Salinomycin suppresses TGF-ß1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer.

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-ß1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-ß1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-ß1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-ß1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-ß1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-ß1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-ß1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Loss of abcd4 in zebrafish leads to vitamin B12-deficiency anemia.

ABCD4, a member of the ATP-binding cassette transporter superfamily, is associated with the transport of vitamin B12 which is crucial for the development of red blood cells (RBCs) and may also be involved in its metabolism. However, the molecular function of ABCD4 during RBC development in zebrafish is mostly unknown. Using a morpholino-based knockdown approach, we found that abcd4-knockdown resulted in abnormal RBCs of irregular shapes and various sizes. o-Dianisidine staining, as an indicator of hemoglobin in RBCs, further confirmed that abcd4 morphants possessed fewer hemoglobinized cells and impaired blood circulation. Multiple protein sequence alignment revealed that the amino acid sequence for residues 13-292, which is the domain of vitamin B12 transport, of the zebrafish Abcd4 was highly conserved compared to that of other species. Accordingly, the abcd4 morphants can be rescued with human ABCD4, demonstrating a conserved role of ABCD4 in vertebrates. Notably, the vitamin B12-deficient phenotype in abcd4 morphants, which causes anemia, was recapitulated in the newly-established abcd4 mutant, indicating the possibility that the abcd4 mutant could be used as a disease model of vitamin B12-deficiency anemia. Our study provides an insight that the analysis of the newly-established abcd4 mutant may contribute to understanding its roles in ABCD4-related vitamin B12-deficiency anemia and the associated pathogeneses in humans.

Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells.

BACKGROUND: Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action. METHODS: Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin. CONCLUSIONS: Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

HNRNPA1, a Splicing Regulator, Is an Effective Target Protein for Cervical Cancer Detection: Comparison With Conventional Tumor Markers.

OBJECTIVE: Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), serine/arginine-rich splicing factor 1 (SRSF1), and SRSF3 are splicing regulators associated with oncogenesis. However, the alterations of SF proteins and their diagnostic values in cervical cancer are unclear. To apply SFs clinically, effective marker selection and characterization of the target organ properties are essential. MATERIALS AND METHODS: We concurrently analyzed HNRNPA1, SRSF1, SRSF3, and the conventional tumor markers squamous cell carcinoma antigen (SCCA) and carcinoembryonic antigen (CEA) in cervical tissue samples (n = 127) using semiquantitative immunoblotting. In addition, we compared them with p16 (cyclin-dependent kinase inhibitor 2A [CDKN2A]), which has shown high diagnostic efficacy in immunohistochemical staining studies and has been proposed as a candidate protein for point-of-care screening biochemical tests of cervical neoplasia. RESULTS: HNRNPA1, higher molecular weight forms of SRSF1 (SRSF1-HMws), SRSF3, CEA, and p16 levels were higher (P < 0.05) in cervical carcinoma tissue samples than in nontumoral cervical tissue samples. However, the levels of SRSF1-Total (sum of SRSF1-HMws and a lower molecular weight form of SRSF1) and SCCA, a commonly used cervical tumor marker, were not different between carcinoma and nontumoral tissue samples. In paired sample comparisons, HNRNPA1 (94%) showed the highest incidence of up-regulation (carcinoma/nontumor, >1.5) in cervical carcinoma, followed by p16 (84%), SRSF1-HMws (69%), SRSF3 (66%), CEA (66 %), SCCA (32%), and SRSF1-Total (31%). HNRNPA1 (92%) and p16 (91%) presented the two highest diagnostic accuracies for cervical carcinoma, which were superior to those of SRSF3 (75%), SRSF1-HMws (72%), CEA (72%), SCCA (59%), and SRSF1-Total (55%). CONCLUSIONS: Our results identified that HNRNPA1 is the best diagnostic marker among the SFs and conventional markers given its excellent diagnostic efficacy for cervical carcinoma, and it has a p16-comparable diagnostic value. We suggest that HNRNPA1 is an additional effective target protein for developing cervical cancer detection tools.

NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction.

