Application of a custom haplotype caller to analyze sequence-based data of 56 microhaplotypes.

Microhaplotypes (microhaps) are recently introduced markers that aim to complement the limitations of conventional forensic markers such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). With the potential of microhaps in forensics becoming clearer through massively parallel sequencing (MPS), MPS-based studies on microhaps are being actively reported. However, simpler workflow schemes for the generation and analysis of MPS data are still required to facilitate the practical application of MPS in forensics. In this study, we developed an in-house MPS panel that simultaneously amplifies 56 microhaps and a custom haplotype caller, Visual Microhap. The developed tool works on a web browser and provides four analysis options to extract SNP-based haplotypes from sequence-based data obtained by STRait Razor 3.0. To demonstrate the utility of the MPS panel and data analysis workflow scheme, we also analyzed 56 microhaps of 286 samples from four populations (African-American, Caucasian, Hispanic, and Korean). The average effective number of alleles (Ae) for the four groups was 3.45, ranging from 1.74 to 6.98. Forensic statistical parameters showed that this microhap panel is more powerful than conventional autosomal STRs for human identification. Meanwhile, the 56-plex panel mostly comprised microhaps with high Ae; however, the four populations were grossly distinguishable from each other by cluster analysis. Consequently, the developed in-house MPS panel for 56 microhaps and the adopted workflow using open-source tools can increase the utility of microhap MPS in forensic research and practice.

Inhibition of STAT5A promotes osteogenesis by DLX5 regulation.

The regulation of osteogenesis is important for bone formation and fracture healing. Despite advances in understanding the molecular mechanisms of osteogenesis, crucial modulators in this process are not well-characterized. Here we demonstrate that suppression of signal transducer and activator of transcription 5A (STAT5A) activates distal-less homeobox 5 (DLX5) in human bone marrow-derived stromal cells (hBMSCs) and enhances osteogenesis in vitro and in vivo. We show that STAT5A negatively regulates expression of Dlx5 in vitro and that STAT5A deletion results in increased trabecular and cortical bone mass and bone mineral density in mice. Additionally, STAT5A deletion prevents age-related bone loss. In a murine fracture model, STAT5A deletion was found to significantly enhance bone remodeling by stimulating the formation of a fracture callus. Our findings indicate that STAT5A inhibition enhances bone formation by promoting osteogenesis of BMSCs.

Inhibition of miR-449a Promotes Cartilage Regeneration and Prevents Progression of Osteoarthritis in In Vivo Rat Models.

Traumatic and degenerative lesions of articular cartilage usually progress to osteoarthritis (OA), a leading cause of disability in humans. MicroRNAs (miRNAs) can regulate the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) and play important roles in the expression of genes related to OA. However, their functional roles in OA remain poorly understood. Here, we have examined miR-449a, which targets sirtuin 1 (SIRT1) and lymphoid enhancer-binding factor-1 (LEF-1), and observed its effects on damaged cartilage. The levels of chondrogenic markers and miR-449a target genes increased during chondrogenesis in anti-miR-449a-transfected hBMSCs. A locked nucleic acid (LNA)-anti-miR-449a increased cartilage regeneration and expression of type II collagen and aggrecan on the regenerated cartilage surface in acute defect and OA models. Furthermore, intra-articular injection of LNA-anti-miR-449a prevented disease progression in the OA model. Our study indicates that miR-449a may be a novel potential therapeutic target for age-related joint diseases like OA.

Genetic characteristics of Y-chromosome short tandem repeat haplotypes from cigarette butt samples presumed to be smoked by North Korean men.

