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There is high demand for forensic age prediction in actual crime investigations. In this study, a novel age prediction model for saliva samples using methylation-sensitive high resolution melting (MS-HRM) was developed. The methylation profiles of ELOVL2 and EDARADD showed high correlations with age and were used to predict age with support vector regression. ELOVL2 was first reported as an age predictive marker for saliva samples. The prediction model showed high accuracy with a mean absolute deviation (MAD) from chronological age of 5.96 years among 197 training samples. The model was further validated with an additional 50 test samples (MAD = 6.25). In addition, the age prediction model was applied to saliva extracted from seven cigarette butts, as in an actual crime scene. The MAD (7.65 years) for these samples was slightly higher than that of intact saliva samples. A smoking habit or the ingredients of cigarettes themselves did not significantly affect the prediction model and could be ignored. MS-HRM provides a quick (2 hours) and cost-effective (95% decreased compared to that of DNA chips) method of analysis. Thus, this study may provide a novel strategy for predicting the age of a person of interest in actual crime scene investigations.

Age prediction with epigenetic information is now edging closer to practical use in forensic community. Many age-related CpG (AR-CpG) sites have proven useful in predicting age in pyrosequencing or DNA chip analyses. In this study, a wide range methylation status in the ELOVL2 and FHL2 promoter regions were detected with methylation-sensitive high resolution melting (MS-HRM) in a labor-, time-, and cost-effective manner. Non-linear-distributions of methylation status and chronological age were newly fitted to the logistic curve. Notably, these distributions were revealed to be similar in 22 living blood samples and 52 dead blood samples. Therefore, the difference of methylation status between living and dead samples suggested to be ignorable by MS-HRM. Additionally, the information from ELOVL2 and FHL2 were integrated into a logistic curve fitting model to develop a final predictive model through the multivariate linear regression of logit-linked methylation rates and chronological age with adjusted R[2] = 0.83. Mean absolute deviation (MAD) was 7.44 for 74 training set and 7.71 for 30 additional independent test set, indicating that the final predicting model is accurate. This suggests that our MS-HRM-based method has great potential in predicting actual forensic age.

We developed a new approach for pairwise kinship analysis in forensic genetics based on chromosomal sharing between two individuals. Here, we defined "index of chromosome sharing" (ICS) calculated using 174, 254 single nucleotide polymorphism (SNP) loci typed by SNP microarray and genetic length of the shared segments from the genotypes of two individuals. To investigate the expected ICS distributions from first- to fifth-degree relatives and unrelated pairs, we used computationally generated genotypes to consider the effect of linkage disequilibrium and recombination. The distributions were used for probabilistic evaluation of the pairwise kinship analysis, such as likelihood ratio (LR) or posterior probability, without allele frequencies and haplotype frequencies. Using our method, all actual sample pairs from volunteers showed significantly high LR values (i.e., ≥ 108); therefore, we can distinguish distant relationships (up to the fifth-degree) from unrelated pairs based on LR. Moreover, we can determine accurate degrees of kinship in up to third-degree relationships with a probability of > 80% using the criterion of posterior probability ≥ 0. 90, even if the kinship of the pair is totally unpredictable. This approach greatly improves pairwise kinship analysis of distant relationships, specifically in cases involving identification of disaster victims or missing persons.

Liver sinusoidal endothelial cells (LSECs) are involved in the transport of nutrients, lipids, and lipoproteins, and LSEC injury occurs in various liver diseases including nonalcoholic fatty liver disease (NAFLD). However, the association between LSEC injury and NAFLD progression remains elusive. Accordingly, in this study, we aimed to elucidate the precise role of LSEC in the pathophysiology of NAFLD using two different mouse models, namely the choline-deficient, L-amino acid-defined and high-fat diet models. Administration of these diets resulted in liver metabolic dysregulation mimicking human NAFLD, such as steatosis, ballooning, lobular inflammation, and fibrosis, as well as central obesity, insulin resistance, and hyperlipidemia. LSEC injury appeared during the simple steatosis phase, and preceded the appearance of activated Kupffer cells and hepatic stellate cells (HSCs). These results indicate that LSEC injury may have a 'gatekeeper' role in the progression from simple steatosis to the early nonalcoholic steatohepatitis (NASH) stage, and LSEC injury may be necessary for the activation of Kupffer cells and HSCs, which in turn results in the development and perpetuation of chronic liver injuries. Taken together, our data provide new insights into the role of LSEC injury in NAFLD/NASH pathogenesis.

著者人数: 36名, Accepted: 2014-12-10

The calculation of likelihood ratios (LRs) for DNA mixture analysis is necessary to establish an appropriate hypothesis based on the estimated number of contributors and known contributor genotypes. In this paper, we recommend a relevant analytical method from the 15 short tandem repeat typing system (the Identifiler multiplex), which is used as a standard in Japanese forensic practice and incorporates a flowchart that facilitates hypothesis formulation. We postulate that: (1) all detected alleles need to be above the analytical threshold (e.g., 150 relative fluorescence unit (RFU)); (2) alleles of all known contributors should be detected in the mixture profile; (3) there should be no contribution from close relatives. Furthermore, we deduce that mixtures of four or more persons should not be interpreted by Identifiler as the LR values of 100, 000 simulated cases have a lower expectation of exceeding our temporal LR threshold (10, 000) which strongly supports the prosecution hypothesis. We validated the method using various computer-based simulations. The estimated number of contributors is most likely equal to the actual number if all alleles detected in the mixture can be assigned to those from the known contributors. By contrast, if an unknown contributor(s) needs to be designated, LRs should be calculated from both two-person and three-person contributions. We also consider some cases in which the unknown contributor(s) is genetically related to the known contributor(s).

[Background]The bile canaliculus is the smallest and first biliary channel and is formed by two or three adjacent hepatocytes. Previous studies of chronic cholangiopathies such as primary sclerosing cholangitis have focused on the bile ductules. However, little is known about the pathological alterations in bile canaliculi in the early phase of cholangiopathies. [Aim]To characterize the bile canalicular morphology in the early phase of sclerosing cholangitis we used 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine-induced mouse model of sclerosing cholangitis. [Methods]Mice were fed a diet with 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (0.1%). Serum biochemical, histological, immunohistochemical, and electron microscopic analyses were performed 1, 2, 4, and 7 days after feeding. [Results]All experimental groups showed significantly increased serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels. From day 1, bile canalicular abnormalities such as dilatation and meandering and loss of microvilli were observed. After bile canalicular abnormalities had appeared, substantial infiltration of inflammatory cells was observed amongst the necrotic cells and periductal region. After these inflammatory changes, cholangiocytes proliferated in the portal area and formed ductular reactions. Finally, periductal fibrosis appeared. [Conclusion]This study provides novel evidence of the occurrence of bile canalicular abnormalities during the early phase of sclerosing cholangitis.