GenoMycAnalyzer: a web-based tool for species and drug resistance prediction for Mycobacterium genomes.

BACKGROUND: Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance prediction, and many clinical laboratories are transitioning to WGS as a routine diagnostic tool. However, user-friendly and high-confidence automated bioinformatics tools are needed to rapidly identify M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), detect drug resistance, and further guide treatment options. RESULTS: We developed GenoMycAnalyzer, a web-based software that integrates functions for identifying MTBC and NTM species, lineage and spoligotype prediction, variant calling, annotation, drug-resistance determination, and data visualization. The accuracy of GenoMycAnalyzer for genotypic drug susceptibility testing (gDST) was evaluated using 5,473 MTBC isolates that underwent phenotypic DST (pDST). The GenoMycAnalyzer database was built to predict the gDST for 15 antituberculosis drugs using the World Health Organization mutational catalogue. Compared to pDST, the sensitivity of drug susceptibilities by the GenoMycAnalyzer for first-line drugs ranged from 95.9% for rifampicin (95% CI 94.8-96.7%) to 79.6% for pyrazinamide (95% CI 76.9-82.2%), whereas those for second-line drugs ranged from 98.2% for levofloxacin (95% CI 90.1-100.0%) to 74.9% for capreomycin (95% CI 69.3-80.0%). Notably, the integration of large deletions of the four resistance-conferring genes increased gDST sensitivity. The specificity of drug susceptibilities by the GenoMycAnalyzer ranged from 98.7% for amikacin (95% CI 97.8-99.3%) to 79.5% for ethionamide (95% CI 76.4-82.3%). The incorporated Kraken2 software identified 1,284 mycobacterial species with an accuracy of 98.8%. GenoMycAnalyzer also perfectly predicted lineages for 1,935 MTBC and spoligotypes for 54 MTBC. CONCLUSIONS: GenoMycAnalyzer offers both web-based and graphical user interfaces, which can help biologists with limited access to high-performance computing systems or limited bioinformatics skills. By streamlining the interpretation of WGS data, the GenoMycAnalyzer has the potential to significantly impact TB management and contribute to global efforts to combat this infectious disease. GenoMycAnalyzer is available at http://www.mycochase.org .

An innovative strategy harnessing self-activating CAR-NK cells to mitigate TGF-ß1-driven immune suppression.

The dysfunction of natural killer (NK) cells, mediated by transforming growth factor ß1 (TGFß1) within the tumor microenvironment, impedes antitumor therapy and contributes to poor clinical outcomes. Our study introduces self-activating chimeric antigen receptor (CAR)-NK cells that block TGFß1 signaling by releasing a specifically designed peptide, P6, which targets mesothelin in pancreatic tumors. P6 originates from the interaction sites between TGFß1 and TGFß receptor 1 and effectively disrupts TGFß1's inhibitory signaling in NK cells. Our analysis demonstrates that P6 treatment interrupts the SMAD2/3 pathway in NK cells, mitigating TGFß1-mediated suppression of NK cell activity, thereby enhancing their metabolic function and cytotoxic response against pancreatic tumors. These CAR-NK cells exhibit potent antitumor capabilities, as evidenced in spheroid cultures with cancer-associated fibroblasts and in vivo mouse models. Our approach marks a substantial advancement in overcoming TGFß1-mediated immune evasion, offering a promising avenue for revolutionizing cancer immunotherapy.

Respiratory protection strategies for the public in emergency response.

A strategy was established to minimize the stochastic effects of internal and external exposure to radioactive substances by wearing respiratory protection equipment (RPE) during an emergency evacuation. During the evacuation of residents in the event of a nuclear power plant accident, the stochastic effects of internal exposure caused by the inhalation of radioactive aerosol and external exposure due to accumulated radioactive particles in the filter medium of a mask must be minimized. The radioactivity concentration along an evacuation route considers atmospheric dispersion and the resuspension of particles deposited on surfaces. The effective dose due to internal exposure is evaluated using inhalation dose coefficients for each particle diameter. When the face seal leakage and filter medium penetration ratio for each particle diameter of the RPE (N95) is taken into consideration, the internal dose is reduced by 97.2%. Furthermore, the accumulated radioactivity in the filter medium decreases by 91.4% when the respirator is replaced every 48 h.

Dead layer estimation of an HPGe detector using MCNP6 and Geant4.