Korea has been divided into South Korea and North Korea for over 70 years. DNA profiles of the North Korean population have never been reported in the Y-chromosome STR Haplotype Reference Database (YHRD; https://yhrd.org ). To investigate genetic features of Y-chromosome STR haplotypes of the North Korean population for the first time. Genomic DNA was isolated from 838 cigarette butts assumed to have been smoked by North Korean men and amplified with PowerPlex Y23 (PPY23) kit. Statistical parameters were calculated using Nei's formula and analysis of molecular variance (AMOVA). Multidimensional scaling (MDS) plot was constructed by the AMOVA tool and neighbor-joining (NJ) tree was constructed by MEGA 6.06. A total of 121 haplotypes were analyzed for PPY23 loci from a sample population. Haplotype diversity and discrimination capacity were 0.9992 and 0.9837, respectively. Genetic diversities ranged from 0.2981 to 0.9716. For the 16 Y-filer loci and eight minimal loci, respectively 90.9 and 82.6% of the matched haplotypes were estimated to belong to haplogroup O, representing the Southeast and East Asian type. The MDS plot and NJ tree indicated that the samples are most closely related to South Korean. In addition, p-value in the pairwise comparison to the South Korean was slightly above statistical significance (p = 0.0534). The Y-STR haplotypes of the samples were unique and highly genetically polymorphic. Despite the separation between North and South Korea for 70 years, they can still be considered a single genetic population, based on Y-STR haplotypes.

Monitoring of post-mortem changes of saliva N-glycosylation by nano LC/MS.

The estimation of post-mortem interval (PMI) is a crucial part for investigations of crime and untimely deaths in forensic science. However, standard methods of PMI estimation are easily confounded by extenuating circumstances and/or environmental factors. Therefore, a panel of PMI markers obtained from a more acceptable and accurate method is necessary to definitely determine time of death. Saliva, one of the vital fluids encountered at crime scenes, contains various glycoproteins that are highly affected by biochemical environment. Here, we investigated saliva N-glycans between live and dead rats to determine the alteration of N-glycans using an animal model system because of the limitation of saliva collection from recently deceased humans. Rat saliva samples were collected both before and after death. N-Glycans were enzymatically released by PNGase F without any glycoprotein extraction. Released native glycans were purified and enriched by PGC-SPE. About 100 N-glycans were identified, profiled, and structurally elucidated by nano LC/MS and tandem MS. Sialylated N-glycans were exclusively present in abundance in live rat saliva whereas non-sialylated N-glycans including LacdiNAc disaccharides were detected in high level following death. Through in-depth investigations using quantitative comparison and statistical analysis, 14 N-glycans that significantly changed after death were identified as the potential marker candidates for PMI estimation. To the best of our knowledge, this is the first study to monitor the post-mortem changes of saliva glycosylation, with obvious forensic applications.

Identification of female-specific blood stains using a 17ß-estradiol-targeted aptamer-based sensor.

Blood stain evidence obtained from a violent crime scene provides decisive clues that can enable a case to be solved through forensic analyses such as genetic identification. However, collected samples usually contain a mixture of biological material from different sources, making genetic identification difficult. To address this issue, we developed an activatable aptamer sensor targeting 17ß-estradiol for detection of female-specific blood in mixed samples. With the sensor, we were able to detect blood originating from females using a variable light source (495 nm). The sensor was especially sensitive to blood from young females (10-40 years) but not to blood from older females (≥ 50 years). Genomic DNA was extracted from the female blood specimens identified by this method and used for quantification and short tandem repeat genotyping. We confirmed that there was no fluorescence interference from the aptamer sensor. These results indicate that this novel aptamer sensor can be used to analyze evidentiary blood samples and thereby facilitate subsequent genetic identification.

Ascorbic acid and vitamin C-containing beverages delay the leucomalachite green reaction to detect latent bloodstains.

