Wildlife forensics using mitochondrial DNA sequences: Species identification based on hairs collected in the field and confiscated tanned Felidae leathers

To identify species based on samples without recognizable morphological characteristics, DNA-based approaches are the best option. Here, we describe two cases of the determination of species and geographical origin of wildlife specimens under the regulation of international treaties and domestic laws related to wildlife management in South Korea. First, hairs of suspected wild or reared endangered Asiatic black bears were analyzed using cytochrome oxidase I and the control region. Confiscated Felidae leathers were also investigated using cytochrome b, but they were proven to be fabricated canine leathers. These results were used as scientific evidence for wildlife-related law enforcement. Our results suggest that unrecognizable wildlife specimens can be identified efficiently using DNA sequence-based analysis. Finally, this study shows that conservation genetics research and its applications can be incorporated into wildlife forensic studies.     

The genetic effect of copy number variations on the risk of type 2 diabetes in a Korean population

Background

Unlike Caucasian populations, genetic factors contributing to the risk of type 2 diabetes mellitus (T2DM) are not well studied in Asian populations. In light of this, and the fact that copy number variation (CNV) is emerging as a new way to understand human genomic variation, the objective of this study was to identify type 2 diabetes–associated CNV in a Korean cohort.

Methodology/Principal Findings

Using the Illumina HumanHap300 BeadChip (317,503 markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 275 patients with type 2 diabetes mellitus (T2DM) and 496 nondiabetic subjects (Total n = 771). To increase the sensitivity of CNV identification, we incorporated multiple factors using PennCNV, a program that is based on the hidden Markov model (HMM). To assess the genetic effect of CNV on T2DM, a multivariate logistic regression model controlling for age and gender was used. We identified a total of 7,478 CNVs (average of 9.7 CNVs per individual) and 2,554 CNV regions (CNVRs; 164 common CNVRs for frequency>1%) in this study. Although we failed to demonstrate robust associations between CNVs and the risk of T2DM, our results revealed a putative association between several CNVRs including chr15:45994758–45999227 (P = 8.6E-04, P^corr = 0.01) and the risk of T2DM. The identified CNVs in this study were validated using overlapping analysis with the Database of Genomic Variants (DGV; 71.7% overlap), and quantitative PCR (qPCR). The identified variations, which encompassed functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the development process, in cell communication, in signal transduction, and in biological regulation.

Conclusion/Significance

We expect that the methods and findings in this study will contribute in particular to genome studies of Asian populations.     

In vitro determination of the contraceptive spermicidal activity of essential oil of Trachyspermum ammi (L.) Sprague ex Turrill fruits

This study was conducted to determine the spermicidal and contraceptive efficacy of essential oil of Trachyspermum ammi on human sperm in vitro. Chemical compositions of the oil were analyzed by GC-MS. Nearly 30 compounds representing 91.39% of the total oil were identified. The minimum effective dose (MED) of essential oil of T. ammi that induced instant immobilization of human spermatozoa in vitro was 125 μg/mL. The motility was also irreversible. All of the human sperms were found to be non viable within 10 min at this concentration. The activity of acrosomal enzyme was reduced and a significant releases of 5'-nucleotidase into the surrounding medium was noted after treatment with MED concentration of essential oil, indicating the plasma membrane degradation of the sperm. The maximum number of human sperm failed to decondense when treated with MED concentration of essential oil. The morphological deformities of sperm plasma membrane were evidenced by SEM, which showed vaculation, detachment of heads and tail coiling. The present research indicates that essential oil of T. ammi possesses appreciable spermicidal potential, which may be explored as an effective constituent of vaginal contraceptive.     

Effects of UCP2 and UCP3 Variants on the Manifestation of Overweight in Korean Children

To investigate the associations of uncoupling protein (UCP)2 and UCP3 gene variants with overweight and related traits, we genotyped UCP2−866G>A, UCP2Ala55Val, and UCP3−55C>T in 737 Korean children and 732 adults and collected data regarding anthropometric status and blood biochemistry. Information concerning the children's lifestyles and dietary habits was collected. The UCP2−866G>A and UCP3−55C>T gene variants showed significant associations with BMI level, waist circumference, and body weight in the children but not in the adults. Compared with −866GG carriers, the −866GA and AA carriers showed a strong decreasing trend in the risk for overweight (odds ratio (OR), 0.67; 95% confidence interval (CI), 0.45–1.01; P = 0.053). In comparison with UCP3−55CC carriers, children carrying −55CT and TT showed a significant reduction in the risk of overweight (OR, 0.67; 95% CI, 0.46–0.98; P = 0.039). There was also evidence of interactions between the effects of the combined UCP2−UCP3 genotype and obesity‐related metabolic traits. The greatest protective effect against overweight was seen in those with the combined genotype non‐UCP2−866GG and non‐UCP3−55CC, as compared with those carrying both UCP2−866GG and UCP3−55CC (OR, 0.60; 95% CI, 0.38–0.95; P = 0.030). In the subgroup with a low level of physical activity, UCP3−55CC carriers had higher BMI values than UCP3−55T carriers (16.6 ± 2.3 kg/m² vs. 16.1 ± 1.9 kg/m², P = 0.016). Low physical activity may aggravate the susceptibility to overweight in UCP2−866GG and UCP3−55CC carriers.     

PSP1, a Phosphatidylserine-Recognizing Peptide,Is Useful for Visualizing Radiation-Induced Apoptosis in Colorectal Cancer In Vitro and In Vivo

Accurate and timely visualization of apoptotic status in response to radiation is necessary for deciding whether to continue radiation or change to another mode of treatment. This is especially critical in patients with colorectal cancer, which requires a delicate combination of surgery, radiation, and chemotherapy in order to achieve optimal outcome. In this study, we investigated the potential of phosphatidylserine-recognizing peptide 1 (PSP1) as an apoptosis-targeting probe, which identifies phosphatidylserine on cell surfaces. We first screened colon cancer cell lines for their sensitivity to radiation and selected two cell lines: HCT116 and HT29. Cell binding assay using fluorescence-activated cell sorting and optical imaging showed that HCT116 cells had better binding to PSP1 than HT29 cells. Thus, mouse xenograft model using HCT116 cells was generated and was topically irradiated with either single or fractionated dose of radiation followed by systemic administration of PSP1 for subsequent molecular optical imaging. We confirmed that the PSP1 probe was selectively bound to apoptosis-induced tumor in a radiation dose-dependent manner. We also observed that fractionated radiation regimen, which is recently being used in clinical situation, was more effective in inducing tumor apoptosis than corresponding single-dose radiation treatment. We then evaluated the correlation between tumor targeting of PSP1 and suppression effect of tumor development and found that tumor volume and fluorescence intensity were correlated before (correlation coefficient r² = 0.534) and after (r² = 0.848) radiation therapy. Our study shows that PSP1 peptide is an efficient index probe for deciding “go or no-go” for radiation therapy in colorectal cancer.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.