👤 Chong Woo Yoo

Cultured human pluripotent stem cells (hPSCs) grow as colonies that require breakdown into small clumps for further propagation. Although cell death mechanism by single-cell dissociation of hPSCs has been well defined, how hPSCs respond to the deadly stimulus and recover the original status remains unclear. Here we show that dissociation of hPSCs immediately activates ERK, which subsequently activates RSK and induces DUSP6, an ERK-specific phosphatase. Although the activation is transient, DUSP6 expression persists days after passaging. DUSP6 depletion using the CRISPR/Cas9 system reveals that DUSP6 suppresses the ERK activity over the long term. Elevated ERK activity by DUSP6 depletion increases both viability of hPSCs after single-cell dissociation and differentiation propensity towards mesoderm and endoderm lineages. These findings provide new insights into how hPSCs respond to dissociation in order to maintain pluripotency. Show less

Obesity is a known risk factor for metabolic diseases and is often associated with chronic inflammation in adipose tissue. We previously identified the polyethoxylated flavonoid Nobiletin (NOB) as a circadian clock modulator that directly binds to and activates the ROR receptors in the core oscillator, markedly improving metabolic fitness in obese mice. Here, we show that NOB enhanced the oscillation of core clock genes in differentiated 3T3-L1 adipocytes, including ROR target genes such as Show less

The prevalence of dyslipidemia has increased steadily in Korea, and the incidence of dyslipidemia differs by sex. In this study, we identified single nucleotide polymorphisms (SNPs) related to dyslipidemia in Korean cohorts through genome-wide association study (GWAS) analysis. Genotyping was conducted to determine the genotypes of 72,298 participants and investigate genotypes for 7,079,946 SNPs. Sex, age, and BMI were set as covariates for GWAS, and significant SNPs were identified in the discovery and replication stages using logistic regression. GWAS of the entire cohort revealed a total of five significant SNPs: rs117026536 (LPL), rs651821 (APOA5), rs9804646 (APOA5), rs9926440 (CETP), and rs429358 (APOE). GWAS of the male subjects revealed a total of four significant SNPs. While rs9804646 (APOA5) and rs429358 (APOE) were significant for all the subjects, rs662799 (APOA5) and rs56156922 (CETP) were significant only for the male subjects. GWAS of the female subjects revealed two significant SNPs, rs651821 (APOA5) and rs9804646 (APOA5), both of which were significant in all the subjects. This is the first study to identify sex-related differences in genetic polymorphisms in Korean populations with dyslipidemia. Further studies considering environmental variables will be needed to elucidate these sex-related genetic differences in dyslipidemia. Show less

Alcohol consumption is associated with a high increased lipid profile and this association may depend on genetic risk factors. In this study, we aimed to assess the effects of genetic variation associated with alcohol consumption on lipid profiles using data from two Korean population studies. We performed a genotype association study using the HEXA (n = 51,349) and KNHANES (n = 9158) data. Genotype analyses of the two sets of Korean population data showed associations of increased total cholesterol and high-density lipoprotein (HDL)-cholesterol with CETP rs708272. The HEXA and KNHANES populations revealed differences in HDL cholesterol according to the presence of CETP rs708272, independent of ALDH2 rs671 and alcohol consumption. In contrast, total cholesterol levels were associated with alcohol consumption and ALDH2 rs671 in men with CETP rs708272 (CT and TT genotypes). Furthermore, in drinkers with ALDH2 rs671 (GA and AA genotypes), higher total cholesterol was associated with the CETP rs708272 TT minor homozygous genotype based on both HEXA and KNHANES data. Our findings demonstrated that alcohol consumption and genetic variation in either CETP or ALDH2 may be associated with cholesterol levels. We hope these findings will provide a better understanding of the relationship between alcohol consumption and cholesterol according to each individual's genetic background. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency affects the first step of urea cycle and is a severe form of urea cycle disorder (UCD). The severity of hyperammonemic encephalopathy determines the clinical course of UCDs. Here, we describe the genetic and clinical characteristics of CPS1 deficiency in Korea. This study included seven patients with CPS1 deficiency genetically confirmed from January 1992 to September 2020. The peak ammonia level during the first crisis, the half time of peak ammonia level, the initial plasma amino acid levels, and neurological outcomes were compared between CPS1 deficiency and two common UCDs (i.e., 17 patients with argininosuccinate synthetase 1 deficiency and 24 patients with ornithine transcarbamylase deficiency). Eleven CPS1 mutations were identified, including 10 novel mutations. Eight mutations were missense. Six patients with CPS1 deficiency had neonatal type. The peak ammonia level, initial glutamate level, and accompanying rate of irreversible neurological damages were highest in patients with CPS1 deficiency. The patient with late-onset CPS1 deficiency responded dramatically to N-carbamylglutamate treatment. The clinical manifestations of CPS1 deficiency were the most severe among UCDs. Considering the high proportion of missense mutations, responsiveness to N-carbamylglutamate would be evaluated in a future study. Show less