In this study, Monte Carlo simulations were used to calculate the full-energy peak efficiency of a p-type coaxial high-purity germanium (HPGe) detector. The HPGe detector was modeled using MCNP6 and Geant4, and the thickness of the dead layer of germanium crystals was estimated for an accurate simulation. The dead layer was divided into front and side components, where a point source and a Marinelli beaker source were used to estimate each dead layer thickness. The model was validated by comparing the simulated as well as experimental results for the standard sources of cylindrical and Marinelli beakers. The Geant4 results and experimental results matched up to 4% in the 59.54-1836.05 keV energy range, while MCNP6 matched up to 6% when adjusted for coincidence summing effects. HPGe detector modeled in Monte Carlo simulations can be utilized for experimental validation and experimental setup prior to using actual HPGe detectors.

Semi-mechanistic analysis of emergency planning zones for 20 MWe lead-cooled fast reactor by hypothetical accidents during Korea's arctic exploration.

Small modular reactors or micro modular reactors have been considered as an alternative power source for merchant ships because of minimal carbon emission and a long lifecycle without refueling. Ahead of the operation, however, their emergency planning zones (EPZs) should be optimally set and approved to ensure both radiological safety and cost efficiency in case of nuclear accidents. Thus, the required size of EPZs was analyzed based on semi-mechanistic assumptions for hypothetical accidents with MicroURANUS, a micro modular fast reactor type of 20-MW lead-bismuth-cooled, used to power an icebreaker during Korea's Arctic missions. For meteorological data, actual icebreaker data acquired in 2020s Artic exploration were utilized. The results showed that EPZ sizes, rationalized in terms of stability class, wind directions, and inherent radioprotection design, ranged within the assumed icebreaker boundary. When comparing various regulations among countries and the International Atomic Energy Agency, dose criteria of Korea (10 mSv/2 d) were found to be strictest. Since major contribution to the whole-body dose was from noble gases (over 96%), a high-quality air filtration system in addition to external shielding would significantly reduce hazards. The emergency situation could be successfully controlled without evacuation and sheltering, avoiding overestimated socioeconomic costs.

Copy number variations in East-Asian population and their evolutionary and functional implications.

Recent discovery of the copy number variation (CNV) in normal individuals has widened our understanding of genomic variation. However, most of the reported CNVs have been identified in Caucasians, which may not be directly applicable to people of different ethnicities. To profile CNV in East-Asian population, we screened CNVs in 3578 healthy, unrelated Korean individuals, using the Affymetrix Genome-Wide Human SNP array 5.0. We identified 144,207 CNVs using a pooled data set of 100 randomly chosen Korean females as a reference. The average number of CNVs per genome was 40.3, which is higher than that of CNVs previously reported using lower resolution platforms. The median size of CNVs was 18.9 kb (range 0.2-5406 kb). Copy number losses were 4.7 times more frequent than copy number gains. CNV regions (CNVRs) were defined by merging overlapping CNVs identified in two or more samples. In total, 4003 CNVRs were defined encompassing 241.9 Mb accounting for approximately 8% of the human genome. A total of 2077 CNVRs (51.9%) were potentially novel. Known CNVRs were larger and more frequent than novel CNVRs. Sixteen percent of the CNVRs were observed in > or =1% of study subjects and 24% overlapped with the OMIM genes. A total of 476 (11.9%) CNVRs were associated with segmental duplications. CNVS/CNVRs identified in this study will be valuable resources for studying human genome diversity and its association with disease.

Strategy for high-fidelity multiplex DNA copy number assay system using capillary electrophoresis devices.

Structural variation of human genome such as duplications and deletions, collectively termed copy number variation (CNV), is one of the major genetic variations. Reliable and efficient measurement of CNV will be essential to develop diagnostic tools for CNV-related diseases. We established a strategy based on multiplex PCR and capillary electrophoresis (CE) for reliable CNV assay. Multiplex-PCR was performed using five primer sets for target loci and a diploid control (DC). We designed primers satisfying three conditions: different size of each PCR product for CE separation, unified annealing temperature for multiplex PCR, and suitability for quantitative PCR (qPCR). We defined the accurate PCR cycles for quantification of copy numbers at which the amplifications for all targets were supposed to be exponential, named maximum doubling cycle. CE was carried out with PCR product and the ratio of the peak areas (target/diploid control) was calculated. Our multiplex PCR-CE analysis reliably determined copy numbers of X chromosome with variable copies ranging from 1 to 5 and showed higher reliability than qPCR (correlation coefficient 0.996 versus 0.898). When measuring the six randomly selected autosomal CNV targets using our multiplex PCR-CE, the results agreed with those from qPCR. In addition, our strategy was validated for the broad application to commonly used CE devices. Taken together, this assay will be useful for accurate analysis of multiple disease-associated CNVs in a clinical setting.

Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma.

BACKGROUND: Since hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms. METHODS: TPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays. RESULTS: Silencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells. CONCLUSIONS: Based on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway.

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array.

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Correlation of Androgen Deprivation Therapy with Cognitive Dysfunction in Patients with Prostate Cancer: A Nationwide Population-Based Study Using the National Health Insurance Service Database.

PURPOSE: The purpose of this study was to evaluate the association of androgen deprivation therapy (ADT) with cognitive dysfunction. MATERIALS AND METHODS: Using the National Health Insurance Service database of the entire Korean adult prostate cancer population (n=236,391), data on ADT and cognitive dysfunction between 2008 and 2015 were analyzed. We excluded patients previously diagnosed with cognitive dysfunction, dementia, or a cerebral event history. We tested the effect of ADT on the risk of cognitive dysfunction using propensity score-matched Cox proportional hazards regression models and Kaplan-Meier survival analysis. Our final cohort comprised of 35,401 individuals with prostate cancer, including 24,567 men (70.6%) who underwent ADT. RESULTS: During a mean follow-up period of 4.1 years, 4,741 patients were newly diagnosed with cognitive dysfunction. A statistically significant association was found between ADT and the risk of cognitive dysfunction (hazard ratio, 1.169; p=0.002). Meanwhile, age (≥ 70 years), diabetes, hypertension, cardiovascular history, and peripheral vascular disease were identified as factors that contribute to the increased risk of cognitive dysfunction. In contrast, the use of statins and aspirin was associated with a lower risk of cognitive dysfunction. Kaplan-Meier analysis demonstrated that patients aged 70 years or older who underwent ADT had the lowest cumulative probability of remaining cognitive dysfunction-free (log-rank p < 0.001). CONCLUSION: Our. RESULTS: revealed an association between the use of ADT for the treatment of prostate cancer and an increased risk of cognitive dysfunction in a nationwide population-based study. This finding should be further evaluated in prospective studies.

Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q.

To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor-related genes, using whole-genome array-CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high-grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1-q32.1 (64.5%), 1q32.1-q44 (59.2%), 8q11.21-q24.3 (48.7%) and a loss in 17p13.3-p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2-p21.1 and gain in 8q11.21-q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC.

Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of lung cancer cells detected by siRNA targeted knock-down analysis.

Molecular mechanism of lung carcinogenesis and its aggressive nature is still largely elusive. To uncover the biomarkers related with tumorigenesis and behavior of lung cancer, we screened novel differentially expressed genes (DEG) in A549 lung cancer cell line by comparison with CCD-25Lu, normal pulmonary epithelial cell line, using annealing control primer(ACP)-based GeneFishing system. Of the DEGs, over-expression of leucyl-tRNA synthetase 1 (LARS1) was prominent and this up-regulation was confirmed by immunoblotting and real-time quantitative RT-PCR analysis. In addition to A549 cell line, primary lung cancer tissues also expressed higher level of LARS1 mRNA than their normal counter tissues. To explore the oncogenic potential of LARS1 over-expression in lung cancer, we knocked-down LARS1 by treating siRNA and observed the tumor behavior. LARS1 knock-down cells showed reduced ability to migrate through transwell membrane and to form colonies in both soft agar and culture plate. Taken together, these findings suggest that LARS1 may play roles in migration and growth of lung cancer cells, which suggest its potential implication in lung tumorigenesis.

CD63 as a biomarker for predicting the clinical outcomes in adenocarcinoma of lung.