The leucomalachite green (LMG) test is one of catalytic tests for the detection of latent bloodstains and generally used in forensic field because of convenience and cost/time-effectiveness. However, contamination of latent bloodstains at crime scenes can interfere with the LMG reaction, resulting in false-negative or false-positive decisions. Herein, we examined if ascorbic acid and vitamin C (l-ascorbic acid or ascorbate)-containing beverages affect the LMG reaction. Ascorbic acid showed the inhibitory activities on the LMG reaction in a dose-dependent manner. Similarly, vitamin C-containing beverages also inhibited the LMG reaction and the inhibitory effects were proportional to the concentrations of vitamin C in beverages. It was also identified that as incubation time after adding LMG reagent to the mixtures of blood and ascorbic acid or beverages was increased, the inhibitory effects of ascorbic acid vitamin C-containing beverages on LMG test were disappeared. These results suggest that the LMG reaction is delayed but not stopped by ascorbic acid and vitamin C-containing beverages. Neither incubation at room temperature around 20-25°C nor the addition of acetic acid affects the inhibitory activity of ascorbic acid on LMG reaction. We also showed that ascorbic acid does not affect DNA stability, allowing us to obtain full short tandem repeat (STR) profiles through amplification of DNA using commercial STR kits. In conclusion, ascorbic acid and vitamin C-containing beverages delayed the LMG reaction, suggesting that it should be considered that negative results of LMG test could be false negative due to contamination of bloodstains with inhibitory factors on LMG test.

Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra.

The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products.

Population genetics of insertion-deletion polymorphisms in South Koreans using Investigator DIPplex kit.

We assessed the applicability of 30 insertion-deletion polymorphisms (INDELs) in forensic use and the level of genetic diversity in South Korea (n=373) using the Investigator DIPplex kit (Qiagen). Allele frequencies, heterozygocities, and forensic efficacy parameters were determined. No deviation from Hardy-Weinberg equilibrium was observed for any of the INDEL markers. A high level of discrimination power was observed (combined power of discrimination: 0.99999999995). The combined match probability value was 2.84 × 10(-11) and the mean typical paternity indices were 0.878. Furthermore, we found one microvariant allele at HLD93 (rs2307570) that has not been reported. We expect that these 30 loci of INDEL markers will be useful for forensic identification and paternity testing in the South Korean population.

Mutation Screening of the γ-Aminobutyric Acid Type-A Receptor Subunit γ2 Gene in Korean Patients with Childhood Absence Epilepsy.

BACKGROUND AND PURPOSE: Since the γ-aminobutyric acid type-A receptor subunit γ2 gene (GABRG2) mutation was discovered in an Australian family with childhood absence epilepsy (CAE) and febrile convulsions, a few screening studies for the GABRG2 mutation have been conducted in sporadic individuals with CAE from other ethnic groups. The aim of this study was to determine whether or not the previously reported genetic mutations and single-nucleotide polymorphisms (SNPs) of GABRG2 can be reproduced in sporadic Korean individuals with CAE, compared to healthy Korean individuals. METHODS: Thirty-five children with CAE in Chonnam National University Hospital and healthy controls (n=207) were enrolled, and the medical records of patients with CAE were reviewed. CAE was diagnosed according to the Classification and Terminology of the International League Against Epilepsy. All nine exons of GABRG2 were directly sequenced. In addition, the two SNPs found in our CAE patients were analyzed: C315T in exon 3 (E3) and C588T in exon 5 (E5). The frequencies of the two SNPs in the CAE patients were compared with data from healthy controls (for E3 and E5) and from previously reported Korean population data (only for E3). RESULTS: No mutation of GABRG2 was found in our CAE patients. In addition, the allele and genotype frequencies of the two polymorphisms did not differ significantly between CAE patients, healthy controls, and the Korean general population (p>0.05). CONCLUSIONS: Our study of sporadic Korean individuals with CAE found no evidence that GABRG2 contributes to the genetic basis of CAE.

Technical note: improved ancient DNA purification for PCR using ion-exchange columns.

A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples.

Comparison of polymerase chain reaction and the indirect particle agglutination antibody test for the diagnosis of Mycoplasma pneumoniae pneumonia in children during two outbreaks.