Genetic variations resulting in the loss of function of the discs large homologs (DLG2)/postsynaptic density protein-93 (PSD-93) gene have been implicated in the increased risk for schizophrenia, intellectual disability, and autism spectrum disorders (ASDs). Previously, we have reported that mice lacking exon 14 of the Show less

The mechanisms contributing to recurrence of glioblastoma (GBM), an aggressive neuroepithelial brain tumor, remain unknown. We have recently shown that nuclear respiratory factor 1 (NRF1) is an oncogenic transcription factor and its transcriptional activity is associated with the progression and prognosis of GBM. Herein, we extend our efforts to (1) identify influential NRF1-driven gene and microRNA (miRNA) expression for the aggressiveness of mesenchymal GBM; and (2) understand the molecular basis for its poor response to therapy. Clinical data and RNA-Seq from four independent GBM cohorts were analyzed by Bayesian Network Inference with Java Objects (BANJO) and Markov chain Monte Carlo (MCMC)-based gene order to identify molecular drivers of mesenchymal GBM as well as prognostic indicators of poor response to radiation and chemotherapy. We are the first to report sex-specific NRF1 motif enriched gene signatures showing increased susceptibility to GBM. Risk estimates for GBM were increased by greater than 100-fold with the joint effect of NRF1-driven gene signatures-CDK4, DUSP6, MSH2, NRF1, and PARK7 in female GBM patients and CDK4, CASP2, H6PD, and NRF1 in male GBM patients. NRF1-driven causal Bayesian network genes were predictive of poor survival and resistance to chemoradiation in IDH1 wild-type mesenchymal GBM patients. NRF1-regulatable miRNAs were also associated with poor response to chemoradiation therapy in female IDH1 wild-type mesenchymal GBM. Stable overexpression of NRF1 reprogramed human astrocytes into neural stem cell-like cells expressing SOX2 and nestin. These cells differentiated into neurons and form tumorospheroids. In summary, our novel discovery shows that NRF1-driven causal genes and miRNAs involved in cancer cell stemness and mesenchymal features contribute to cancer aggressiveness and recurrence of aggressive therapy-resistant glioblastoma. Show less

Hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) is heterogeneous and frequently contains multifocal tumors, but how the multifocal tumors relate to each other in terms of HBV integration and other genomic patterns is not clear. To interrogate heterogeneity of HBV-HCC, we developed a HBV genome enriched single cell sequencing (HGE-scSeq) procedure and a computational method to identify HBV integration sites and infer DNA copy number variations (CNVs). We performed HGE-scSeq on 269 cells from four tumor sites and two tumor thrombi of a HBV-HCC patient. HBV integrations were identified in 142 out of 269 (53%) cells sequenced, and were enriched in two HBV integration hotspots chr1:34,397,059 (CSMD2) and chr8:118,557,327 (MED30/EXT1). There were also 162 rare integration sites. HBV integration sites were enriched in DNA fragile sites and sequences around HBV integration sites were enriched for microhomologous sequences between human and HBV genomes. CNVs were inferred for each individual cell and cells were grouped into four clonal groups based on their CNVs. Cells in different clonal groups had different degrees of HBV integration heterogeneity. All of 269 cells carried chromosome 1q amplification, a recurrent feature of HCC tumors, suggesting that 1q amplification occurred before HBV integration events in this case study. Further, we performed simulation studies to demonstrate that the sequential events (HBV infecting transformed cells) could result in the observed phenotype with biologically reasonable parameters. Our HGE-scSeq data reveals high heterogeneity of HCC tumor cells in terms of both HBV integrations and CNVs. There were two HBV integration hotspots across cells, and cells from multiple tumor sites shared some HBV integration and CNV patterns. Show less

ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type. Show less

Transforming growth factor-beta (TGF-β) has a dichotomous role, functioning as a tumor suppressor and tumor promoter. TGF-β signatures, explored in mouse hepatocytes, have been reported to predict the clinical outcomes of hepatocellular carcinoma (HCC) patients; HCCs exhibiting early TGF-β signatures showed a better prognosis than those with late TGF-β signatures. The expression status of early and late TGF-β signatures remains unclear in defined lesions of human B-viral multistep hepatocarcinogenesis. The expression of TGF-β signatures, early and late responsive signatures of TGF-β were investigated and analyzed for their correlation in cirrhosis, low-grade dysplastic nodules (DNs), high-grade DNs, early HCCs and progressed HCCs (pHCCs) by real-time PCR and immunohistochemistry. The expression levels of TGF-β signaling genes ( The enrichment of the late responsive signatures of TGF-β with induction of stemness is considered to be involved in the progression of the late stage of multistep hepatocarcinogenesis, whereas the early responsive signatures of TGF-β are suggested to have tumor-suppressive roles in precancerous lesions of the early stage of multistep hepatocarcinogenesis. Show less

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice. Show less

Using a machine learning method, this study aimed to identify unique causal networks of genes associated with bone, brain, and lung metastasis of breast cancer. Bayesian network analysis identified differentially expressed genes in primary breast cancer tissues, in bone, brain, and lung breast cancer metastatic tissues, and the clinicopathological features of patients obtained from the Gene Expression Omnibus microarray datasets. We evaluated the causal Bayesian networks of breast metastasis to distant sites (bone, brain, or lung) by (i) measuring how well the structures of each specific type of breast cancer metastasis fit the data, (ii) comparing the structures with known experimental evidence, and (iii) reporting predictive capabilities of the structures. We report for the first time that the molecular gene signatures are specific to the different types of breast cancer metastasis. Several genes, including CHPF, ARC, ANGPTL4, NR2E1, SH2D1A, CTSW, POLR2J4, SPTLC1, ILK, ALDH3B1, PDE6A, SCTR, ADM, HEY1, KCNF1, and UVRAG, were found to be predictors of the risk for site-specific metastasis of breast cancer. Expression of POLR2JA, SPTLC1, ILK, ALDH3B1, and the estrogen receptor was significantly associated with breast cancer bone metastasis. Expression of PDE6A and NR2E1 was causally linked to breast cancer brain metastasis. Expression of HEY1, KCNF1, UVRAG, and the estrogen and progesterone receptors was strongly associated with breast cancer lung metastasis. The causal Bayesian network structures of these genes identify potential interactions among the genes in distant metastases of breast cancer, including to the bone, brain, and lung, and may serve as target candidates for treatment of breast cancer metastasis. Show less