NSrp70 (nuclear speckle-related protein 70), a recently discovered protein and it belongs to the serine/arginine (SR) rich related protein family. NSrp70 is recognized as an important splicing factor comprising RNA recognition motif (RRM) and arginine/serine (RS)-like regions at the N- and C-terminus respectively, along with two coiled coil domains at each terminus. However, other functions of NSrp70 remain unelucidated. In this study, we investigated the role of NSrp70 in Xenopus embryogenesis and found that its maternal expression plays a critical role in embryonic development. Knockdown of NSrp70 resulted in dramatic reduction in the length of developing tadpoles and mild to severe malformation in Xenopus embryos. In addition, knockdown of NSrp70 resulted in an extremely short axis by blocking gastrulation and convergent extension. Further, animal cap assays along with activin A treatment revealed that NSrp70 is an essential factor for dorsal mesoderm induction as knockdown of NSrp70 caused a dramatic down-regulation of dorsal mesoderm specific genes and its loss significantly shortened the elongation region of animal caps. In conclusion, NSrp70 is crucial for early embryonic development, influencing gastrulation and mesoderm induction.

The splicing factor SRSF1 modulates pattern formation by inhibiting transcription of tissue specific genes during embryogenesis.

Alternative splicing is a major mechanism regulating pattern of gene expression through the production of multiple mRNAs from a single gene transcript. Any misregulation can cause various human diseases and also have severe effects on embryogenesis. SRSF1 is one of the critical factors regulating alternative splicing at many stages of vertebrate development and any disturbance in SRSF1 leads to serious consequences. In current study, we investigated the effects of loss of the SRSF1 gene using antisense morpholino oligonucleotides (MO) in Xenopus embryogenesis. It is evident from the results of RT-PCR and whole-mount in situ hybridization that SRSF1 is a maternal gene having strong expression in head, eyes and central nervous system. Moreover, SRSF1 morphants exhibited malformed phenotypes, including miscoiled guts, heart and cartilage formation, edema in the head and heart, and small eyes. Especially, in SRSF1 morphants, bone cartilage formation was reduced in the brain and Nkx-2.5 expression was dramatically reduced in the heart of SRSF1 morphants. In addition, a dramatic reduction in functional chordin RNA in SRSF1 morphants was observed suggesting that chordin is one of the targets of SRSF1. Thus, we concluded that SRSF1 is an essential factor for pattern formation including heart, cartilage and germ layers through the regulation of specific genes.

Comparative expression patterns and diagnostic efficacies of SR splicing factors and HNRNPA1 in gastric and colorectal cancer.

BACKGROUND: Serine/arginine-rich splicing factors (SRSFs) and HNRNPA1 have oncogenic properties. However, their proteomic expressions and practical priority in gastric cancer (GC) and colorectal cancer (CRC) are mostly unknown. To apply SFs in clinics, effective marker selection and characterization of properties in the target organ are essential. METHODS: We concurrently analyzed SRSF1, 3, and 5-7, and HNRNPA1, together with the conventional tumor marker carcinoembryonic antigen (CEA), in stomach and colorectal tissue samples (n = 420) using semiquantitative immunoblot, subcellular fractionation, and quantitative real-time polymerase chain reaction methods. RESULTS: In the semiquantitative immunoblot analysis, HNRNPA1 and SRSF7 levels were significantly higher in GC than in gastric normal mucosa, and SRSF7 levels were higher in intestinal-type compared with diffuse-type of gastric adenocarcinoma. Of the SFs, only HNRNPA1 presented greater than 50 % upregulation (cancer/normal mucosa > 2-fold) incidences and CEA-comparable, acceptable (>70 %) detection accuracy (74 %) for GC. All SF protein levels were significantly higher in CRC than in colorectal normal mucosa, and HNRNPA1 levels were higher in low-stage CRC compared with high-stage CRC. Among the SFs, HNRNPA1 and SRSF3 presented the two highest upregulation incidences (88 % and 74 %, respectively) and detection accuracy (90 % and 84 %, respectively) for CRC. The detection accuracy of HNRNPA1 was comparable to that of CEA in low (≤ II)-stage CRC but was inferior to that of CEA in high (>II)-stage CRC. Extranuclear distributions of HNRNPA1 and SRSF6 (cytosol/microsome) differed from those of other SRSFs (membrane/organelle) in both cancers. In an analysis of the six SF mRNAs, all mRNAs presented unacceptable detection accuracies (≤70 %) in both cancers, and all mRNAs except SRSF6 were disproportionate to the corresponding protein levels in GC. CONCLUSION: Our results provide a comprehensive insight into the six SF expression profiles in GC and indicate that, among the SFs, HNRNPA1, but not HNRNPA1 mRNA, is the most effective, novel GC marker. Regardless of the good to excellent detection accuracy of SRSF3 and HNRNPA1 in CRC, the SFs have lower practical priority than CEA, especially for high-stage CRC detection.

