Witness for resolution: post-conflict quadratic affiliation in semi-free ranging pigs.

In social mammals, post-conflict resolution can involve the reunion of former opponents (reconciliation), spontaneous/solicited post-conflict affiliation of a third party with either opponent (triadic contacts), and affiliation between other individuals (hereafter bystanders; quadratic contacts). Quadratic contacts-possibly informing complex cognitive abilities-have been neglected in post-conflict studies. We investigated quadratic affiliation in semi-free ranging pigs Sus scrofa, at the ethical farm Parva-Domus (Cavagnolo, Italy). Kinship was known. We collected behavioral data on adult pigs (n = 104) via video recordings (43 h) followed by video analyses. Affiliative and anxiety behaviors between bystanders were collected under post-conflict (PC; following a conflict between non-bystanders) and matched-control (MC; no conflict) conditions. Quadratic affiliation was present in pigs, as bystanders affiliated more in PC than MC, and such affiliation was followed by a decrease in the anxiety behaviors of both the interacting bystanders. Thus, quadratic contacts may be partly aimed at reducing one's own anxiety (intrinsic regulation). Quadratic affiliation was highest between closely related bystanders, which suggests that such affiliation may be most effective when close kin is involved. Quadratic affiliation was lowest after reconciliation and spontaneous triadic contacts. This suggests that direct peacemaking between opponents and spontaneous triadic contacts with close kin may most likely replace quadratic affiliation. Hence, pigs can be influenced by the negative events that affect other pigs-but not themselves-and their response may be modulated by social factors. Such non-random quadratic affiliation may point toward the presence of elements of social appraisal abilities in pigs.

Privacy and ethical challenges of the Amelogenin sex test in forensic paternity/kinship analysis: Insights from a 13-year case history.

The Amelogenin sex test included in forensic DNA typing kits has the potential to identify congenital conditions such as differences/disorders of sex development (DSD). It can also reveal mismatches between genotypic sex and gender marker in identity documents of transgender persons who obtained legal gender recognition. In a 13-year case history of paternity/kinship tests, involving n = 962 females and n = 1001 males, two mismatches between Amelogenin sex test (male) and gender marker (female), and three cases of chromosomal DSD (Klinefelter syndrome) were observed. The concrete risk of observing Amelogenin anomalies, their potential causes, and the context in which they occur (forensic, i.e. non-medical) mean that laboratory operators are called to strike a complex balance between privacy interests and individual health rights when providing preliminary information and reporting Amelogenin incidental findings. This case history argues for the need of a more responsible approach towards the Amelogenin sex test in the forensic community.

Recombulator-X: A fast and user-friendly tool for estimating X chromosome recombination rates in forensic genetics.

Genetic markers (especially short tandem repeats or STRs) located on the X chromosome are a valuable resource to solve complex kinship cases in forensic genetics in addition or alternatively to autosomal STRs. Groups of tightly linked markers are combined into haplotypes, thus increasing the discriminating power of tests. However, this approach requires precise knowledge of the recombination rates between adjacent markers. The International Society of Forensic Genetics recommends that recombination rate estimation on the X chromosome is performed from pedigree genetic data while taking into account the confounding effect of mutations. However, implementations that satisfy these requirements have several drawbacks: they were never publicly released, they are very slow and/or need cluster-level hardware and strong computational expertise to use. In order to address these key concerns we developed Recombulator-X, a new open-source Python tool. The most challenging issue, namely the running time, was addressed with dynamic programming techniques to greatly reduce the computational complexity of the algorithm. Compared to the previous methods, Recombulator-X reduces the estimation times from weeks or months to less than one hour for typical datasets. Moreover, the estimation process, including preprocessing, has been streamlined and packaged into a simple command-line tool that can be run on a normal PC. Where previous approaches were limited to small panels of STR markers (up to 15), our tool can handle greater numbers (up to 100) of mixed STR and non-STR markers. In conclusion, Recombulator-X makes the estimation process much simpler, faster and accessible to researchers without a computational background, hopefully spurring increased adoption of best practices.

An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

Domestic pigs (Sus scrofa) engage in non-random post-conflict affiliation with third parties: cognitive and functional implications.