DLG2, also known as postsynaptic density protein-93 (PSD-93) or chapsyn-110, is an excitatory postsynaptic scaffolding protein that interacts with synaptic surface receptors and signaling molecules. A recent study has demonstrated that mutations in the DLG2 promoter region are significantly associated with autism spectrum disorder (ASD). Although DLG2 is well known as a schizophrenia-susceptibility gene, the mechanisms that link DLG2 gene disruption with ASD-like behaviors remain unclear. Mice lacking exon 14 of the Dlg2 gene (Dlg2 Dlg2 These results suggest that homozygous Dlg2 deletion in mice leads to ASD-like behavioral phenotypes, including social deficits and increased repetitive behaviors, as well as reductions in excitatory synaptic input onto dorsolateral spiny projection neurons, implying that the dorsal striatum is one of the brain regions vulnerable to the developmental dysregulation of DLG2. Show less

Assessment of differentiation potential is a basic requirement to obtain qualified human pluripotent stem cells (hPSCs). Here, we report a simple differentiation method using fetal bovine serum (FBS) to estimate differentiation potential and propensity of hPSCs. PluriTest using RNA-sequencing showed that cells differentiated after treatment with 5% FBS. Expression patterns of three germ layer markers revealed that cells cultured in Knockout Serum Replacement-containing medium (KSR) with mouse feeder cells had higher differentiation potential than cells cultured in a chemically defined medium (E8) with recombinant matrix proteins, especially into the mesoderm and endoderm lineages. Analysis of differentially expressed genes between KSR and E8 identified DUSP6 as a marker for where cells had been cultured. Expression of DUSP6 correlated with FGF-ERK signaling activity. Fine-tuning of FGF-ERK signaling activity to a range that can shut down DUSP6 transcription but sustain NANOG transcription partially increased the differentiation potential. Our data suggest that differentiation with 5% FBS is good to estimate differentiation potential and propensity at the early stage, and that DUSP6 is an excellent marker to monitor ERK signaling activity. Show less

Although the changes in DNA methylation are assumed to be due to the association between adverse intrauterine conditions and adult metabolic health, evidence from human studies is rare. Little is known about the changes in DNA methylation present at birth that affect metabolic profiles in childhood. Previous studies have shown that the melanocortin 4 receptor (MC4R) and hepatocyte nuclear factor 4 alpha (HNF4α) genes are associated with obesity and metabolic disorders. Thus, we investigated the associations of the DNA methylation statuses of MC4R and HNF4α in cord blood with metabolic profiles in childhood.We collected data from 90 children 7 to 9 years of age included in the Ewha Birth & Growth Cohort Study in Korea. DNA methylation was analyzed by pyrosequencing. The children were split into 2 groups according to the cutoff triglyceride (TG) levels (<110 and ≥110 mg/dL).The methylation statuses of MC4R and HNF4α at birth were significantly associated with the TG level in childhood (P < .05). It was interesting to note that the methylation statuses of MC4R and HNF4α in cord blood were significantly decreased, whereas childhood body mass index was significantly increased, in children with high TG levels compared with children with low TG levels (P < .05).Our findings show that the methylation statuses of MC4R and HNF4α at birth are associated with metabolic profiles in childhood. These epigenetic modifications occurring in early life may contribute to subsequent metabolic-related disorders. Thus, we suggest that DNA methylation status in cord blood may be predictive of the risk of developing metabolic syndrome. Show less