BACKGROUND: Diagnosis of Mycoplasma pneumoniae pneumonia is challenging because of the lack of standardized rapid tests. Many serologic tests and polymerase chain reaction (PCR) based methods are used with different diagnostic criteria. METHODS: This retrospective study was conducted to compare the diagnostic values of the indirect particle agglutination test and nested PCR of nasopharyngeal aspirates for the diagnosis of M. pneumoniae pneumonia in children. These assays were evaluated in 234 hospitalized children with community-acquired lower respiratory tract infections during 2 outbreaks of M. pneumoniae pneumonia in 2000 and 2003. RESULTS: The cumulative PCR positive rate was 26.7% in patients with maximum antibody titers of < or =1:320 and 78.2% in those with titers of > or =1:640. Based on these data, a positive PCR, a 4-fold increase in antibody titer, or a single titer > or =1:640 were considered to indicate acute M. pneumoniae infection. Overall, 152 children were diagnosed to have M. pneumoniae pneumonia; 27 (18%) by serology only, 26 (17%) by PCR only, and 99 (65%) by both methods. Children who were diagnosed by PCR only were significantly younger (P = 0.003) and were more often immunocompromised (P = 0.019) than those that were PCR negative. Duration of cough before PCR diagnosis was shorter in cases diagnosed by PCR only than those that were PCR negative (P = 0.045). CONCLUSIONS: In conclusion, during the 2 outbreaks of M. pneumoniae infection, we found that the PCR test may be useful for the rapid diagnosis of M. pneumoniae pneumonia, particularly in young children and in immunocompromised patients and in early stage disease.

Single nucleotide polymorphisms in the multidrug resistance 1 gene in Korean epileptics.

PURPOSE: P-glycoprotein 170 encoded by the multidrug resistance 1 (MDR1) gene exports various antiepileptic drugs out of the CNS, which leads to multidrug resistance. This study was performed to elucidate the relationship between single nucleotide polymorphisms (SNPs) in the MDR1 gene and drug resistance in Koreans with epilepsy. SUBJECTS AND METHODS: Three SNPs at nucleotide position 1236 in exon 12, 2677 in exon 21 and 3435 in exon 26 of the MDR1 gene were genotyped in 207 Korean epileptics. Subjects were classified according to whether they had drug-resistant (RS group; N=99) or drug-responsive epilepsy (RP group; N=108). The frequencies of genotype and haplotype were compared between the RS and RP groups. RESULTS: The frequencies of genotype and haplotype in the RS group were not statistically different from those in the RP group. CONCLUSIONS: In Korean epileptics, there was no significant relationship between three known SNPs in MDR1 and drug resistance. And there was no association of MDR1 haplotype based on above three sites with pharmacoresistance.

Effects of nutritional supplement with herbal extract on bone mineral density and height in prepubescent children - a preliminary study.

The aim of this study was to investigate the efficacy of a nutritional supplement with herbal extracts on height and on the bone mineral density (BMD) development of prepubescent children who were in the 25th percentile of standard height of their age. All children were administered a supplement for 6 months, with height and BMD measured. The supplement increased significantly the height and BMD, compared with the baseline in both boys and girls. The annual growth rate was higher than the standard rate. These preliminary data indicate that supplements enhanced the rate of BMD and height development, although this result must be replicated in a large population-based study and placebo-controlled trials to confirm the conclusions.

Effects of multifunctional fabrics on cardiac autonomic tone and psychological state.

Heart rate variability was compared in 20 subjects wearing multifunctionalfabric (experimental sessions) and cotton (control sessions) clothing. Anxiety, depression, fatigue, and stress levels were lower and emotional levels were higher during the experimental sessions than in the control sessions. Multifunctional fabrics reduced the low-frequency/high-frequency power ratio of heart rate variability. These results support the hypothesis that multifunctional fabrics increase cardiac parasympathetic tone. In addition, subjects had lower heart rates during the experimental sessions compared with controls, suggesting a stabilizing effect on the autonomic nervous system. In conclusion, multifunctional fabrics may act to stabilize both the autonomic nervous system and psychological state.

