Primer information of STR loci included in the new studied kit.

<p>Primer information of STR loci included in the new studied kit.</p

A New Multiplex Assay of 17 Autosomal STRs and Amelogenin for Forensic Application

<div><p>This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 17 autosomal loci (D1S1656, D2S441, D3S1358, D3S3045, D6S477, D7S3048, D8S1132, D10S1435, D10S1248, D11S2368, D13S325, D14S608, D15S659, D17S1290, D18S535, D19S253 and D22-GATA198B05) and Amelogenin. Primers for the loci were designed and optimized so that all of the amplicons were distributed from 50 base pairs (bp) to less than 500 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. Strategies were developed to overcome challenges that encountered in creating the final assay. The limits of the multiplex were tested, resulting in the successful amplification of genomic DNA range from 0.25–4 ng with 30 PCR cycles. A total of 681 individuals from the Chinese Han population were studied and forensic genetic data were present. No significant deviations from Hardy–Weinberg equilibrium were observed. A total of 180 alleles were detected for the 17 autosomal STRs. The cumulative mean exclusion chance in duos (CMEC_D) was 0.999967, and cumulative mean exclusion chance in trios (CMEC_T) was 0.99999995. We conclude that the present 17plex autosomal STRs assay provides an additional powerful tool for forensic applications.</p> </div

The genotyping profile of positive control DNA of 9947A with the 17plex assay.

<p>The genotyping profile of positive control DNA of 9947A with the 17plex assay.</p

The genotyping profile of ladder with the 17plex assay.

<p>The genotyping profile of ladder with the 17plex assay.</p

Intra-Monozygotic Twin Pair Discordance and Longitudinal Variation of Whole-Genome Scale DNA Methylation in Adults

<div><p>Monozygotic twins share identical genomic DNA and are indistinguishable using conventional genetic markers. Increasing evidence indicates that monozygotic twins are epigenetically distinct, suggesting that a comparison between DNA methylation patterns might be useful to approach this forensic problem. However, the extent of epigenetic discordance between healthy adult monozygotic twins and the stability of CpG loci within the same individual over a short time span at the whole-genome scale are not well understood. Here, we used Infinium HumanMethylation450 Beadchips to compare DNA methylation profiles using blood collected from 10 pairs of monozygotic twins and 8 individuals sampled at 0, 3, 6, and 9 months. Using an effective and unbiased method for calling differentially methylated (DM) CpG sites, we showed that 0.087%–1.530% of the CpG sites exhibit differential methylation in monozygotic twin pairs. We further demonstrated that, on whole-genome level, there has been no significant epigenetic drift within the same individuals for up to 9 months, including one monozygotic twin pair. However, we did identify a subset of CpG sites that vary in DNA methylation over the 9-month period. The magnitude of the intra-pair or longitudinal methylation discordance of the CpG sites inside the CpG islands is greater than those outside the CpG islands. The CpG sites located on shores appear to be more suitable for distinguishing between MZ twins.</p></div

Massively parallel sequencing of 231 autosomal SNPs with a custom panel: a SNP typing assay developed for human identification with Ion Torrent PGM

<p>The custom-designed single nucleotide polymorphism (SNP) panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing (MPS) technology and Ion Torrent personal genome machine (PGM). SNPs were chosen from SNPforID, IISNP, HapMap, dbSNP, and related published literatures. Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls. Ten SNPs (rs4606077, rs334355, rs430046, rs2920816, rs4530059, rs1478829, rs1498553, rs7141285, rs12714757 and rs2189011) with low coverage or heterozygote imbalance should be optimized or excluded from the panel. Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS–SNP panel. A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA. Mixture testing with this panel is possible through analysis of the <i>F</i>_MAR (frequency of major allele reads) values at most loci with enough high coverage depth and low level of sequencing noise. These results indicate the potential advantage of the custom MPS–SNP assays and Ion Torrent PGM platform for forensic study.</p

EGb761 induces partially Caspase-Dependent Apoptosis in Melanoma Cells.