BACKGROUND: The prognosis of lung cancer is still poor, since there are few early detection tools available yet. So, it is important to identify more efficient and clinically applicable biomarkers associated with the prognosis in as earlier stages as possible. PATIENTS AND METHODS: In this study, we observed the expression of CD63 in 90 cases of non-small cell lung cancer (NSCLC) to explore the potential of this molecule as a prognostic biomarker for lung cancer subtypes using real-time quantitative RT-PCR and tissue microarray based immunohistochemistry. RESULTS: Majority of NSCLCs (75.8%) showed lower CD63 RNA level (less than half) than normal tissue. Notably, all SqCs showed low-expression except one case, while AdCs showed diverse range of expression. CD63 protein expression level was largely compatible with RNA level. Totally, 63.3% of NSCLC were CD63 protein expression negative. All SqCs were negative, while majority (70.2%) of adenocarcinomas (AdCs) were positive. CD63 protein negativity was significantly associated with SqCs type, larger tumor size, and advanced stage. In AdCs, CD63 negativity was associated with poor survival (p=0.008). This association was also significant in earlier stage (I and II) AdCs (p=0.041), but not in advanced stage AdCs. After being adjusted for age and sex by Cox regression and stratified by stages, CD63 negativity still showed borderline association with poor survival as an independent predictor (p=0.076, HR=2.3). CONCLUSIONS: Taken together, these results suggest that CD63 can be a biomarker for predicting the prognosis in earlier stage of lung AdCs. Our findings can be a clue to investigate the role of CD63 in tumorigenesis of AdCs of lung and other cancers.

DNA copy number alterations and expression of relevant genes in mouse thymic lymphomas induced by gamma-irradiation and N-methyl-N-nitrosourea.

The genetic mechanism for the development and progression of a lymphoma is unclear. This study investigated the alterations in the DNA copy number and the expression profiles of the genes located in the altered regions in mouse thymic lymphomas that were induced by two mutagens, gamma-irradiation and N-methyl-N-nitrosourea (MNU). Microarray-based comparative genomic hybridization was used to precisely delineate the boundaries of the altered region. The copy number gains of chromosomes 4 and 5 were observed only in the radiation-induced lymphomas, and gains of chromosomes 10 and 14 were observed only in the MNU-induced lymphomas. Regional copy number losses in chromosomes 11, 16, and 19 appeared frequently in the radiation-induced lymphomas. The cancer-related genes Pten, Ikaros/Znfn1a1, Ercc4, and Top3b were located in the minimal deletion regions. In particular, the expression levels of the Pten, Top3b, and Ikaros genes were downregulated in both lymphoma groups, but the expression level of Ercc4 was downregulated only in the MNU group. This study also examined the expression levels of Sparc, Cxcl1, and Myc (synonym: c-Myc), which are located in the copy number gained chromosomes. Sparc was upregulated specifically in the radiation group, and Cxcl1 in the MNU group. c-Myc was upregulated in both groups. There was limited correlation between the DNA copy number profiles and the expression of the cancer-related genes in mouse lymphomagenesis. The chromosome aberrations and novel expression profiles of the cancer-related genes within the altered regions may provide important clues to the genetic mechanism for the development of lymphoma.

Effects of ginseng on peripheral blood mitochondrial DNA copy number and hormones in men with metabolic syndrome: A randomized clinical and pilot study.

BACKGROUND: It has been observed that mitochondrial dysfunction is associated with an increased risk of metabolic syndrome. There is growing evidence that hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and hormone (testosterone and growth hormone) deficiency may lead to metabolic syndrome. Recent studies have reported that ginseng treatment improves mitochondrial and HPA-axis function and increases anabolic hormone secretion. OBJECTIVES: The objective of this study was to investigate the effect of red ginseng (RG) on metabolic syndrome, hormones, and mitochondrial function using leukocyte mitochondrial DNA copy number in men with metabolic syndrome. METHODS: We performed a randomized, double-blind, placebo-controlled study in 62 subjects who were not taking drugs that could affect their metabolic function. A total of 62 men with metabolic syndrome were randomly assigned to either an RG group (3.0g/day) or a placebo group for 4 weeks. We analyzed changes in metabolic syndrome components, leukocyte mitochondrial DNA copy number, hormones (total testosterone, IGF-1, cortisol, and DHEAS) and inflammatory markers (C-reactive protein, ferritin) from baseline to 4 weeks. RESULTS: Significant improvement in mitochondrial function (95% CI -44.9 to -1.3) and an increase in total testosterone (95% CI -70.1 to -1.0) and IGF-1(P=0.01) levels were observed in the RG group when compared with the placebo group. Diastolic blood pressure (95% CI 2.0-9.4) and serum cortisol (95% CI 1.1-5.5) significantly decreased in the RG group. CONCLUSIONS: We found evidence that RG had a favorable effect on mitochondrial function and hormones in men with metabolic syndrome.