Effects of a Korean traditional herbal remedy on psychoneuroendocrine responses to examination stress in medical students: a randomized placebo-controlled trial.

The aim of this study was to investigate the efficacy of Ondamtanggamibang, a Korean traditional herbal remedy, as a treatment of stress-related psychophysiological variables in healthy medical students experiencing examination stress. Forty volunteers were randomly assigned to either an herbal remedy group (n = 20) or a placebo control group (n = 20). After treatment, systolic blood pressure and plasma concentrations of norepinephrine and cortisol concentrations decreased significantly in the herbal remedy group. The herbal remedy group also exhibited beneficial changes in psychological variables such as anxiety, depression, stress and emotional state. These results suggest that a Korean traditional herbal remedy may reduce systolic blood pressure and catecholamine levels, possibly by stabilizing the sympathetic nervous system. This herbal remedy also reduced the negative psychological symptoms, stress and heightened the emotional state experienced by medical students during examinations.

Effect of multi-functional fabric on sleep stages and growth hormone levels during sleep.

Nine young girls participated in cross-over sessions, sleeping with either multi-functional fabric (experimental session) or cotton (control session). The relative duration of slow-wave sleep (SWS) was 1.89-fold higher in the experimental session than in the control session. The peak growth hormone (GH) secretion in the experimental session was more than 2.4-fold higher than during the control session (p <.001). The quality of sleep during the experimental session was significantly better than in the control session (p <.01). These results suggest that multi-functional fabric wear is effective in inducing deep sleep, increasing GH, and improving the quality of sleep.

Hibiscus extract inhibits the lipid droplet accumulation and adipogenic transcription factors expression of 3T3-L1 preadipocytes.

OBJECTIVES: This study was designed to investigate the effect of hibiscus (Hibiscus sabdariffa) on adipogenic differentiation of 3T3-L1 cells at the cellular and molecular levels. DESIGN: Various concentrations of hibiscus extract were added to confluent 3T3-L1 preadipocytes at the outset of the differentiation program and further incubated for 36 hours. Cells were maintained in postdifferentiation medium containing insulin with hibiscus extract in complete culture medium. RESULTS: Hibiscus extract inhibited the adipocyte differentiation of 3T3-L1 preadipocytes induced by insulin, dexamethasone, and isobutylmethylxanthine (IBMX) in a dose-dependent manner. Hibiscus blocked the cytoplasmic lipid accumulation when administered at the onset of differentiation and 4 days after induction of differentiation. The inhibitory effect of hibiscus on adipogenic lipid accumulation of preadipocytes was significant (p < 0.01) between control cells and cells treated with hibiscus. Hibiscus extract significantly attenuated the expression of key adipogenic transcription factors, including CCAAT element binding protein (C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR)gamma at protein levels. CONCLUSION: These results suggest that hibiscus extract blocks adipogenesis, in part, by its suppression on the expression of adipogenic transcription factors, including C/EBPalpha and PPARgamma.

Qi-training enhances respiratory burst function and adhesive capacity of neutrophils in young adults: a preliminary study.

The main objective of this study is to examine the effect of Qi-training on the immune system, especially neutrophil bactericidal function. Nine healthy male subjects were studied for the effects of one bout of ChunDoSunBup (CDSB) Qi-training on superoxide (O2- production and adhesion capacity of neutrophils at times immediately after (Post I) and 2 hours after the Qi-training (Post II). The Qi-training enhanced O2- production, reaction velocity and neutrophil adhesion capacity and there were significant differences at Post I compared to before Qi-training (Pre). In addition, the number of white blood cells (WBC), monocytes and lymphocytes were changed significantly through Qi-training.Therefore, it seems that CDSB Qi-training may increase the resistance of trained individuals against common infection and inflammation.