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR.

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of ß-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-ß-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-ß-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib-simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/ß-catenin signaling-dependent down-regulation of survivin and apoptosis induction.

Analysis of patient health questionnaire-9 (PHQ-9) based depression prevalence according to a discordance between quantitative urinary cotinine levels and self-report of second-hand smoke exposure among adults: A cross-sectional study.

Background: Second-hand smoke (SHS) exposure appears to be more common among individuals with depression. However, self-report of SHS exposure is an inaccurate classification compared to confirming SHS exposure using urinary cotinine (UC). Additionally, the dose-response relationship between depression and UC is controversial. Methods: The severe stress rate and depression prevalence was estimated among 14530 Korean participants aged ≥19 years using data patient health questionnaire-9 (PHQ-9) and on UC from the Korean National Health and Nutrition Examination Survey. Measured UCs were divided into four categories: UC- (≤0.3 µg/L), UC± (0.4 µg/L-0.9 µg/L), UC+ (1.0 µg/L-11.9 µg/L), and UC++ (≥12.0 µg/L). Results: About 55.0 % participants were female and participants' mean age was 51.1 years. Non-smokers were 80.3 %. Among non-smokers, non-SHS exposure participants (SR-) and SHS exposure participants (SR+) were 83.0 % and 17.0 %, respectively. When UC- was used as the reference subgroup, the UC++ subgroup showed a higher depression prevalence, whereas the UC ± subgroup showed a lower prevalence. In the same UC categories, the depression prevalence and severe stress rate were higher among females than among males. Furthermore, the SR + subgroup had a higher severe stress rate than the SR- subgroup. Conclusions: Our study showed a paradoxical reduction in the depression prevalence and severe stress rate in the UC ± subgroup compared to the UC- subgroup. Additionally, the dose-response relationship between the SHS exposure biomarker and the depression prevalence was not linear. Our study indicates that an emotional stress-based model may be more appropriate for explaining the relationship between depression and SHS exposure.

Fenofibrate, a peroxisome proliferator-activated receptor α ligand, prevents abnormal liver function induced by a fasting-refeeding process.

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is an anti-hyperlipidemic agent that has been widely used in the treatment of dyslipidemia. In this study, we examined the effect of fenofibrate on liver damage caused by refeeding a high-fat diet (HFD) in mice after 24h fasting. Here, we showed that refeeding HFD after fasting causes liver damage in mice determined by liver morphology and liver cell death. A detailed analysis revealed that hepatic lipid droplet formation is enhanced and triglyceride levels in liver are increased by refeeding HFD after starvation for 24h. Also, NF-κB is activated and consequently induces the expression of TNF-α, IL1-ß, COX-2, and NOS2. However, treating with fenofibrate attenuates the liver damage and triglyceride accumulation caused by the fasting-refeeding HFD process. Fenofibrate reduces the expression of NF-κB target genes but induces genes for peroxisomal fatty acid oxidation, peroxisome biogenesis and mitochondrial fatty acid oxidation. These results strongly suggest that the treatment of fenofibrate ameliorates the liver damage induced by fasting-refeeding HFD, possibly through the activation of fatty acid oxidation.

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase.

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Levels of YKL-40 in pleural effusions and blood from patients with pulmonary or pleural disease.