Increased viability of PC12 cells exposed to amyloid-beta peptide by transduction with human TAT-methionine sulfoxide reductase.

Methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide to methionine, which is able to scavenge oxidatively damaged proteins. Oxidative stress has been linked to the pathophysiology of Alzheimer's disease, and a decrease in MsrA activity has also been implicated in Alzheimer's disease. The transactivator of transcription (TAT) protein from human immunodeficiency virus 1 has been used to deliver full-length proteins into mammalian cells. We produced genetic in-frame TAT-MsrA fusion protein and successfully transduced it into PC12 cells, where it showed enzymatic activity. We showed that transduction of TAT-MsrA increased cell viability and reduced DNA fragmentation in PC12 cells treated with amyloid-beta (A beta). We suggest that MsrA transduction could reduce the oxidative damage caused to cellular proteins by A beta and could play a role in the treatment of Alzheimer's disease.

Zn2+-induced cell death is mediated by the induction of intracellular ROS in ARPE-19 cells.

PURPOSE: Recent studies have shown that Zn2+ induced cell death in retinal pigment epithelial cells. Here we sought to investigate the mode of Zn2+-induced cell death and the role of reactive oxygen species (ROS) in human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: Cell viability was measured by MTT assay. Cell death of ARPE-19 cells was measured by annexin V-fluorescein isothiocyanate (FITC) binding assay, TUNEL assay. The formation of intracellular ROS was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The activation of mitogen-activated protein kinase (MAPK) was examined by Western blot analysis. RESULTS: This study demonstrated that Zn2+ treatment induced both necrosis and apoptosis in ARPE-19 cells. Exposure of ARPE-19 cells to Zn2+ led to the activation of ERK1/2, JNK1/2/3, and p38 MAPKs. The activation of these MAPKs was blocked by treatment with the antioxidant, N-acetylcystein (NAC). More importantly, inhibition of ROS production by NAC completely prevented Zn2+-induced cell death in RPE cells. CONCLUSIONS: This study suggests that Zn2+ induces both apoptosis and necrosis in ARPE-19 cells and that its cytotoxicity may depend on the induction of intracellular ROS.

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27(Kip1) protein level specifically increased after KIF14 knockdown. The increase in p27(Kip1) was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27(Kip1) accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27(Kip1) for degradation by the 26S proteasome, leading to accumulation of p27(Kip1). The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis.

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 × 10-7). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 × 10-5). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 × 10-10). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.

The potential role of VPREB1 gene copy number variation in susceptibility to rheumatoid arthritis.

Although the etiology of rheumatoid arthritis (RA) remains unknown, it has been widely suggested that RA has a genetic background. In humans, a copy number loss of 22q11.2, a region harboring the VPREB1 gene, has been suggested to be associated with several immunologic disorders, but there has been no study on the copy number variation (CNV) of the VPREB1 and its potential association with RA. Here, we explored the association between the RA and the CNV of the VPREB1 gene by performing genomic quantitative PCR and quantification of B cell subsets in RA patients and controls. The proportion of the individuals with <2 copies of the VPREB1 gene was significantly higher in the patient group than that in the controls (12.9% vs 0.9%, p<0.0001), while that of the individuals with >2 copies was lower in the patient group than that in the controls (1.7% vs 18.9%, p<0.0001). The odds ratio (OR) of the individuals with <2 copies was significantly higher compared with the odds ratio of those individuals with 2 copies (OR=12.1, 95% confidence interval (CI) 2.8-51.6). Likewise, the OR of the individuals with >2 copies was significantly lower than the OR of those individuals with 2 copies (OR=0.09, 95% CI 0.03-0.3). We also found that the proportion of CD21⁻CD23⁻ B cells was significantly higher in the RA patients compared with that of the controls (11.9% vs 5.7%, p=0.002), but the proportion of CD21+CD23+ cells was significantly lower in the RA patients (26.2% in RA vs 34.9% in the controls, p=0.005). To the best of our knowledge, this is the first evidence showing the association between a low copy number of the VPREB1 gene and RA, and this may help understanding the pathogenesis of RA and other autoimmune disorders.