In social mammals, conflict resolution involves the reunion of former opponents (aggressor and victim) after an aggressive event (reconciliation) or post-conflict triadic contacts with a third party, started by either opponent (solicited-TSC) or spontaneously offered by the third party (unsolicited-TUC). These post-conflict strategies can serve different functions, including consolation (specifically when TUCs reduce the victim's anxiety). We investigated the possible presence and modulating factors of such strategies on semi-free ranging pigs (Sus scrofa; N = 104), housed at the ethical farm Parva Domus (Cavagnolo, Italy). Kinship was known. Reconciliation was present and mainly occurred between weakly related pigs to possibly improve tolerant cohabitation. Triadic contacts (all present except aggressor TSCs) mostly occurred between close kin. TSCs enacted by victims reduced neither their post-conflict anxiety behaviors nor further attacks by the previous aggressor, possibly because TSCs remained largely unreciprocated. TUCs towards aggressors did not reduce aggressor post-conflict anxiety but limited aggression redirection towards third parties. TUCs towards the victim reduced the victim but not the third-party's anxiety. However, TUCs may also provide inclusive fitness benefits to third parties by benefiting close kin. In sum, pigs engaged in non-random solicited/unsolicited triadic contacts, which suggests that pigs might possess socio-emotional regulation abilities to change their own or others' experience and elements of social appraisal, necessary to detect the emotional arousal of relevant others and (in case of TUCs) take the agency to restore homeostasis.

Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples.

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1-5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

Evaluation of the Effects of Different Sample Collection Strategies on DNA/RNA Co-Analysis of Forensic Stains.

The aim of this study was to evaluate the impact of different moistening agents (RNase-free water, absolute anhydrous ethanol, RNAlater®) applied to collection swabs on DNA/RNA retrieval and integrity for capillary electrophoresis applications (STR typing, cell type identification by mRNA profiling). Analyses were conducted on whole blood, luminol-treated diluted blood, saliva, semen, and mock skin stains. The effects of swab storage temperature and the time interval between sample collection and DNA/RNA extraction were also investigated. Water provided significantly higher DNA yields than ethanol in whole blood and semen samples, while ethanol and RNAlater® significantly outperformed water in skin samples, with full STR profiles obtained from over 98% of the skin samples collected with either ethanol or RNAlater®, compared to 71% of those collected with water. A significant difference in mRNA profiling success rates was observed in whole blood samples between swabs treated with either ethanol or RNAlater® (100%) and water (37.5%). Longer swab storage times before processing significantly affected mRNA profiling in saliva stains, with the success rate decreasing from 91.7% after 1 day of storage to 25% after 7 days. These results may contribute to the future development of optimal procedures for the collection of different types of biological traces.

Sweet tooth: DNA profiling of a cranium from an isolated retained root fragment.

Sampling of healthy multi-rooted teeth is recommended for the genetic identification of human skeletal remains. However, this may not always be possible, as in the reported case consisting of an isolated human cranium found in an aggregate crushing and processing plant in Piedmont, Northwest Italy. The cranium displayed significant weathering, suggesting a post-mortem interval of several years, and was edentulous with the exception of the apical root fragment of the upper left canine, consequence of an antemortem horizontal fracture. Prolonged decalcification of the root fragment followed by powder-free DNA extraction from ~10 mg of root tip tissue led to the recovery of >10 ng of high molecular weight human DNA, in comparison with ~0.01 ng of DNA per mg of bone powder obtained from the petrous portion of the temporal bone. Quantity and quality of DNA isolated from apical tooth tissue enabled multiple genotyping, including a reportable female STR profile, mitochondrial DNA analysis, and ancestry-informative insertion/deletion polymorphisms. Although the cranium remained unidentified after DNA comparisons, our findings confirm that apical tooth tissue is a promising source of DNA, easily obtained through a powder-free extraction protocol. Results also indicate that root tips should not be overlooked in challenging identification cases, even in the presence of compromised tooth specimens.

Yawn contagion in domestic pigs (Sus scrofa).

