Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

15 STR loci frequencies in the population from Santa Catarina, Southern Brazil

Allele frequencies for 15 short tandem repeats (STR) loci were determined with a sample of 3000 unrelated individuals from the population of Santa Catarina, Southern Brazil. The loci are most commonly used in forensic and paternity testing, being analyzed by the Powerplex 16 (Promega) commercial kit. The data shows that most polymorphic loci were Penta E and FGA. The distributions of the genotypes in the evaluated loci are in Hardy–Weinberg equilibrium. Comparative analyses between our population data and other Brazilian populations are presented. The calculated forensic parameters showed that the loci are useful for the solution of forensic problems in Brazilian Southern region.     

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.     

“The big sleep: Elucidating the sequence of events in the first hours of death to determine the postmortem interval”

Recent developments on postmortem interval estimation (PMI) take an advantage of the autolysis process, pointing out to the analysis of the expression of apoptosis and autophagy genes towards this purpose. Oxidative stress plays a role in this signaling as a regulatory mechanism and/or as a consequence of cell death. Additionally, melatonin has been implicated on apoptosis and autophagy signaling, making melatonin a suitable target for PMI determination. The aim of this study was to investigate the early PMI through the analysis of the expression of autophagy genes as well as oxidative stress and melatonin receptor. Our results demonstrated a rapidly increased on the expression of autophagy genes according to the expected sequence of events, then a marked decrease in this expression, matched with the switch to the apoptosis signaling. These results revealed potential candidates to analyze the PMI in the first hours of death, helping to estimate the time-since-death.     

High‐Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high‐resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual‐dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.     

Extraction and isolation of cannabinoids from marijuana seizures and characterization by 1H NMR allied to chemometric tools

Marijuana, a drug derived from the Cannabis sativa L. plant, is the world's most consumed illicit drug. In this paper, a total of 156 marijuana samples seized in the state of Espírito Santo (ES), Brazil were studied and analysed by proton nuclear magnetic resonance (¹H NMR) spectroscopy to identify the major cannabinoids present. A crude extract of all samples was purified using high performance liquid chromatography so that these compounds could serve as reference substances. Nine fractions were obtained and analysed by ¹H NMR and gas chromatography–mass spectrometry (GC–MS), with five presented cannabinoids. ?⁹-THC (Δ⁹-trans-tetrahydrocannabinol), ?⁹-THCA (?⁹-tetrahydrocannabinolic acid), ?⁸-THC (?⁸-tetrahydrocannabinol), 11-hydroxycannabinol, CBV (cannabivarin), and CBN (cannabinol) were found, and their chemical structures were confirmed by GC–MS. The latter compound was obtained with high purity (≈100%), while the others were obtained as less complex mixtures with purity higher than 75% (except for Δ⁸-THC). Principal component analysis (PCA) was used on the ¹H NMR spectra of the 156 samples, and it was found that the samples were grouped according to the months, differentiating into two groups (from July 2014 to January 2015 and from February 2015 to July 2015), where non-grouping was observed from four macro-regions of the ES state (North, Central, Metropolitan, and South). The chemical profile of the seized samples was correlated to the ¹H NMR spectrum of an isolated CBN sub-fraction, in which the group formed by samples seized in the year 2015 presented lower CBN content in the chemical composition. From the PCA score plot, two groups of samples were confirmed using the partial least squares discriminant analysis and orthogonal projections to latent structures classification methods.     

Female student athletes’ perceptions of acceptability and the occurrence of sexual-related behaviour by their coaches in India

The current study assessed the perceptions of acceptable sexual behaviour of coaches and the occurrence of sexual harassment among female student in India. A sample of 180 Indian female student-athletes at intercollegiate and inter-university levels with male coaches participated in this study. A questionnaire on sport-specific Touch and Behaviour versus Unwanted Intimacy from coaches (Vanden Auweele et al., 2008), consisting of 41 items on a 5-point rating scale was used. The participants were asked to indicate the acceptability of specific coaching behaviour as well as the occurrence of the behaviour represented in each item. The acceptability of the behaviour was determined by means and standard deviations, while the occurrence was determined by frequencies. Factor analysis was performed to determine the structure of the coaches’ behaviour and athletes’ acceptability thereof, which yielded four factors. Cronbach’s alpha was used to determine the internal consistency of the extracted items on each factor. Two factors, namely, unwanted sexual behaviour and inappropriate verbal and physical sexual behaviour, were regarded by athletes as very serious and unacceptable coach behaviour, while a third factor represented context-dependent suspicious behaviour (a grey area in which athletes differ in their opinion) and was perceived to be serious and unacceptable. A fourth factor represented acceptable behaviour. The occurrence of very serious and unacceptable behaviour was reported by 31% of the female athletes.     

Lawyers,law schools and social change – defining the challenges of academic legal education in the late modernity

This article argues that due to their position and task in society, legal professionals are confronted with specific difficulties connected to contemporary circumstances. To outline these circumstances, this article draws on the work of Ulrich Beck and Zygmunt Bauman and places both theories within the late modernity. Lawyers need to be able to deal with the difficulties late modernity poses and are therefore in need of appropriate knowledge and skills. Law schools should offer relevant schooling so that their students are equipped to deal with the difficulties confronting them in late modernity’s society. This article offers a first inquiry into the challenges that lawyers currently face, alongside anticipating alteration of academic law school programs by clarifying the challenges caused by two societal processes in late modernity, namely (1) the increase of technological possibilities and, simultaneously, the demystification of science; and (2) globalization. These processes lead to a complex society ruled by uncertainty that faces the challenge of allocating responsibility. In addition, some initial suggestions are presented regarding the conceivable adjustments to academic legal education in late modernity.     

Start-EU-up! Lessons from international incubation practices to address the challenges faced by Western European start-ups

Despite the continued efforts of policy makers, Western European start-ups are still struggling. Further, as questions are being raised about the effectiveness of incubators, there is a growing call for incubators around the world to learn from each other and improve themselves. Our paper enables Western European incubators to learn from their foreign peers by qualitatively exploring the challenges faced by Western European start-ups and the practices that incubators around the world use to address these challenges. Our study thereby takes a two-step approach. First, using the components of the entrepreneurial ecosystem to structure data coming from 90 qualitative interviews with Western European entrepreneurs and incubator managers, we explore five interrelated challenges that constrain the ability of Western European start-ups to grow into high-impact businesses. In the second part of our study, we conduct a total of 191 interviews in Silicon Valley, the greater Boston area, Israel and Australia to identify practices that incubators in these regions use to address the five challenges identified in the first study. Our findings suggest that, rather than addressing the underlying institutions that cause challenges in the entrepreneurial ecosystem, incubators provide symptomatic solutions by creating an environment that protects start-ups from unfavorable institutions. Accordingly, we conclude that existing incubators have only limited potential to strengthen entrepreneurial ecosystems, and we end our paper with a call for a new generation of ‘systemic incubators’ that aim to transform or create institutions to address the institutional challenges that constrain start-up activity.