In the present study, two tissue-specific and seven age-correlated CpG sites were selected from publicly available data from the Illumina HumanMethylation 450 BeadChip and bibliographic searches, to help build a tissue-dependent, and an age-prediction model, respectively. For the development of both models, a total of 184 samples (N = 91 saliva and N = 93 buccal cells) ranging from 21 to 86 years old were used. Validation of the models was performed using either k-fold cross-validation and an additional set of 184 samples (N = 93 saliva and N = 91 buccal cells, 21-86 years old). The tissue prediction model was developed using two CpG sites (HUNK and RUNX1) based on logistic regression that produced a correct classification rate for saliva and buccal swab samples of 88.59 % for the training set, and 83.69 % for the testing set. Despite these high success rates, a combined age prediction model was developed covering both saliva and buccal cells, using seven CpG sites (cg10501210, LHFPL4, ELOVL2, PDE4C, HOXC4, OTUD7A and EDARADD) based on multivariate quantile regression giving a median absolute error (MAE): ± 3.54 years and a correct classification rate ( %CP±PI) of 76.08 % for the training set, and an MAE of ± 3.66 years and a %CP±PI of 71.19 % for the testing set. The addition of tissue-of origin as a co-variate to the model was assessed, but no improvement was detected in age predictions. Finally, considering the limitations usually faced by forensic DNA analyses, the robustness of the model and the minimum recommended amount of input DNA for bisulfite conversion were evaluated, considering up to 10 ng of genomic DNA for reproducible results. The final multivariate quantile regression age predictor based on the models we developed has been placed in the open-access Snipper forensic classification website.

A compilation of tri-allelic SNPs from 1000 Genomes and use of the most polymorphic loci for a large-scale human identification panel.

In a directed search of 1000 Genomes Phase III variation data, 271,934 tri-allelic single nucleotide polymorphisms (SNPs) were identified amongst the genotypes of 2,504 individuals from 26 populations. The majority of tri-allelic SNPs have three nucleotide substitution-based alleles at the same position, while a much smaller proportion, which we did not compile, have a nucleotide insertion/deletion plus substitution alleles. SNPs with three alleles have higher discrimination power than binary loci but keep the same characteristic of optimum amplification of the fragmented DNA found in highly degraded forensic samples. Although most of the tri-allelic SNPs identified had one or two alleles at low frequencies, often single observations, we present a full compilation of the genome positions, rs-numbers and genotypes of all tri-allelic SNPs detected by the 1000 Genomes project from the more detailed analyses it applied to Phase III sequence data. A total of 8,705 tri-allelic SNPs had overall heterozygosities (averaged across all 1000 Genomes populations) higher than the binary SNP maximum value of 0.5. Of these, 1,637 displayed the highest average heterozygosity values of 0.6-0.666. The most informative tri-allelic SNPs we identified were used to construct a large-scale human identification panel for massively parallel sequencing, designed for the identification of missing persons. The large-scale MPS identification panel comprised: 1,241 autosomal tri-allelic SNPs and 29 X tri-allelic SNPs (plus 46 microhaplotypes adapted for genotyping from reduced length sequences). Allele frequency estimates are detailed for African, European, South Asian and East Asian population groups plus the Peruvian population sampled by 1000 Genomes for the 1,270 tri-allelic SNPs of the final MPS panel. We describe the selection criteria, kinship simulation experiments and genomic analyses used to select the tri-allelic SNP components of the panel. Approximately 5 % of the tri-allelic SNPs selected for the large-scale MPS identification panel gave three-genotype patterns in single individual samples or discordant genotypes for genomic control DNAs. A likely explanation for some of these unreliably genotyped loci is that they map to multiple sites in the genome - highlighting the need for caution and detailed scrutiny of multiple-allele variant data when designing future forensic SNP panels, as such patterns can arise from common structural variation in the genome, such as segmental duplications.

Effect of pH(24), NaCl content and proteolysis index on the relationship between water content and texture parameters in biceps femoris and semimembranosus muscles in dry-cured ham.

The aim of the present study was to determine the effect of pH level and NaCl content on the relationship between water content and texture parameters in semimembranosus and biceps femoris muscles in dry-cured ham. The experiment was undertaken using 18 hams, selected in a commercial slaughterhouse. Half of the hams had a pH<5.7 and the rest a pH>6.2, measured in the semimembranosus muscle at 24-h post mortem (pH(SM24)). The hams were treated with 20, 50 or 80g of NaCl per kg of ham. At the end of the aging process nine samples from semimembranosus and biceps femoris muscles were dried to different levels of water content covering the range from 22.4% to 58.5%. At the end of the drying period, a Texture Profile Analysis was used to determine textural parameters. Samples from biceps femoris muscle and samples from hams with low pH(SM24) showed a higher proteolysis index (100×non-protein nitrogen/total nitrogen) than samples from semimembranosus muscle and samples from hams with high pH(SM24), respectively. The proteolysis index decreased when the added NaCl amount increased. The proteolysis index was the parameter that best explained the modifications in the relationship between water content and the texture parameters (hardness, cohesiveness and springiness) of dry-cured ham muscles and it would be considered in order to predict the texture in dry-cured ham at different drying levels. Dry-cured hams with a lower proteolysis index were more prone to present harder texture at low water contents, which is typical of hams with crustiness problems.