Contrary to spontaneous yawning-an ancient phenomenon common to vertebrates-contagious yawning (elicited by others' yawns) has been found only in highly social species and may reflect an emotional inter-individual connection. We investigated yawn contagion in the domestic pig, Sus scrofa. Owing to the complex socio-emotional and cognitive abilities of Sus scrofa, we posited that yawn contagion could be present in this species (Prediction 1) and influenced by individual/social factors (Prediction 2). In June-November 2018, on 104 semi-free ranging adolescent/adult pigs, 224 videos were recorded for video analysis on yawning. Kinship information was refined via genetic analyses. Statistical elaboration was conducted via GLMMs and non-parametric/randomization/cross-tabulation tests. We found yawn contagion in Sus scrofa, as it was more likely that pigs yawned when perceiving rather than not perceiving (yawning/control condition) others' yawns (response peak in the first out of three minutes). Yawn contagion was more likely: (1) in response to males' yawns; (2) as the age increased; (3) within short distance (1 m); (4) between full siblings, with no significant association between kinship and distance. The influence of kinship suggests that-as also hypothesized for Homo sapiens-yawn contagion might be linked with emotional communication and possibly contagion.

Assessment of the Precision ID Identity Panel kit on challenging forensic samples.

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the "same donor" or from a "first degree relative" was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10-13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping.

Mitochondrial DNA variation in Sub-Saharan Africa: Forensic data from a mixed West African sample, Côte d'Ivoire (Ivory Coast), and Rwanda.

This study provides 398 novel complete mitochondrial control region sequences that augment the still underrepresented data from Africa by three datasets: a mixed West African sample set deriving from 12 countries (n = 145) and datasets from Côte d'Ivoire (Ivory Coast) (n = 100) as well as Rwanda (n = 153). The analysis of mtDNA variation and genetic comparisons with published data revealed low random match probabilities in all three datasets and typical West African and East African diversity, respectively. Genetic parameters indicate that the presented mixed West African dataset may serve as first forensic mtDNA control region database for West Africa in general. In addition, a strategy for responsible forensic application of precious mtDNA population samples potentially containing close maternal relatives is outlined. The datasets will be uploaded to the forensic mtDNA database EMPOP (https://empop.online) upon publication.

58 cases of sexual violence bearing forensic interest: congruence between the victim's report and the data from laboratory analyses.

The assistance provided by specialised healthcare personnel to victims of a sexual violence cannot focus just on the clinical intervention appropriate for the lesions suffered by the patient, but must also take legal and forensic needs into account. Anamnestic data represents a crucial step towards the finding of forensic evidence. Our retrospective study aims to analyse the congruence between verbal reports from abused women and the laboratory data to the end of identifying ways for enhancing the gathering of anamnestic data. We considered 960 medical records related to sexual violence that reached the Rape Centre "Soccorso Violenza Sessuale" of Turin between 2003 and 2013. Having consulted the register of evidence, we selected the cases for which the local judicial authority had asked for expert advice on biological material. The selected cases have been gathered in two different categories depending on whether the victim could or could not recall the events. Then, we looked at the results of the cytological analysis performed to identify the presence of sperm cells, at the results of the body fluid identification, and at the results of the DNA quantitation. Our findings strongly suggest that forensic investigations should be carried out independently from the presence of memories of the traumatic events on the victim's part. Moreover, they suggest that forensic investigations should also be pursued in the presence of a negative cytologic examination.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Spotting expertise in the eyes: billiards knowledge as revealed by gaze shifts in a dynamic visual prediction task.

In sports, as in other activities and knowledge domains, expertise is a highly valuable asset. We assessed whether expertise in billiards is associated with specific patterns of eye movements in a visual prediction task. Professional players and novices were presented a number of simplified billiard shots on a computer screen, previously filmed in a real set, with the last part of the ball trajectory occluded. They had to predict whether or not the ball would have hit the central skittle. Experts performed better than novices, in terms of both accuracy and response time. By analyzing eye movements, we found that during occlusion, experts rarely extrapolated with the gaze the occluded part of the ball trajectory-a behavior that was widely diffused in novices-even when the unseen path was long and with two bounces interposed. Rather, they looked selectively at specific diagnostic points on the cushions along the ball's visible trajectory, in accordance with a formal metrical system used by professional players to calculate the shot coordinates. Thus, the eye movements of expert observers contained a clear signature of billiard expertise and documented empirically a strategy upgrade in visual problem solving from dynamic, analog simulation in imagery to more efficient rule-based, conceptual knowledge.

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome.

