Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population.

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent-child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39-1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38-1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells.

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

Surgical technique of two-step pelvic and para-aortic sentinel lymph node mapping in early-stage endometrial cancer: laparoscopic, robotic, and open method.

Since sentinel lymph node mapping in endometrial cancer is becoming more widely used, the need of standardizing surgical technique is growing [1, 2]. The objective of this surgical video is to describe the procedure of two-step pelvic and para-aortic sentinel lymph node mapping using indocyanine green and fluorescent camera in endometrial cancer, in three versions of surgical modality of laparoscopic, robotic, and open laparotomy. The patients in the surgical video are diagnosed with biopsy-proven endometrial cancer in its early stage determined by the preoperative imaging study. After collecting washing cytology, bilateral salpinges were clamped with Endo Clip™ to minimize tumor spillage. Gauze packing in posterior cul-de-sac was done to minimize the spillage of indocyanine green dye during paraaortic sentinel lymph node mapping. Indocyanine green dye was injected in bilateral uterine fundus, to detect isolated paraaortic sentinel lymph node pathway. After bilateral paraaortic sentinel lymph node was sampled, cervical injection of Indocyanine green dye was done in 3 o'clock and 9 o'clock directions, both superficially and deeply, 2 mL in each side. After dissecting off the obliterated umbilical ligament, para-vesical and para-rectal spaces were developed. The ureter, uterine artery, and internal and external iliac vessels were identified before bilateral pelvic sentinel lymph nodes were sampled. Asan Medical Center's Institutional Review Board exempted this project. Sentinel paraaortic and pelvic lymph nodes were successfully harvested by two-step method of sentinel lymph node mapping through laparoscopic, robotic, and open laparotomy methods. This surgical video provides specific steps of pelvic and para-aortic sentinel lymph node mapping.

Analysis of mutation rates and haplotypes of 23 Y-chromosomal STRs in Korean father-son pairs.

Y-chromosomal short tandem repeats (Y-STRs) have been widely used in forensic genetics, and accurate knowledge of mutation rates at Y-STR loci is essential in kinship analysis. The main aim of this study was to estimate Y-STR mutation rates in Korean males. To obtain locus-specific mutations and haplotypes at 23 Y-STRs, we analyzed samples from 620 Korean father-son pairs. In addition, we also analyzed 476 unrelated individuals using the PowerPlex® Y23 System, with the aim of augmenting the available data for the Korean population. The PowerPlex® Y23 system facilitates analysis of the 23 Y-STR loci (DYS576, DYS570, DYS458, DYS635, DYS389 II, DYS549, DYS385, DYS481, DYS439, DYS456, DYS389 I, DYS19, DYS393, DYS391, DYS533, DYS437, DYS390, Y GATA H4, DYS448, DYS438, DYS392, and DYS643). Locus-specific mutation rate estimates varied from 0.00 to 8.06 × 10-3 per generation, with an average mutation rate of 2.17 × 10-3 (95% CI, 1.5-3.1 × 10-3). To obtain comprehensive genetic values for the Korean population, we combined data obtained in this study with previously reported values, thereby enabling us to estimate the locus-specific mutation rates regarding 22,711 allele transmissions. By combining these data, we obtained an overall average mutation rate of 2.91 × 10-3 (95% CI, 2.3-3.7 × 10-3). In addition, among the 476 unrelated Korean males, we detected 467 different haplotypes, with an overall haplotype diversity value of 0.9999. By extracting haplotypes of Y-STRs described in previous literature on 23 Y-STR reported in Korea, we obtained gene diversities for 1133 Korean individuals. We believe that the values and characteristics of the 23 Y-STRs analyzed in this study will contribute to establishing criteria for forensic genetic interpretation, including kinship analysis.

Promotion of tumorigenesis by miR-1260b-targeting CASP8: Potential diagnostic and prognostic marker for breast cancer.

MicroRNAs are reported as promising biomarkers for the diagnosis and treatment of breast cancer. miR-1260b is identified as a tumor-associated noncoding microRNA in other cancers, although the role of miR-1260b and its clinical relevance in breast cancer remain unclear. In this study, miR-1260b as a potential prognostic biomarker was observed by univariate and multivariate Cox regression analyses in 102 breast tumor tissues. The tumorigenic role of miR-1260b in terms of proliferation, apoptosis, and migration of breast cancer cells was investigated using gain- and loss-of-function assays in vitro. Additionally, the potential early diagnosis and treatment monitoring marker of miR-1260b was validated in 129 plasma samples. We found that high miR-1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high-miR-1260b-expression group showed shorter overall survival than the low-miR-1260b-expression group. The inhibition of oncogenic miR-1260b induced apoptosis and decreased migration and invasion of MDA-MB-231 cells. CASP8 was revealed as a direct target gene of miR-1260b, which is closely related to apoptosis. Furthermore, miR-1260b expression levels in plasma were significantly higher in patients with breast cancer than in healthy controls. The patients who tested positive for miR-1260b showed 16.3- and 18.2-fold higher risks in the early stage and locally advanced stage, respectively, compared with healthy controls, and the risk was decreased 6.2-fold after neoadjuvant chemotherapy. Taken together, miR-1260b may be a potential novel diagnostic, prognostic, and therapeutic target in breast cancer.

Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell.

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The carbonyl products generated from singlet oxygen-mediated photooxidation of Cy5 undergo a sequence of carbon-carbon bond-breaking and -forming events to bring about the novel dye-to-dye transformation. We also show that the deletion of a two-methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647) and Cyanine5.5. The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. We demonstrate that the potentially deleterious photoconversion, however, can be exploited to develop a new photoactivation method for high-density single-particle tracking in a living cell without using UV illumination and cell-toxic additives.

Validation and assessment of the Investigator® 24plex QS kit for forensic casework application: Comparison with the PowerPlex® fusion system and GlobalFiler™ PCR amplification kits.

Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/µL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples.

Comparison of the Prognostic Factors of Fetuses With Congenital Pulmonary Airway Malformations According to Type.

OBJECTIVES: To compare the prognostic factors of fetuses with microcystic and macrocystic congenital pulmonary airway malformations (CPAMs). METHODS: We retrospectively evaluated fetuses with CPAMs at Asan Medical Center. The CPAM size, mass effect, and maximum cyst size in macrocystic CPAMs were evaluated prenatally. The adverse postnatal outcomes, including respiratory symptoms, mechanical ventilation, and surgery, were evaluated. RESULTS: In 118 cases, 2 fetal deaths and 1 neonatal death occurred. All cases of fetal hydrops and complete regression after birth were in the macrocystic and microcystic CPAM groups, respectively. Twenty-four neonates (20.7%) had respiratory symptoms, and 18 (15.5%) required mechanical ventilation. Sixty-three neonates (54.3%) underwent surgery, of whom 21 (33.3%) required surgery in the neonatal period. The maximum congenital pulmonary airway malformation volume ratio was significantly associated with all postnatal outcomes (P < .05), and the optimal cutoff values were lower for respiratory symptoms, mechanical ventilation, and neonatal surgery in the macrocystic CPAMs. The maximum cyst size was also associated with all postnatal outcomes in macrocystic CPAMs (P < .05). CONCLUSIONS: Different cutoff values for the maximum congenital pulmonary airway malformation volume ratio should be applied according to the CPAM type for the prediction of postnatal outcomes. The maximum cyst size can also be a useful prognostic factor in macrocystic CPAMs.

Inhibitory Effects of Menadione on Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation via NF-κB Inhibition.

H. pylori is classified as a group I carcinogen by WHO because of its involvement in gastric cancer development. Several reports have suggested anti-bacterial effects of menadione, although the effect of menadione on major virulence factors of H. pylori and H. pylori-induced inflammation is yet to be elucidated. In this study, therefore, we demonstrated that menadione has anti-H. pylori and anti-inflammatory effects. Menadione inhibited growth of H. pylori reference strains and clinical isolates. Menadione reduced expression of vacA in H. pylori, and translocation of VacA protein into AGS (gastric adenocarcinoma cell) was also decreased by menadione treatment. This result was concordant with decreased apoptosis in AGS cells infected with H. pylori. Moreover, cytotoxin-associated protein A (CagA) translocation into H. pylori-infected AGS cells was also decreased by menadione. Menadione inhibited expression of several type IV secretion system (T4SS) components, including virB2, virB7, virB8, and virB10, that are responsible for translocation of CagA into host cells. In particular, menadione inhibited nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and thereby reduced expression of the proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α in AGS as well as in THP-1 (monocytic leukemia cell) cell lines. Collectively, these results suggest the anti-bacterial and anti-inflammatory effects of menadione against H. pylori.

Validation of Reduced Reagent Volumes in the Implementation of the Quantifiler® Trio Quantification Kit.

The Quantifiler® Trio Quantification Kit has been developed to quantify the total amount of amplifiable and human male DNA in samples and to estimate the extent of DNA degradation. To minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/10-volume) using DNA samples of varying types and concentrations. Our results demonstrated concordance between the manufacturer's method and the low-volume method for DNA quantification, DNA degradation index estimation, and human male DNA quantification. We confirmed the practical utility of the low-volume method with 109 casework samples by evaluating short tandem repeat (STR) profiling success with respect to DNA quantity and quality. We also defined a cutoff value for DNA quantity to ensure reliable STR results. Using a reduced volume of reagents, 10 times more reactions per kit are possible; accordingly, this method reduces the cost of DNA quantification, while maintaining performance.

A triple-function nanotube as a reactant reservoir, reaction platform, and byproduct scavenger for photo-cyclopropanation.

Herein, we report the advanced-concept triple-functionality of a metal-organic nanotube (MONT), which acts as a reservoir for unstable reactants, a photoreaction platform, and a scavenger for byproduct iodine. Self-assembly of CdI2 with a new Y-type ligand (L) produces the substantial 1D MOF, [CdI2(L)], thus forming a thick nanotube with a 1.4 nm diameter.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1.

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase.

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.

Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues.

Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.