Texture parameters of dry-cured ham m. biceps femoris samples dried at different levels as a function of water activity and water content.

Instrumental texture parameters of m. biceps femoris (BF) samples from six commercial dry-cured hams, dried to a different degree, were related to water activity and water content. Samples were carved into cubes and Texture Profile Analysis (compression 50%, 10 mm high samples), water activity (a(w) 25 °C) and water content analyses were performed. A negative non-linear relationship between hardness and water content and water activity was observed. From this relationship, critical X and a(w) values, below which there is a dramatic increase in hardness, can be found (around 0.55 kg H(2)O/kg dry matter and around 0.70, respectively). Cohesiveness and springiness showed a positive linear relationship with water content and water activity.

Profiles of water content, water activity and texture in crusted dry-cured loin and in non-crusted dry-cured loin.

This study compares the profiles of water activity (a(w)), water content (X) and texture in loin with crusted surface versus those profiles in loin without crust, and establishes a mathematical model able to describe hardness based on a(w) and/or X. Two loins (m. longissimus dorsi) were dry-cured, aged and then each one was divided into four pieces. Two of them were dried at 15±2 °C, 50±3% RH (CL treatment) and the other two were dried at 2±2 °C, 80±3% RH (NCL treatment). The pieces of CL were dried under more severe conditions in order to develop a crust on the surface. Three-millimetre thick slices were taken from the most external part towards the inner part. The slices were prepared (10×10×3 mm) for texture profile analysis (TPA). Measurements of a(w) and X were carried out on each slice. Variance analyses and non-linear regression analyses were performed to create a model for loin hardness prediction through X and/or a(w) and a linear regression model for cohesiveness and springiness. CL loins showed a higher hardness and chewiness and lower cohesiveness at the surface (3-mm thickness) than the NCL loins. Hardness and chewiness, fitted with a non-linear model, were better described by X than by a(w). Springiness showed a low relationship with X and a(w). The on-line monitoring of X and a(w) at the surface of the product would enable an estimation of the profiles of water content, a(w) and texture and it could be, therefore, a useful tool to avoid crusting.

Relationship between water content, NaCl content, pH and texture parameters in dry-cured muscles.

The aim of the present study was to describe the effect of NaCl and pH on the relationship between water content and hardness, cohesiveness and springiness in dry-cured muscles. The experiment was undertaken using 18 hams, selected in a commercial slaughterhouse. Half of the hams had a pH<5.7 and the rest a pH>6.2, measured on the semimembranosus muscle at 24-h postmortem. The semimembranosus and biceps femoris muscles were cut from hams, cured and individually packaged in bags and were laid in trays in a room at 2±2°C for 45days. Thereafter nine samples from each muscle were shaped like a parallelepiped and dried until different levels of drying, ranging from 28.5% to 59.7% water content, were attained. The rest of the muscle was ground and packaged until its subsequent physicochemical analysis. At the end of the drying period, a Texture Profile Analysis was used to determine textural parameters. The results indicated that for a range of X (kg H(2)O/kg dry matter) between 0.8 and 1.3 the hardness remains practically unchanged while for X<0.6 the hardness increases substantially. The samples from hams with low pH(SM) had greater hardness, cohesiveness and springiness than those from hams with high pH(SM). Dry-cured muscles with lower NaCl content showed lower hardness, cohesiveness and springiness, especially in those with pH(SM)>6.2. At X values lower than 0.6 the hardness was more influenced by water content than by NaCl content or pH(SM).

[Exogoninae (Polychaeta: Syllidae) from the Mexican Caribbean region with a key for the Gran Caribbean species]. / Exogoninae (Polychaeta: Syllidae) del Caribe mexicano con una clave para las especies del Gran Caribe.

This paper identifies the Exogoninae (Syllidae) from the Mexican Caribbean coasts and includes a key to identify all the species recorded from the Grand Caribbean Sea. The classification of the family and the composition of Exogoninae are briefly examined; the correct names of the subfamilies are Syllinae Grube, 1850, Eusyllinae Malaquin, 1893, Autolytinae Malaquin, 1893 and Exogoninae Langerhans, 1879. Exogoninae includes Anguillosyllis Day, 1963, Brania de Quatrefages, 1866, Braniella Hartman, 1963, Exogone Ørsted, 1845, Exogonella Hartman, 1961, Exogonoides Day, 1963, Parapionosyllis Fauvel, 1923, Psammosyllis Westheide, 1990, Spermosyllis Claparède, 1864, and Sphaerosyllis Claparède, 1863. Pseudexogone Augener, 1922, formerly included in the group, is not a syllid; it belongs to Pilargidae. We collected 814 specimens belonging to 3 genera, 3 subgenera and 13 species as Brania (4), Exogone (4) and Sphaerosyllis (5); five new species are described: Brania russelli n. sp, Brania uebelackerae n. sp, Brania westheidei n. sp., Exogone (Exogone) bondi n. sp. and Exogone (Parexogone) sanmartini n. sp. For each species, selected references, diagnostic features, observations on morphological variability, distribution and illustrations are provided; new species also have an english diagnosis. Most abundant species were B. uebelackerae n. sp. (295), S. taylori Perkins (169), E. (E.) dispar Webster (76), and E. (E.) bondi n sp. (72).