We aimed to evaluate whether the longitudinal interaction between APOA5-1131C variants and overweight could accelerate age-related increases in arterial stiffness and circulating triglycerides in healthy subjects. This 3-year prospective cohort study included 503 healthy subjects. Brachial-ankle pulse wave velocity (baPWV), triglycerides, APOA5 -1131T > C, apolipoprotein (apo) A-V level, and low-density lipoprotein (LDL) particle size were measured at baseline and within a mean follow-up period of 3 years. At the 3-year follow-up, in the overweight group, subjects with the C allele showed increases in triglycerides and baPWV relative to baseline. Additionally, in the overweight group, there was a genotype effect on changes in triglycerides: subjects with the C allele had greater increases in triglyceride concentrations than subjects with the TT genotype. Furthermore, overweight subjects with the C allele had greater increases in triglyceride concentrations than normal-weight subjects with the C allele (P-interaction = 0.013). Overweight subjects with the C allele had greater increases in baPWV than normal-weight subjects with the C allele (P-interaction = 0.047). Changes in baPWV were affected by age, baseline baPWV, and changes in systolic blood pressure (BP) and triglycerides. Changes in triglycerides were affected by APOA5 -1131T > C genotype, age, baseline triglyceride level, and changes in BMI and apo A-V. In the overweight group, changes in baPWV were affected by changes in systolic BP, LDL particle size, and triglycerides. This prospective study shows that the interactive effect between APOA5 -1131C variants and overweight can accelerate age-related increase in arterial stiffness via the regulation of circulating triglycerides in healthy subjects. Show less

We performed a genome-wide association study to find genetic variants associated with high-density lipoprotein (HDL)-cholesterol levels in a Korean population and verified two apolipoprotein A5 (APOA5) gene variants, rs662799 (-1131T>C) and rs2075291 (c.553G>T), in 612 subjects with low HDL-cholesterol (cases) and 1536 subjects with normal HDL-cholesterol (controls). To explain this association, we compared clinical outcomes according to their genotype in normal (control) and low HDL (case) groups. In both the case and control groups, the rare alleles of rs662799 and rs2075291 were associated with higher triglyceride and lower HDL-cholesterol levels. In the subjects with the rs662799 CC genotype, lower levels of apoA-I and apoA-V and a smaller low-density lipoprotein (LDL) particle size were detected in both the case and control groups. In the case group, APOA5 rs662799 single nucleotide polymorphisms (SNPs) were associated with lower adiponectin and higher brachial-ankle pulse wave velocity (ba-PWV). Our results show that two APOA5 variants, rs662799 (-1131T>C) and rs2075291 (c.553G>T), are associated with HDL-cholesterol levels in a Korean population, and suggest that individuals with an APOA5 rs662799 CC genotype are at higher risk of atherosclerosis, particularly when they have low HDL-cholesterol, and this association is related to adiponectin levels. Show less

We aimed to evaluate the longitudinal interaction effects between the minor allele of FADS1 rs174547 and overweight on n-3 and n-6 long-chain polyunsaturated fatty acid (PUFA) levels and pulse wave velocity (PWV). Plasma PUFA levels were measured via GC-MS, and arterial stiffness was determined as brachial-ankle PWV (ba-PWV) at baseline and after a mean follow-up of 3 years. The FADS1 rs174547 T > C genotype was analyzed. At 3-years of follow-up, after adjustment for age, sex, smoking and drinking, there were interaction effects between the FADS1 rs174547 T > C genotype and baseline BMI on the changes (from baseline) in plasma arachidonic acid (AA) levels, in the eicosapentaenoic acid (EPA)/AA ratio, and in ba-PWV (p for interaction = 0.036, 0.022, and 0.001, respectively). There were smaller increases in AA levels from baseline among normal-weight C allele carriers (n = 112) and overweight TT subjects (n = 47) than among normal-weight TT subjects (n = 91). Overweight C allele carriers (n = 37) showed greater reductions in the plasma EPA/AA ratio and greater increases in ba-PWV than the 3 other populations studied. The minor allele of the FADS1 rs174547 polymorphism is associated with age-related decreases in the EPA/AA ratio and increases in ba-PWV among overweight subjects. Show less

