DNA methylation-based organ tissue identification: Marker identification, SNaPshot multiplex assay development, and interlaboratory comparison.

Identifying body fluids and organ tissues is highly significant as they can offer crucial evidence in criminal investigations and aid the court in making informed decisions, primarily through evaluating the biological source and possibly at the activity level up to death or fatal damage. In this study, organ tissue-specific CpG markers were identified from Illumina's methylation EPIC array data of nine organ tissues, including epidermis, dermis, heart, skeletal muscle, blood, kidney, brain, lung, and liver, from autopsies of 10 Koreans. Through the validation test using 43 samples, 18 hypomethylation markers, with two markers for each organ tissue type, were selected to construct a SNaPshot assay. Two multiplex assays involving forward and reverse SBE primers were designed to help investigators accurately determine the organ origin of the analyzed tissue samples through repeated analysis of the same PCR products for markers. The developed multiplex demonstrated high accuracy, achieving 100.0 % correct detection of the presence of nine organ tissue types in 88 samples from autopsies of 10 Asians. However, two lung samples showed additional positive indications of the presence of blood. An interlaboratory comparison using 80 autopsy samples (heart, skeletal muscle, blood, kidney cortex, kidney medulla, brain, lung, and liver) from 10 individuals in Germany revealed overall comparable results with correct detection of the presence of eight organ tissue types in 92.5 % samples (74 of 80 samples). In the case of six samples, it was impossible to determine the correct tissue successfully due to drop-outs of unmethylation signals at target tissue marker loci. One of these lung samples revealed only non-intended off-target signals for blood. The observed differences might be due to differences in sample collection during routine autopsy, technical differences due to the PCR cycler, and the threshold used for signal calling. Indicating the presence of additional tissue type and off-target unmethylation signals seems alleviated by applying more stringent hypomethylation thresholds. Therefore, the developed SNaPshot multiplex assays will be valuable for forensic investigators dealing with organ tissue identification, as well as for prosecutors and defense aiming to establish the circumstances that occurred at the crime scene.

Forensic genetic analysis of single-nucleotide polymorphisms and microhaplotypes in Koreans through next-generation sequencing using precision ID identity panel.

BACKGROUND: Forensic DNA analysis has seen remarkable advancements with the advent of Next Generation Sequencing (NGS). In particular, NGS analysis of single nucleotide polymorphisms (SNPs) offers significant advantages in the analysis of challenging samples compared to conventional STR analysis. OBJECTIVE: This study aimed to investigate the SNPs of the Precision ID Identity Panel, a commercially available NGS panel for personal identification, by generating genetic profiles of 298 Koreans and comparing them with other global populations. METHODS: A total of 124 SNPs, including 90 autosomal and 34 Y-SNPs, were analyzed using the Precision ID Identity Panel, and forensic parameters, microhaplotypes, and population differences were investigated. RESULTS: The NGS data were successfully obtained from 298 Koreans. The analysis of forensic parameters exhibited a low combined match probability of 1.532 × 10- 34, which is comparable to that obtained from commonly used STR analysis. Additionally, the microhaplotype analysis revealed that the use of 16 microhaplotypes provided higher discriminatory power compared to single target SNPs. Furthermore, the adoption of microhaplotype data resulted in an increase of over 20% in expected heterozygosity at five loci. Inter-population analysis showed a close genetic relationship between Koreans and individuals from China and Myanmar in East and Southeast Asia, which are geographically adjacent to Korea. CONCLUSIONS: The results of this study show that the Precision ID Identity panel can be a useful alternative where traditional STR typing is not feasible. Also, the data from our study will be useful as a reference for Koreans in forensic investigations and the prosecution of criminal justice.

A Hepatitis B Virus-Derived Peptide Exerts an Anticancer Effect via TNF/iNOS-producing Dendritic Cells in Tumor-Bearing Mouse Model.