[Bone mineral density and bone mineral content variations in various communities]. / Variaciones de la densidad y de la concentración mineral ósea entre distintas comunidades.

OBJECTIVE: To study the bone mineral density (DMO) and the bone mineral concentration (CMO) in lumbar spine (L2 and L4) by dual-energy x-ray absorptiometry with a lunar DPX (DEXA) in a children sample of the community of Madrid; to relate the values obtained with the age, sex and pubertal development; and to compare the values of DMO found with the publications of other autonomous communities. MATERIALS AND METHODS: 351 children, 184 boys and 167 girls selected at random in our environment. The age range oscillated between 6 months and 20 years. Grouped in intervals of a year according to the sex; and in accordance with the pubertal development. The bone mineral content was measured by dual-energy x-ray absorptiometry with a lunar DPX in the lumbar spine at level of L2-L4. The statistic analysis has been accomplished with the SPSS version 6.0.1. They have been obtained the average and deviation standard for each group from studied age, and also according to pubertal development; the DMO and CMO of the boys and girls have been compared by groups of age; the DMO and CMO within each sex between a group of age and the immediately superior have been compared through an analysis of the variance; the effect that the age has on the DMO and the CMO has been evaluated and finally through multivariant analysis techniques the regression models have been estimated between the age and the DMO, and the age with the CMO. CONCLUSION: The DMO shows variations between the various communities and even in different samples of a same community, in Carrascosa study the DMO is highest, expressed in SD score, in many several groups of age studied with respect to our values and with the values of Moreno et al and Armadá et al; the values of DMO in our study are greater than the ones found by Moreno et al and Armadá et al; the first four years and the adolescence are the periods of maximum increase of the DMO, but it also increases in an oscillatory way in the intermediate stages; the girls present some highest levels of DMO in the groups of age of 12-13 and 14-15 years, probably in relationship to a most precocious beginning of the puberty; and finally the regression line of the DMO as compared to the age for both sexes are parallel and have equal court point, what means that they are coincident.

[The solubility and in vivo-in vitro correlation of naltrexone]. / Estudio de disolucion y correlacion in vivo/in vitro de naltrexona.

The dissolution of three dosages of Naltrexone tablets was studied, there being no monograph of this active principle in the most commonplace pharmacopoeia. The dissolution parameters were calculated "in vitro", then a correlation model was applied to determine the relationship between these parameters and the "in vivo" pharmacokinetic parameters of Naltrexone at each dosage level and also their degree of association. The regression lines between the "in vivo" and "in vitro" parameters were then plotted. Three C level correlations were obtained as per USP 23 between the pharmacokinetic parameter Cmax and the dissolution parameters 1/T50, E.D.30 and kd. The dissolution test conditions were also validated.

[Cholesterol content in longissimus muscle beef from slaughter cattle in Venezuela]. / Contenido de colesterol en el músculo longissimus de bovinos venezolanos.

An observational study was conducted with 149 cattle, raised under tropical conditions of Venezuela (mostly grassfed), to study the relationships of sex class (62 bulls, 67 steers, 20 heifers), age by dentition (2.5; 3.0; 3.5 and 4.0 yr), physiological maturity (A or B), cattle type (17 Dairy or 132 Zebu type crossbreds), Venezuelan carcass grade (Optima, Excelente, Selecta or Superior), marbling level (four levels from "None" to "Small quantity"), carcass fat cover (four level: "Even", "Uneven", "patch-like" and "Devoid") and subcutaneous fat thickness (SFT) over the ribeye (1 = 0.1-0.2 cm; 2 = 0.3-0.4 cm; 3 = 0.5-0.9 cm and 4 = > 1.0 cm) on cholesterol content (mg/ 100 g wet weight) of longissimus muscle. Cholesterol content, as determined colormetrically, did not vary in response to the differences in sex class, age, maturity level, carcass grade, marbling level or SFT represented in the present survey. However, cattle type affected (P = 0.08) cholesterol content. Least square means analysis showed that dairy type contained 12.2 mg more of cholesterol/100 g of muscle than Zebu type. The overall mean (+/- SD) muscle cholesterol for the kind of cattle sampled herein (66.6 +/- 16 mg/100 g) was not considered to be different from those of cattle fed in other latitudes.