<p>(A) EGb761 induces caspase activation. Whole cell lysates from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for the indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. (B) Mel-RM and Mel-AT cells with or without treatment with EGb761 (400 μg/ml) for 16 h were subjected to flow cytometry analysis of caspase-3 activation using an antibody that specifically recognizes the activated form of caspase-3. The data shown are representative of three individual experiments. (C) Mel-RM and Mel-AT cells were pretreated with the pan-caspase inhibitor z-VAD-fmk (20 μmol/L), the caspase-3 inhibitor z-DEVD-fmk (30 μmol/L), or the caspase-9 inhibitor z-LEHD-fmk (30 μmol/L) 1h before adding EGb761 (400 μg/ml) for another 24h, respectively. Apoptosis was measured by the propidium iodide method using flow cytometry. Data are the mean ± SEM of three individual experiments. * Present p<0.05 vs control.</p

EGb761 regulates Bcl-2 family proteins expression in melanoma cells.

<p>(A) EGb761 alters the expression levels of anti- and pro-apoptotic Bcl-2 family proteins in melanoma cell lines. Whole cell lysates from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. (B) 5% ethanol as control vehicle did not alter the expression levels of Mcl-1. Mel-AT cells with 5% ethanol for increasing periods. Whole cell lysates from Mel-AT cells treated were subjected to Western blot analysis. The data shown are representative of three individual experiments.(C) Mel-RM and Mel-AT cells were treated with EGb761 (400 μg/ml) or 5% ethanol for the indicated periods. Total RNA was isolated and subjected to real-time PCR analysis for Mcl-1. The relative abundance of mRNA expression treated with 5% ethanol was arbitrarily designated as 1. Columns, mean of three individual experiments; bars, SEM. * Present p<0.05 vs control.(D) Relative expression of anti-apoptosis Bcl-2 family proteins in melanoma cell lines Mel-RM and Mel-AT without treatment. Quantitative expression levels of Mcl-1, Bcl-2 and Bcl-X_L were normalized to GAPDH.(E) Relative expression of pro-apoptosis Bcl-2 family proteins in melanoma cell lines Mel-RM and Mel-AT without treatment. Quantitative expression levels of Bax, Bid, Noxa, PUMA, Bim, Bad and Bak were normalized to GAPDH.</p

EGb761 inhibits cell growth and induces apoptosis in melanoma cells in vitro.

<p>(A) Effects of EGb761 on proliferation in Mel-RM and Mel-AT cells. After EGb761 treatment at indicated concentration (100-800ug/ml) or 5% ethanol treatment as the vehicle control, Mel-RM and Mel-AT cells proliferation was assessed in a 96-well plate at 72 hours by MTT assay. The data shown are the mean ± SEM of three individual experiments. (B) Representative dot plots showing fluorescence channel analyses of melanoma cells after dual staining with Annexin V and propidium iodide. Melanocytes, Mel-RM and Mel-AT cells were treated with EGb761 at 400 μg/ml for 24h (5% ethanol treatment as the vehicle control) and then stained with FITC-conjugated Annexin V (green fluorescence, horizontal axis) and PI(red fluorescence, vertical axis), and analyzed using flow cytometry. The data shown are representative of three individual experiments. (C) EGb761 induces apoptosis of melanoma cells. Mel-RM and Mel-AT cells treated with EGb761 (100–800 μg/ml) or 5% ethanol treatment as the vehicle control for indicated intervals were subjected to measurement of apoptosis by the Annexin V/PI staining method using flow cytometry. The data shown are the mean ± SEM of three individual experiments. (D) Induction of apoptosis by EGb761 in a panel of melanoma cell lines and melanocytes. Cells treated with EGb761 (400 μg/ml) or 5% ethanol for 24h were subjected to measurement of sub-G1content by the propidiumiodide method using flow cytometry. Columns, mean of three individual experiments; Bars, SEM.</p

Apoptosis of melanoma induced by EGb761 involves activation of Bax and Bak and changes in mitochondria.

<p>(A) EGb761 induces reduction in ΔΨm. Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for the indicated intervals were subjected to measurement of ΔΨm by JC-1staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. The data shown are representative of three individual experiments. (B) EGb761 induces activation of Bax and Bak. Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for the indicated intervals were subjected to measurement of activation of Bax and Bak by flow cytometry using antibodies that specifically recognize activated Bax and Bak, respectively. The filled and open histograms were generated from control (5% ethanol treated) (C) and EGb761-treated cells (E), respectively. The numbers represent MFIs. The data shown are representative of three individual experiments. (C) EGb761 induces mitochondrial release of cytochrome c and AIF. Mitochondrial and cytosolic fractions from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for 16h were subjected to Western blot analysis. COX IV or GAPDH levels were included to show relative purity of the mitochondrial or cytosolic fractions. The data shown are representative of three individual experiments.</p