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

Allele and haplotype diversity of X-chromosomal STRs in Ivory Coast.

Twenty-one X-chromosomal short tandem repeat (STR) loci, including the six clusters of linked markers DXS10148-DXS10135-DXS8378 (Xp22), DXS7132-DXS10079-DXS10074 (Xq12), DXS6801-DXS6809-DXS6789 (Xq21), DXS7424-DXS101 (Xq22), DXS10103-HPRTB-DXS10101 (Xq26), DXS8377-DXS10146-DXS10134-DXS7423 (Xq28) and the loci DXS6800, GATA172D05 and DXS10011 were typed in a population sample from Ivory Coast (n=125; 51 men and 74 women). Allele and haplotype frequencies as well as linkage disequilibrium data for kinship calculations are provided. On the whole, no significant differences in the genetic variability of X-STR markers were observed between Ivorians and other sub-Saharan African populations belonging to the Niger-Kordofanian linguistic group.

Linkage and linkage disequilibrium analysis of X-STRs in Italian families.

Twenty X-chromosomal short tandem repeat (STR) loci were typed in 80 families of Italian descent, composed by mother and two or more sons, for a total of 93 meiosis. The analyzed X-STR panel included six clusters of closely linked markers (each spanning<3cM): DXS10135-DXS10148-DXS8378 (Xp22); DXS7132-DXS10074-DXS10079 (Xq12); DXS6801-DXS6809-DXS6789 (Xq21); DXS7424-DXS101 (Xq22); DXS10103-HPRTB-DXS10101 (Xq26); DXS8377-DXS10134-DXS7423-DXS10146 (Xq28). Recombination fractions between pairs of markers calculated by pedigree analysis were compared with those obtained from the second-generation Rutgers combined linkage-physical map of the human genome. The observed differences confirm that recombination is not homogeneous along the X chromosome and that the conventional subdivision of X-STRs in four groups of completely unlinked markers cannot be regarded as true. Significant linkage disequilibrium was found between markers DXS6801 and DXS6809 (p=0.017). The effect on likelihood calculations of inferring haplotype frequencies from allele distributions rather than haplotype count in the relevant population was evaluated.

Analysis of 11 tetrameric STRs in wild boars for forensic purposes.

STR profiling of animal species has a wide range of applications, including forensic identification, wildlife preservation, veterinary public health protection and food safety. We tested the efficacy of a multiplex PCR-based assay including 11 porcine-specific tetrameric STRs in a population sample of wild boars (n=142) originating from Piedmont (North West Italy). Multiple deviations from Hardy-Weinberg expectations were observed, mostly due to a reduction in observed heterozygosity indicative of a high degree of inbreeding. A value of θ of 0.046 and an inbreeding coefficient of 0.089 were estimated. Combined power of discrimination and probability of exclusion values for the STR panel were 0.9999999999996 and 0.99989. In order to test the suitability of the method for meat traceability purposes, a domestic pig reference sample (n=412), consisting of commercial lines commonly used in the meat production process, was also typed. A Bayesian cluster analysis carried out using the observed genotypes, showed a percentage of correct subspecies assignment of individual samples of 0.974 for wild boars and 0.991 for pigs, thus demonstrating the usefulness of the multiplex STR-typing system for discrimination purposes.

Analysis of 21 X-chromosomal STRs in an Algerian population sample.

Twenty-one X-chromosomal short tandem repeat loci, including the six clusters of linked markers DXS10148-DXS10135-DXS8378 (Xp22), DXS7132-DXS10074-DXS10079 (Xq12), DXS6801-DXS6809-DXS6789 (Xq21), DXS7424-DXS101 (Xq22), DXS10103-HPRTB-DXS10101 (Xq26), DXS8377-DXS10146-DXS10134-DXS7423-DXS10011 (Xq28), and the loci DXS6800 and GATA172D05 were typed in a northwestern Algerian population sample (n = 210; 104 men and 106 women). Allele and haplotype frequencies were calculated. No evidence of linkage disequilibrium was observed between pairs of loci within clusters of linked markers. At locus DXS10148, sequence analysis of a subset of alleles displaying unusual amplicon length (>/= 36 repeat units) and anomalous electrophoretic mobility showed that this marker has a complex molecular structure with different repeat variants within alleles of identical amplicon size.