Exposure to metabolic stress has been suggested to influence the susceptibility to metabolic disorders in offspring according to epidemiological and animal studies. Nevertheless, molecular mechanisms remain unclear. We investigated impacts of diet-induced paternal obesity on metabolic phenotypes in offspring and its underlying molecular mechanism. Male founder mice (F0), fed with control diet (CD) or high-fat diet (HFD), were mated with CD-fed females. F1 progenies were mated with outbred mice to generate F2 mice. All offspring were maintained on CD. Metabolic phenotypes, metabolism-related gene expression and endoplasmic reticulum (ER) stress markers were measured in serum or relevant tissues of F2 mice. DNA methylation in sperm and testis of the founder and in the liver of F2 mice was investigated. Male founder obesity, instigated by HFD, led to glucose dysregulation transmitted down to F2. We found that F2 males to HFD founders were overweight and had a high fasting glucose relative to F2 to CD founders. F2 females to HFD founders, in contrast, had a reduced bodyweight relative to F2 to CD founders and exhibited an early onset of impaired glucose homeostasis. The sex-specific difference was associated with distinct transcriptional patterns in metabolism-related organs, showing altered hepatic glycolysis and decreased adipose Glucose transporter 4 (Glut4) in males and increased gluconeogenesis and lipid synthesis in females. Furthermore, the changes in females were linked to hepatic ER stress, leading to suppressed insulin signaling and non-obese hyperglycemic phenotypes. DNA methylation analysis revealed that the Nr1h3 locus was sensitive to HFD at founder germ cells and the alteration was also detected in the liver of F2 female. Our findings demonstrate that male founder obesity influences impaired glucose regulation in F2 progeny possibly via ER stress in a sex-specific manner and it is, in part, contributed by altered DNA methylation at the Nr1h3 locus. Show less

Hypertriglyceridemia is recognized as an independent risk factor for coronary artery disease. The apolipoprotein A5 gene (APOA5) is a key regulator of triglyceride levels. We aimed to evaluate the associations of single nucleotide polymorphisms (SNPs) in APOA5, including -1131T>C and c.553G>T, with hypertriglyceridemia, apoA5 concentrations, atherogenic LDL cholesterol levels, and arterial stiffness in hypertriglyceridemic patients. The study population included 599 hypertriglyceridemic patients (case) and 1,549 untreated normotriglyceridemic subjects (control). We genotyped two APOA5 variants, -1131T>C (rs662799) and c.553G>T (rs2075291). The frequencies of the CC genotype of -1131T>C (0.165) and the T allele of c.553G>T (0.119) were significantly higher in hypertriglyceridemic patients than in normotriglyceridemic subjects (0.061 and 0.070, respectively; all p<0.001). In the control and case groups, both the -1131T>C and c.553G>T variants were associated with higher triglyceride and lower HDL cholesterol levels. Controls with the -1131CC variant had lower apoA5 concentrations than controls with the -1131TT variant. Similar effects of the -1131T>C variant on apoA5 were observed in the cases. In the hypertriglyceridemic group, the -1131T>C variant was associated with a smaller LDL particle size, higher levels of oxidized LDL and malondialdehyde, and higher brachial-ankle pulse wave velocity. The -1131T>C and c.553G>T polymorphisms were associated with hypertriglyceridemia in the study population, but only the -1131T>C polymorphism directly affected apoA5 concentrations. Hypertriglyceridemic patients carrying the APOA5 -1131T>C polymorphism exhibited increased atherogenic LDL levels and arterial stiffness, probably due to an effect of the -1131T>C polymorphism on apoA5 concentrations. Show less

