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

Sex-related morbidity and mortality in non-adult individuals from the Early Medieval site of Valdaro (Italy): the contribution of dental enamel peptide analysis

In this work, osteological and paleopathological analyses are combined with liquid-chromatography mass spectrometry to study life and death of 30 non-adult individuals from an Early Medieval Italian funerary context (Valdaro, 7th-8th cent. AD). We estimated individual sex by exploiting sexual differences in enamel-bounded peptides. Enamel proteins were extracted through an acid etching of the whole tooth crowns for 4 samples\ud and through a partial digestion of small enamel chunks for the remaining 26 samples. Both protocols were informative on the sex of the individuals through the identification of amelogenin isoforms (AMELX and AMELY). In addition, low-mineralized tooth germs were analysed and they provided reliable information on the infants’ sex. We observed the presence of 13 males and 17 females among the non-adults of Valdaro, not significantly different from a random sample with an equal frequency of males and females. Cribra cranii and endocranial lesion occurrence showed an association with sex, with higher frequencies in male individuals

INVESTIGATIONS ON THE OCULAR PHARMACOKINETICS OF BENDAZAC IN RABBITS

The ocular pharmacokinetics of bendazac were studied in rabbits, following intravenous administration of bendazac lysine. The compound and its 5-hydroxyderivative were determined in different eye compartments and plasma by radioassay, using [14C]bendazac, and HPLC. The highest concentrations were found in the iris and in descending order in the ciliary body, retina, cornea, tears, aqueous humor, vitreous, and lens. The time course of concentrations in the plasma, aqueous and vitreous humor, ciliary body, and retina showed kinetics described by the exponential equation y = ae(bx) with a half-life of 2.47, 4.56, 3.59, and 3.22 hr, respectively; in the lens the half-life was 17.77 hr

A comparative study of Atropa belladonna and atropine on an animal model of urinary retention

The ability of Atropa belladonna L. and atropine to produce urinary retention has been studied in the rat. Our results suggest that A. belladonna is more effective than expected on the basis of its alkaloidic content

Analysis of lonidamine in rat serum and testis by high performance liquid chromatography.

The pharmacokinetics and the serum and testicular levels of the drug up to 120 h after the administration of one single dose (100 mg/kg body weight) of lonidamine to Sprague-Dawley rats have been studied. Results were highly variable, as previously reported, but a very good Linear correlation was found between the serum and the testicular levels, suggesting that, in the rat, and possibly in the human, testicular levels could be estimated based on the serum concentrations

Intracellular signal transduction pathways as targets for neurotoxicants

The multiple cascades of signal transduction pathways that lead from receptors on the cell membrane to the nucleus, thus translating extracellular signals into changes in gene expression, may represent important targets for neurotoxic compounds. Among the biochemical steps and pathways that have been investigated are the metabolism of cyclic nucleotides, the formation of nitric oxide, the metabolism of membrane phospholipids, the activation of a multitude of protein kinases and the induction of transcription factors. This brief review will focus on the interactions of three known neurotoxicants, lead, ethanol and polychlorinated biphenyls, with signal transduction pathways, particularly the family of protein kinase C isozymes, and discusses how such effects may be involved in their neurotoxicity. © 2001 Elsevier Science Ireland Ltd

Effects of lonidamine on testicular and epididymal proteins in the rat.

Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis

Extended criteria grafts and emerging therapeutics strategy in liver transplantation. The unstable balance between damage and repair

Due to increasing demand for donor organs, "extended criteria" donors are increasingly considered for liver transplantation, including elderly donors and donors after cardiac death. The grafts of this subgroup of donors share a major risk to develop significant features of ischemia reperfusion injury, that may eventually lead to graft failure. Ex-situ machine perfusion technology has gained much interest in liver transplantation, because represents both a useful tool for improving graft quality before transplantation and a platform for the delivery of therapeutics directly to the organ. In this review, we survey ongoing clinical evidences supporting the use of elderly and DCD donors in liver transplantation, and the underlying mechanistic aspects of liver aging and ischemia reperfusion injury that influence graft quality and transplant outcome. Finally, we highlight evidences in the field of new therapeutics to test in MP in the context of recent findings of basic and translational research

ChIP analysis of Yna1p and Yna2p occupying the <i>YNT1</i> gene promoter in different physiological conditions and genetic backgrounds.