BACKGROUND: YKL-40 (a chitinase-like protein) is an inflammatory biomarker that is associated with lung injury pathogenesis. We aimed to identify the diagnostic values of YKL-40 in pleural effusions and to evaluate circulating YKL-40 levels during multiple etiological pulmonary/pleural diseases and the role of YKL-40 as a monitoring marker of inflammatory pulmonary disease. METHODS: Pleural YKL-40 (n=197), YKL-39 (the most homologous chitinase-like protein to human YKL-40), and conventional pleural marker levels were measured in patients with pulmonary/pleural disease. Additionally, serum YKL-40 and YKL-39 levels were analyzed in both patients and controls (n=432) and serially monitored in patients with asthma (n=27) or pneumonia (n=22). RESULTS: Pleural YKL-40 levels were higher than those in the serum and highest in tuberculous pleural effusions (TPEs; 1181 ng/mL), followed by parapneumonic, malignant, and cardiogenic effusions (560 ng/mL). The diagnostic accuracy of pleural YKL-40 (0.78) for discriminating between tuberculous and malignant effusion was comparable to or greater than those of YKL-39, total protein, C-reactive protein and CYFRA 21-1, and lower than those of adenosine deaminase (p<0.05) and carcinoembriogenic antigen (p=0.05). Serum YKL-40 levels were higher in the pneumonia group than in the cancer, asthma, or control groups. Following treatment, serum YKL-40 levels were more greatly reduced in pneumonia patients than in asthma patients. Serum YKL-39 levels did not differ between patients and controls. CONCLUSIONS: Pleural YKL-40 levels are elevated in TPEs and have fairly good diagnostic efficacy for detecting TPEs. However, adenosine deaminase is more efficient for detecting TPEs than pleural YKL-40. Serum YKL-40 levels are highest during pneumonia compared to common pulmonary/pleural diseases and are more useful for monitoring pneumonia than asthma.

Tuberculous otitis media with endobronchial tuberculosis.

Although tuberculosis can affect various organs and tissues, the lung is the site most commonly involved. Extrapulmonary tuberculosis (EPTB) involves relatively inaccessible and variable sites and is consequently often overlooked by clinicians. The ear is a notably very rare site of EPTB, and the diagnosis is difficult because of the variable and confusing signs and symptoms. To our knowledge, this is the first case in which tuberculous otitis media and endobronchial tuberculosis coexisted.

Effect of Second-Hand Smoke Exposure on Establishing Urinary Cotinine-Based Optimal Cut-Off Values for Smoking Status Classification in Korean Adults.

Regulations for banning smoking in indoor public places and workplaces have increased worldwide in recent years. A consecutive Korean National Health and Nutrition Examination Survey (KNHANES) between 2008 and 2018 showed a trend toward significant decreases in self-reported tobacco smoke exposure and measured urinary cotinine concentrations. We established and compared each optimal cut-off value for assessing the effect of second-hand smoke (SHS) exposure on establishing urinary cotinine-based cut-off values for smoking status classification in a population setting controlled for racial and cultural diversity, using four KNHANES datasets consisting of the 2008, 2011, 2014, and 2018 surveys. A total of 18,229 Korean participants aged >19 years with measured urinary cotinine concentrations were enrolled. Self-reports of current smoking status showed that the prevalence of current smokers decreased from 22.9% to 18.2% between 2008 and 2018. During this period, the median value of urinary cotinine in nonsmokers decreased from 5.86 µg/L to 0.48 µg/L, whereas the median value showed no remarkable decrease in current smokers. The AUC-based optimal cut-off values of urinary cotinine concentration for distinguishing current smokers from nonsmokers decreased from 86.5 µg/L to 11.5 µg/L. Our study showed that decreased SHS exposure would result in decreased optimal cut-off values for distinguishing current smokers from nonsmokers. In addition, the study suggests that the range of urinary cotinine concentration to define SHS exposure for the trend monitoring of populationof SHS exposure is appropriate between 0.30 µg/L and 100 µg/L. In addition, our study showed the importance of determination of cotinine concentration, which would have allowed us to avoid mistakes in qualification to the study group in an increased use of e-cigarette setting.

Anti-Obesity Effects of Traditional and Commercial Kochujang in Overweight and Obese Adults: A Randomized Controlled Trial.