Wnt/β-catenin signaling has a pivotal role in the pathogenesis of hepatocellular carcinoma (HCC). The present study aimed to determine whether genetic variation in the Wnt/β-catenin signaling pathway is associated with the development and/or progression of HCC and the survival of patients with hepatitis B virus (HBV)-associated HCC. We assessed seven single nucleotide polymorphisms (SNPs) of the AXIN1, AXIN2, CTNNB1, and WNT2 genes in 245 patients with HBV-associated HCC and 483 chronic HBV carriers without HCC. We analyzed the association of each SNP with HCC development or progression and overall survival. The CTNNB1 rs3864004 A allele was associated with a decreased risk of HCC development (P=0.049). Haplotype analysis revealed a significantly higher frequency of CTNNB1 G-A/G-A haplotype at rs3864004 and rs4135385 positions in patients with HCC than in chronic HBV carriers without HCC (P=0.042). The AXIN1 rs1805105 T>C SNP was associated with small tumor size and early tumor stage and the WNT2 rs39315 G allele was associated with advanced tumor stage in HCC. In Kaplan-Meier analysis, carriers of the AXIN1 rs214252 C allele showed longer survival than those with the TT genotype (P=0.020). In multivariate Cox regression analysis, absence of CTNNB1 haplotype A-A at rs3864004 and rs4135385 positions and advanced tumor stage were independent poor predictors of patient survival in patients with HCC. These findings suggest that the genetic polymorphisms in CTNNB1 gene might affect tumor development and survival in patients with HBV-associated HCC. Show less

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS3-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties. Show less

Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism. C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1. NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPβ. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition. NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms. Show less

We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. Show less

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR). Show less

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β1–42 (Aβ42)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ42 enhanced autophagy flux in neuronal cells. Aβ42-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ42. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ42, defining a novel role of PRNP in the regulation of autophagy. Show less

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1-5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1-3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development. Show less

Vacuolar protein sorting plays crucial roles in the traffic of molecules between cellular organelles. Although involvement of vacuolar protein sorting proteins in cancer is known, genetic alterations of VPS genes have not been reported in cancers. We found that VPS4B, VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS37D, VPS41, and VPS54 have mononucleotide repeats in their coding sequences. To see whether these genes are mutated in cancers with microsatellite instability, we analyzed the mononucleotide repeats in 30 gastric cancers with high microsatellite instability, 13 gastric cancers with low microsatellite instability, and 45 gastric cancers with stable microsatellites and 40 colorectal cancers with high microsatellite instability, 14 colorectal cancers with low microsatellite instability, and 45 colorectal cancers with stable microsatellites by single-strand conformation polymorphism. We found mutations of VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS41, and VPS54 in 9, 3, 12, 3, 5, 9, 2, and 2 cancers, respectively, all in cancers with high microsatellite instability. The gastric cancers and colorectal cancers with high microsatellite instability harbored one or more mutations of the VPS genes in 53.3% and 50.0%, respectively. Loss of Vps13A expression was observed in 30% of the gastric cancers and 35% of the colorectal cancers, whereas loss of Vps35 was observed in 55% of the gastric cancers and 55% of the colorectal cancers. Our data indicate that frameshift mutations of VPS genes and losses of expression of Vps13A and Vps35 proteins are common in gastric cancers and colorectal cancers with high microsatellite instability and suggest that these alterations might contribute to development of cancers with high microsatellite instability by deregulating vacuolar protein sorting proteins. Show less

Height is a complex genetic trait that involves multiple genetic loci. Recently, 44 loci associated with height were identified in Caucasian individuals by large-scale genome-wide association (GWA) studies. To identify genetic variants influencing height in the Korean population, we analyzed GWA data from 8842 Korean individuals and identified 15 genomic regions with one or more sequence variants associated with height (P<1 x 10(-5)). Of these, eight loci were newly identified in Koreans (SUPT3H, EXT1, FREM1, PALM2-AKAP2, NUP37-PMCH, IGF1, KRT20 and ANKRD60). The 15 significant loci account for approximately 1.0% of height variation, with a 3.7-cm difference between individuals with < or =8 height-increasing alleles (5.1%) and > or =19 height-increasing alleles (4.2%). We also examined the associations between height loci and idiopathic short stature (ISS). Five loci (SPAG17, KBTBD8, HHIP, HIST1H1D and ACAN) were significantly associated with ISS (uncorrected P<0.05), indicating that height-associated genes in the adult population are involved in extreme cases of short stature in children. This study validates previous reports of loci associated with human height and identified novel candidate regions involved in human growth and development. Show less