<p><b>A</b>. GFP-tagged Yna1p expressed from its own nitrate-inducible promoter in the wild type (Yna1::GFP) and <i>YNA2Δ</i> (Yna1::GFP-<i>YNA2Δ</i>) background was analysed by ChIP in cells grown under nitrate-inducing (I) or glutamine-repressing (R) conditions. <b>B</b>. Experimental set-up for the GFP-tagged Yna2p in the wild ytpe (Yna2^OE::GFP) and <i>YNA1Δ</i> background (Yna2^OE::GFP-<i>YNA1Δ</i>) was identical to the one described for Yna1p with the exception that the Yna2p-GFP fusion protein was expressed from the strong MOX promoter in order to be able to analyse Yna2p occupancy also in a <i>YNA1Δ</i> background in which <i>YNA2</i> is normally not expressed. All values shown in this figure represent the amount of <i>YNT1</i> promoter DNA relative to the amount of input DNA in the same sample. Experiments were done in two biological repetitions and in each experiment ChIP analysis was duplicated. Error bars thus represent the standard deviations of four measurements in each condition and strain.</p

Yna1p and Yna2p bind <i>in vitro</i> and <i>in vivo</i> to a region containing a conserved DNA motif present in all nitrate-responsive promoters.

<p><b>A.</b> Alignment of the promoter regions of genes belonging to the nitrate cluster detects a conserved motif (5′CGGAGA) indicated by six consecutive asterisks. This motif could represent a putative upstream activating sequence (UAS) bound by Yna1p and Ynap2. Probes used in the band shift assays are indicated as YNT1 probe 1 and YNT1 probe 2; the two different derivatives of YNT1 probe 2 containing the indicated changes in conserved nucleotides are shown as m1 and m2. <b>B.</b><i>In vitro</i> interaction experiments using electric mobility shift assays (EMSA). <b>FP*, labelled probe (double-stranded oligonucleotide as indicated in A) was incubated without recombinant proteins; P*, labelled probe incubated with the indicated recombinant protein but no unlabelled, specific competitor DNA has been added.; +1P, same conditions as in P* but an equimolar amount of the unlabelled double-stranded oligonucleotide was present in the incubation mixture as specific competitor for complex formation; +2P, same conditions as in +1P but double molar amount of the unlabelled double-stranded oligonucleotide was present in the incubation mixture as specific competitor.</b> Radiolabelled probes described in panel A were incubated with recombinant <i>HIS</i>-Yna1p_(1–185) and <i>HIS</i>-Yna2p_(1–180) proteins expressed in <i>E</i>. <i>coli</i> and purified (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135416#pone.0135416.s006" target="_blank">S6 Fig</a>). <b>While Yna1p</b>_{<b>(1–185)</b>}<b>binds both</b><i>YNT1</i> probes 1 and 2, Yna2p_(1–180) doesn’t interact with the <i>YNT1</i> probe 2. Exchanging 3 nucleotides in the conserved 5′-CGGAGA-3′strech of YNT1 probe 2 disrupts the <b>Yna1p</b>_{<b>(1–185)</b>}<b>binding capability (m2). Likewise, exchange in an upstream conserved residue potentially forming a partial inverted repeat with the CGGAGA motif also disrupts complex formation (m1). This indicates that both motifs are involved in binding Yna1p. C.</b><i>In vivo footprinting</i> methylation protection profile of the <i>YNT1</i> gene promoter. Cells from the wild type strain (wt) as well as from the <i>yna</i> mutant strains (y1, corresponding to <b>L1-<i>yna1</i></b>^{<b><i>I6632</i></b>}<b>, and y2, corresponding to L1-<i>yna2</i></b>^{<b><i>Δ12274–12595</i></b>}<b>) have been incubated at the standard physiological conditions</b><i>(I</i>, <i>nitrate; NI</i>, <i>proline</i>) and subjected to ligation-mediated methylation protection footprinting as detailed in Materials and Methods. The sequence pattern of a control reaction containing <i>in vitro</i> methylated DNA is shown in lane V and the corresponding sequence derived from the <i>YNT1</i> promoter is aligned to the left of the autoradiograph. The sequence corresponding to the banding pattern in the autoradiograph lies between the diagonal dotted lines. In some cases guanines are not properly resolved in the gel but appear as compressed regions. Adenines can become hypersensitive and eventually appear as strong as guanines, mainly in the <i>in vivo</i> lanes. For a better orientation, vertical dotted lines next to the V lane (<i>in vitro</i> control reaction) and next to the sequence correspond to each other. Protected guanines are marked with a triangle and hypersensitive adenines are marked with circles. Asterisks on the displayed vertical sequence correspond to the asterisks marking the conserved residues in the <i>YNT1</i> promoter region used for designing <i>YNT1</i> probes 1 and 2. Note that the sequence displayed next to the autoradiograph is complementary to the sequence in panel A. Below the figure, the scheme shows the location of protected regions (white rectangles) in the <i>YNT1</i> promoter. The conserved 5′-CGGAGA-3′ sequence is indicated by the grey rectangle and asterisks.</p