Kochujang shows anti-obesity effects in cell and animal models. Kochujang is traditionally prepared via slow fermentation or commercially using Aspergillus oryzae. We analyze the anti-obesity effects of two types of Kochujang in overweight and obese adults. The analyses included the following groups: traditional Kochujang containing either a high-dose (HTK; n = 19), or a low-dose of beneficial microbes (LTK; n = 18), and commercial Kochujang (CK; n = 17). Waist circumference decreased significantly in the HTK and CK groups. Total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels decreased in the HTK and LTK groups. Visceral fat is significantly reduced in the HTK group. The population of beneficial microorganisms in stool samples increased in all groups. Consumption of Kochujang reduces visceral fat content and improves the lipid profile, which can be enhanced by enrichment with beneficial microbes. These results suggest that Kochujang has the potential for application in obesity prevention.

Apoptotic induction by simvastatin in human lung cancer A549 cells via Akt signaling dependent down-regulation of survivin.

Statins, HMG-CoA reductase inhibitors have been studied for their antiproliferative and proapototic effects. Recently, statin-induced apoptosis has been associated with down-regulation of survivin expression in cancer cells. However, the mechanism of deregulated survivin by simvastatin on lung cancer is still unclear. Herein, we demonstrated that simvastatin induced caspase-dependent apoptosis in A549 lung cancer cells. Simvastatin also resulted in a decrease in the expression of phosphorylated Akt. In addition, simvastatin effectively down-regulated survivin mRNA and protein, but not cIAP-1 and cIAP-2. The combination of simvastatin and 10 µM LY294002 (non-toxic dose) augmented apoptosis significantly, as evidenced by cleavage of PARP. The immunoreactive band of survivin was markedly decreased in cells treated with 50 µM LY294002 (toxic dose) as well as by the combination of simvastatin and 10 µM LY294002. Moreover, survivin down-regulation by RNA interference induced apoptosis accompanied by an increase in hypodiploid DNA content. Taken together, these data suggest that the anti-cancer effect of simvastatin via induction of apoptosis is related to Akt signaling dependent down-regulation of survivin in lung cancer A549 cells.

[Distribution of hepatitis B virus genotypes in Korea].

BACKGROUND/AIMS: Considering the incidence of prevailing hepatitis B virus (HBV) genotypes in neighboring nations, the predominance of genotype C in Korea is exceptional and needs to be confirmed by nationwide investigation. METHODS: A total of 510 HBsAg (+) or HBeAg (+) serum samples was collected from subjects in several cities and harbors throughout the Korean peninsula for genotype (A-G)-specific multiplex PCR analysis. Another 40 serum samples from chronic HBV carriers from Iksan city were selected for sequencing of the entire HBV genome. Phylogenetic analysis was performed with 22 whole genomic sequences of Korean HBV strains enrolled in GenBank. RESULTS: An amplicon was found in 377 specimens and genotype C occupied 98.1% (370 cases); none of the other genotypes were found. A mixed pattern of genotypes B and C was seen in seven specimens (1.9%), of which five were tested using PCR targeting the X fragment; no genotype B bands were found. With the exception of 1 case, which was subgenotype A2, whole sequences of Korean HBV strains (n=62) belonged to subgenotype C2. CONCLUSIONS: The prevailing HBV genotype in Korea is C2; the other genotypes occur only rarely. Future studies should include confirmation of the detection of genotypes other than C.

Comparison of cobas EGFR Mutation Test v2 and PANAMutyper-R-EGFR for Detection and Semi-Quantification of Epidermal Growth Factor Receptor Mutations in Plasma and Pleural Effusion Supernatant.

BACKGROUND: Plasma epidermal growth factor receptor (EGFR) mutation tests are less invasive than tissue EGFR mutation tests. We determined which of two kits is more efficient: cobas EGFR Mutation test v2 (cobasv2; Roche Molecular Systems, Pleasanton, CA, USA) or PANAMutyper-R-EGFR (Mutyper; Panagene, Daejeon, Korea). We also evaluated whether pleural effusion supernatant (PE-SUP) samples are assayable, similar to plasma samples, using these two kits. METHODS: We analyzed 156 plasma and PE-SUP samples (31 paired samples) from 116 individuals. We compared the kits in terms of accuracy, assessed genotype concordance (weighted κ with 95% confidence intervals), and calculated Spearman's rho between semi-quantitatively measured EGFR-mutant levels (SQIs) measured by each kit. We also compared sensitivity using 47 EGFR-mutant harboring samples divided into more-dilute and less-dilute samples (dilution ratio: ≥ or <1:1,000). RESULTS: cobasv2 tended to have higher accuracy than Mutyper (73% vs 69%, P=0.53), and PE-SUP samples had significantly higher accuracy than plasma samples (97% vs 55-71%) for both kits. Genotype concordance was 98% (κ=0.92, 0.88-0.96). SQIs showed strong positive correlations (P<0.0001). In less-dilute samples, accuracy and sensitivity did not differ significantly between kits. In more-dilute samples, cobasv2 tended to have higher sensitivity than Mutyper (43% vs 20%, P=0.07). CONCLUSIONS: The kits have similar performance in terms of EGFR mutation detection and semi-quantification in plasma and PE-SUP samples. cobasv2 tends to outperform Mutyper in detecting less-abundant EGFR-mutants. PE-SUP samples are assayable using either kit.

