Cross-species amplification from non-human primate DNAs in commercial human DNA assay kits.

Species specificity of commercial human DNA quantification kits and short tandem repeat (STR) profiling kits was examined using primate DNA samples. These samples comprised 33 individuals from eight primate species, each with gender and kinship data, including human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) of Hominidae family, and Japanese macaque (Macaca fuscata), long-tailed macaque (Macaca fascicularis), hamadryas baboon (Papio hamadryas), and savannah monkey (Chlorocebus sp.) of Cercopithecidae family. The findings revealed varying levels of cross-species amplifications in all non-human DNA samples that correlated with their evolutionary proximity to humans, both kit types. Moreover, cross-species amplification, including female DNA samples, was observed in a Y-chromosomal STR profiling kit. Additionally, species specificity differed among the commercial kits examined. The cross-species amplification data presented in this study offer valuable assistance in interpreting the results of individual human identification in forensic cases involving non-human primates.

A practical workflow for forensic species identification using direct sequencing of real-time PCR products.

BACKGROUND: Forensic scientists are often required to identify species of unknown biological samples. Although methods based on sequencing of DNA barcode regions are the gold standard for species identification in single-source forensic samples, they are cumbersome to implement as routine work in forensic laboratories that perform many tests, including human DNA typing. We have developed a species identification workflow that incorporates direct sequencing with real-time PCR products (real-time PCR-direct sequencing) as the technical trick for easy testing in forensic practice. METHOD AND RESULTS: Following our workflow, DNA samples from vertebrates, such as mammals, amphibians, reptiles, birds, and fish, were subjected to species identification using vertebrate universal primers targeting each of the four DNA barcode regions. In real-time PCR melting curve analysis, humans and animals (nonhuman) could be differentiated by comparing melting temperatures, and subsequent real-time PCR-direct sequencing contributed to simplified sequencing. Searches against public DNA databases using the obtained sequences were compatible with the origin of the samples, indicating that this method might be used to identify animal species at the genus level. Furthermore, this workflow was effective in actual casework, which provided rapid test results according to the needs of the investigating agencies. CONCLUSIONS: The species identification workflow will simply sequence as much as possible and can be integrated into routine forensic practice. The real-time PCR-direct sequencing used in this workflow might be beneficial not only for species identification but also for DNA sequencing by using the Sanger method for a variety of life sciences.

Advantages of filtration method for sperm-DNA genotyping in sexual assault cases.

Differential extraction (DE) is a conventional method to isolate sperms from forensic semen samples (e.g. vaginal swab containing semen) for sperm-DNA genotyping. Subsequent to selective digestion of somatic cells in a mixture sample, sperms are collected and purified as a pellet by repetitive centrifugation based on the specific gravity of sperm heads. However, the centrifugation operation requires a technical proficiency and an extensive time to prevent a loss of sperms from the pellet as much as possible. Therefore, we devised a "filtration method (FM)", in which a vacuum filtration operation based on the size of sperm heads is adapted, instead of DE, for isolation of sperms without any loss in principle. Sperms are collected and purified on a polycarbonate membrane filter. In this study, we have compared results of forensic assays by DE and FM for sperm-DNA genotyping from forensic semen samples. Consequently, FM had advantages of easy operation, timesaving, and high yield of sperms from semen samples compared with DE, although FM had a comparable ability to DE for a purification of sperms from mixture samples. Thus, we present that FM could simply lead to success of sperm-DNA genotyping and has a possibility to supersede DE as a gold-standard method.

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions.

Identifying vaginal secretions attaching or adhering to a suspect's belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample. In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.

A simple and versatile authenticity assay of coffee products by single-nucleotide polymorphism genotyping.

Interspecific single-nucleotide polymorphisms (SNPs) in the rbcL DNA barcode have been strictly validated and adopted as a designed SNP genotyping maker to discriminate between two major coffee species, Coffea arabica and C. canephora, and to estimate the mixing ratio of DNA from C. arabica/C. canephora in this study. The SNP genotyping is applicable to not only green (unroasted) coffee beans, but also processed coffee products (roasted coffee beans and instant coffee powder), in which genomic DNA is degraded, because the genotyping developed in this study requires only 10 copies of 63-bp-long DNA fragments of rbcL gene. The authenticity assay established in this study has several advantages: a high versatility to DNA sample conditions; simple and rapid procedures (only two steps; DNA extraction and SNP genotyping); the feasibility in coffee business for practical use to prevent false advertising and provide quality control. Abbreviations: SNP: single-nucleotide polymorphism; SBS: single base substitution; ISR: intergenic spacer region; INDEL: insertion-deletion.

A simple identification method for vaginal secretions using relative quantification of Lactobacillus DNA.

In criminal investigations there are some cases in which identifying the presence of vaginal secretions provides crucial evidence in proving sexual assault. However, there are no methods for definitively identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal secretions and developed a novel identification method for vaginal secretions by relative quantification based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as an endogenous control for relative quantification. As a result, the ΔCt (ΔCt=Ct[LCP]-Ct[HSP]) values of vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin surface samples, and it was possible to discriminate between vaginal secretions and other body fluids. For the one remaining sample, it was confirmed that the predominant species in the microflora was not of the Lactobacillus genus. The ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt values of samples up to 6-months-old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that the simple ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells.

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the ß-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.

Worldwide sequence conservation of transmission-blocking vaccine candidate Pvs230 in Plasmodium vivax.

Pfs230, surface protein of gametocyte/gamete of the human malaria parasite, Plasmodium falciparum, is a prime candidate of malaria transmission-blocking vaccine. Plasmodium vivax has an ortholog of Pfs230 (Pvs230), however, there has been no study in any aspects on Pvs230 to date. To investigate whether Pvs230 can be a vivax malaria transmission-blocking vaccine, we performed evolutionary and population genetic analysis of the Pvs230 gene (pvs230: PVX_003905). Our analysis of Pvs230 and its orthologs in eight Plasmodium species revealed two distinctive parts: an interspecies variable part (IVP) containing species-specific oligopeptide repeats at the N-terminus and a 7.5kb interspecies conserved part (ICP) containing 14 cysteine-rich domains. Pvs230 was closely related to its orthologs, Pks230 and Pcys230, in monkey malaria parasites. Analysis of 113 pvs230 sequences obtained from worldwide, showed that nucleotide diversity is remarkably low in the non-repeat 8-kb region of pvs230 (θπ=0.00118) with 77 polymorphic nucleotide sites, 40 of which results in amino acid replacements. A signature of purifying selection but not of balancing selection was seen on pvs230. Functional and/or structural constraints may limit the level of polymorphism in pvs230. The observed limited polymorphism in pvs230 should ground for utilization of Pvs230 as an effective transmission-blocking vaccine.

On-bead screening of a library to detect host-guest complexation by an aniline reporter.

We developed a new labeling reagent and a color assay system in water to detect binding between target molecules and library members on beads, which is free of label-induced artifacts that can cause misleading results.