Recently, we reported a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, that exerts antiviral effects against human immunodeficiency virus type 1 (HIV-1). Here, we explored the immunotherapeutic potential of Poly6 via its administration into dendritic cells (DCs) in a mouse model. Our data revealed that Poly6 treatment led to enhanced production of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner via the induction of mitochondrial stress. Poly6 treatment in mice implanted with MC38 cells, a murine colon adenocarcinoma line, led to attenuated tumor formation, primarily due to direct cell death induced by Tip-DC mediated nitric oxide (NO) production and indirect killing by Tip-DC mediated cluster of differentiation 8 (CD8) cytotoxic T lymphocyte (CTL) activation via CD40 activation. Moreover, Poly6 treatment demonstrated an enhanced anticancer effect with one of the checkpoint inhibitors, the anti PD-L1 antibody. In conclusion, our data reveal that Poly6 treatment elicits an antitumor immune response in mice, possibly through NO-mediated oncolytic activity via Tip-DC activation and Tip-DC mediated CTL activation. This suggests that Poly6 represents a potential adjuvant for cancer immunotherapy by enhancing the anticancer effects of immune checkpoint inhibitors.

Heat-killed Mycobacterium paragordonae therapy exerts an anti-cancer immune response via enhanced immune cell mediated oncolytic activity in xenograft mice model.

A therapeutic strategy capable of skewing toward a Th1-type immune response is crucial for cancer treatment. Recently, we reported Mycobacterium paragordonae (Mpg) as a potential live vaccine for mycobacterium infections. In this study, we explored the immunotherapeutic potential of heat-killed Mpg (HK-Mpg) in a mouse tumor xenograft model and elucidated its underlying antitumor mechanisms. MC38 cells derived from murine colon adenocarcinoma were implanted by subcutaneously injecting mice. The anticancer effects of HK-Mpg therapy were compared with HK-M. bovis BCG, an effective adjuvant for cancer immunotherapy. HK-Mpg treatment enhanced tumor reduction and mouse survival. Furthermore, HK-Mpg treatment synergistically enhanced the anticancer therapeutic effect of cisplatin. In addition, HK-Mpg enhanced inflammatory cytokine production and recruitment of immune cell into tumor-infiltrating sites and splenocytes in vaccinated mice. Our mechanistic study demonstrates that HK-Mpg therapy elicits a strong antitumor immune response in mice, mainly through natural killer cell-mediated oncolytic activity via the activation of dendritic cells (DCs) and by enhancing inflammatory cytokines production such as IL-12 from DC. Hence, HK-Mpg can be a potential immunotherapy adjuvant, enhancing the effect of cancer chemotherapy.

rt269I Type of Hepatitis B Virus (HBV) Leads to HBV e Antigen Negative Infections and Liver Disease Progression via Mitochondrial Stress Mediated Type I Interferon Production in Chronic Patients With Genotype C Infections.

Hepatitis B virus infection is a serious global health problem and causes life-threatening liver disease. In particular, genotype C shows high prevalence and severe liver disease compared with other genotypes. However, the underlying mechanisms regarding virological traits still remain unclear. This study investigated the clinical factors and capacity to modulate Type I interferon (IFN-I) between two HBV polymerase polymorphisms rt269L and rt269I in genotype C. This report compared clinical factors between rt269L and rt269I in 220 Korean chronic patients with genotype C infections. The prevalence of preC mutations between rt269L and rt269I was compared using this study's cohort and the GenBank database. For in vitro and in vivo experiments, transient transfection using HBV genome plasmid and HBV virion infection using HepG2-hNTCP-C4 and HepaRG systems and hydrodynamic injection of HBV genome into mice tails were conducted, respectively. This report's clinical data indicated that rt269I vs. rt269L was more significantly related to HBV e antigen (HBeAg) negative serostatus, lower levels of HBV DNA and HBsAg, and disease progression. Our epidemiological study showed HBeAg negative infections of rt269I infections were attributed to a higher frequency of preC mutations at 1896 (G to A). Our in vitro and in vivo studies also found that rt269I could lead to mitochondrial stress mediated STING dependent IFN-I production, resulting in decreasing HBV replication via the induction of heme-oxygenase-1. In addition, we also found that rt269I could lead to enhanced iNOS mediated NO production in an IFN-I dependent manner. These data demonstrated that rt269I can contribute to HBeAg negative infections and liver disease progression in chronic patients with genotype C infections via mitochondrial stress mediated IFN-I production.