Correlation between insulin resistance and intracranial atherosclerosis in patients with ischemic stroke without diabetes.

BACKGROUND: Insulin resistance (IR) is associated with an increased risk for cardiovascular morbidity and mortality including ischemic stroke. Its final complications are cardiovascular and cerebrovascular disease caused by atherosclerosis. However, few studies on the relationship between IR and intracranial (IC) atherosclerosis have been reported. METHODS: We analyzed 110 patients with acute stroke without diabetes who underwent brain magnetic resonance angiography and cerebral angiography. Patients were divided into 3 equal groups according to the tertiles of homeostasis model assessment of IR (HOMA-IR): group I (n = 36; HOMA-IR < 0.92), group II (n = 37; 0.92 or= 1.55). Cerebral artery atherosclerosis was classified as either IC or extracranial (EC). RESULTS: Patients with IC or EC atherosclerosis showed higher level of HOMA-IR than those without. When the association was assessed according to the site of atherosclerosis, HOMA-IR showed a significant association with the site of atherosclerosis (IC + EC > IC > EC, P < .01). Multivariate analysis revealed that HOMA-IR was an independent predictor of IC atherosclerosis. CONCLUSIONS: Although the association between IR and stroke patterns in patients with atherosclerosis remains uncertain, IR is associated with IC atherosclerosis in patients with acute ischemic stroke without diabetes.

Expression Profile of Three Splicing Factors in Pleural Cells Based on the Underlying Etiology and Its Clinical Values in Patients with Pleural Effusion.

Splicing factors (SFs) are involved in oncogenesis or immune modulation, the common underlying processes giving rise to pleural effusion (PE). The expression profiles of three SFs (HNRNPA1, SRSF1, and SRSF3) and their clinical values have never been assessed in PE. The three SFs (in pellets of PE) and conventional tumor markers were analyzed using PE samples in patients with PE (N = 336). The sum of higher-molecular weight (Mw) forms of HNRNPA1 (Sum-HMws-HNRNPA1) and SRSF1 (Sum-HMws-SRSF1) and SRSF3 levels were upregulated in malignant PE (MPE) compared to benign PE (BPE); they were highest in cytology-positive MPE, followed by tuberculous PE and parapneumonic PE. Meanwhile, the lowest-Mw HNRNPA1 (LMw-HNRNPA1) and SRSF1 (LMw-SRSF1) levels were not upregulated in MPE. Sum-HMws-HNRNPA1, Sum-HMws-SRSF1, and SRSF3, but neither LMw-HNRNPA1 nor LMw-SRSF1, showed positive correlations with cancer cell percentages in MPE. The detection accuracy for MPE was high in the order of carcinoembryonic antigen (CEA, 85%), Sum-HMws-HNRNPA1 (76%), Sum-HMws-SRSF1 (68%), SRSF3, cytokeratin-19 fragments (CYFRA 21-1), LMw-HNRNPA1, and LMw-SRSF1. Sum-HMws-HNRNPA1 detected more than half of the MPE cases that were undetected by cytology and CEA. Sum-HMws-HNRNPA1, but not other SFs or conventional tumor markers, showed an association with longer overall survival among patients with MPE receiving chemotherapy. Our results demonstrated different levels of the three SFs with their Mw-specific profiles depending on the etiology of PE. We suggest that Sum-HMws-HNRNPA1 is a supplementary diagnostic marker for MPE and a favorable prognostic indicator for patients with MPE receiving chemotherapy.