A transcriptome-Based Deep Neural Network Classifier for Identifying the Site of Origin in Mucinous Cancer.

Purpose: There is a lack of tools for identifying the site of origin in mucinous cancer. This study aimed to evaluate the performance of a transcriptome-based classifier for identifying the site of origin in mucinous cancer. Materials And Methods: Transcriptomic data of 1878 non-mucinous and 82 mucinous cancer specimens, with 7 sites of origin, namely, the uterine cervix (CESC), colon (COAD), pancreas (PAAD), stomach (STAD), uterine endometrium (UCEC), uterine carcinosarcoma (UCS), and ovary (OV), obtained from The Cancer Genome Atlas, were used as the training and validation sets, respectively. Transcriptomic data of 14 mucinous cancer specimens from a tissue archive were used as the test set. For identifying the site of origin, a set of 100 differentially expressed genes for each site of origin was selected. After removing multiple iterations of the same gene, 427 genes were chosen, and their RNA expression profiles, at each site of origin, were used to train the deep neural network classifier. The performance of the classifier was estimated using the training, validation, and test sets. Results: The accuracy of the model in the training set was 0.998, while that in the validation set was 0.939 (77/82). In the test set which is newly sequenced from a tissue archive, the model showed an accuracy of 0.857 (12/14). t-SNE analysis revealed that samples in the test set were part of the clusters obtained for the training set. Conclusion: Although limited by small sample size, we showed that a transcriptome-based classifier could correctly identify the site of origin of mucinous cancer.

Integrated analysis of ascites and plasma extracellular vesicles identifies a miRNA-based diagnostic signature in ovarian cancer.

Ovarian cancer is mostly diagnosed at advantaged stages due to the lack of early diagnostic biomarkers. The common metastasis pattern is characterized by peritoneal dissemination with a formation of malignant ascites. Extracellular vesicles (EVs) are emerging as promising clinical biomarkers in liquid biopsy. Here, we aimed to investigate robust liquid biopsy-based EV miRNA biomarkers for ovarian cancer diagnosis and metastasis regulation. EVs were isolated from malignant ascites and plasma of ovarian cancer patients as well as the benign control counterparts of patients with benign gynecologic diseases. EV small RNA sequencing identified a panel of eight miRNAs (miR-1246, miR-1290, miR-483, miR-429, miR-34b-3p, miR-34c-5p, miR-145-5p, miR-449a) based on dysregulated miRNAs overlapped in the ascites and plasma subset. The ovarian cancer EV miRNA (OCEM) signature developed based on these eight miRNAs demonstrated high diagnostic accuracy in our in-house dataset and multiple public datasets across diverse clinical samples (blood, tissue and urine). In addition, malignant ascites-derived EVs could significantly facilitate the aggressive property of ovarian cancer cells and boost the growth of ascites-derived organoids. Notably, miR-1246 and miR-1290 shuttled in malignant ascites-derived EVs were identified to promote the invasion and migration of ovarian cancer cells through regulating a common target RORα.

Classification and Functional Analysis between Cancer and Normal Tissues Using Explainable Pathway Deep Learning through RNA-Sequencing Gene Expression.

Deep learning has proven advantageous in solving cancer diagnostic or classification problems. However, it cannot explain the rationale behind human decisions. Biological pathway databases provide well-studied relationships between genes and their pathways. As pathways comprise knowledge frameworks widely used by human researchers, representing gene-to-pathway relationships in deep learning structures may aid in their comprehension. Here, we propose a deep neural network (PathDeep), which implements gene-to-pathway relationships in its structure. We also provide an application framework measuring the contribution of pathways and genes in deep neural networks in a classification problem. We applied PathDeep to classify cancer and normal tissues based on the publicly available, large gene expression dataset. PathDeep showed higher accuracy than fully connected neural networks in distinguishing cancer from normal tissues (accuracy = 0.994) in 32 tissue samples. We identified 42 pathways related to 32 cancer tissues and 57 associated genes contributing highly to the biological functions of cancer. The most significant pathway was G-protein-coupled receptor signaling, and the most enriched function was the G1/S transition of the mitotic cell cycle, suggesting that these biological functions were the most common cancer characteristics in the 32 tissues.

Changes in Plasma Choline and the Betaine-to-Choline Ratio in Response to 6-Month Lifestyle Intervention Are Associated with the Changes of Lipid Profiles and Intestinal Microbiota: The ICAAN Study.

Trimethylamine N-oxide (TMAO) and its precursors, including choline, betaine, and L-carnitine, are gut microbiota-related metabolites associated with the risk of obesity. We aimed (1) to comprehensively examine whether the changes in plasma TMAO and its precursors induced by lifestyle intervention are associated with the improvements in plasma metabolic parameters; and (2) to identify the fecal microbiome profiles and nutrient intakes associated with these metabolites and metabolic index. Data from 40 participants (obese children and adolescents) having the plasma metabolites data related to the changes in BMI z-scores after 6-month lifestyle intervention were analyzed. In this study, we observed that choline and the betaine-to-choline ratio (B/C) showed different patterns depending on the changes in BMI z-scores by the response to lifestyle intervention. During the 6 months, an increase in choline and a decrease in B/C were observed in non-responders. We also found that changes in choline and B/C were associated with the improvements in plasma lipid levels. Individuals who showed reduced choline or increased B/C from the baseline to 6 months had a significant decrease in LDL-cholesterol over 6 months compared to those with increased choline or decreased B/C, respectively. In addition, the increase in choline or decrease in B/C was associated with the increase in plasma triglycerides. The distribution of gut microbiota belonging to the Firmicutes, such as Clostridia, Clostridiales, Peptostreptococcaceae, Romboutsia, and Romboutsia timonensis was altered to be lower during the 6 months both as choline decreased and B/C increased. Moreover, the decrease in choline and the increase in B/C were associated with reduced fat intake and increased fiber intake after the 6-month intervention. Finally, lower abundance of Romboutsia showed the association with lower LDL-cholesterol and higher intake of fiber. In summary, we demonstrated that reduced choline and increased B/C by lifestyle intervention were associated with the improvements of LDL-cholesterol and triglycerides, low-fat and high-fiber intakes, and low abundance of Firmicutes. These indicate that changes to circulating choline and B/C could predict individuals' changes in metabolic compositions in response to the lifestyle intervention.

Metabolomic Signatures for the Effects of Weight Loss Interventions on Severe Obesity in Children and Adolescents.

Childhood obesity has increased worldwide, and many clinical and public interventions have attempted to reduce morbidity. We aimed to determine the metabolomic signatures associated with weight control interventions in children with obesity. Forty children from the "Intervention for Children and Adolescent Obesity via Activity and Nutrition (ICAAN)" cohort were selected according to intervention responses. Based on changes in body mass index z-scores, 20 were responders and the remaining non-responders. Their serum metabolites were quantitatively analyzed using capillary electrophoresis time-of-flight mass spectrometry at baseline and after 6 and 18 months of intervention. After 18 months of intervention, the metabolite cluster changes in the responders and non-responders showed a difference on the heatmap, but significant metabolites were not clear. However, regardless of the responses, 13 and 49 metabolites were significant in the group of children with obesity intervention at 6 months and 18 months post-intervention compared to baseline. In addition, the top five metabolic pathways (D-glutamine and D-glutamate metabolism; arginine biosynthesis; alanine, aspartate, and glutamate metabolism; TCA cycle (tricarboxylic acid cycle); valine, leucine, and isoleucine biosynthesis) including several amino acids in the metabolites of obese children after 18 months were significantly changed. Our study showed significantly different metabolomic profiles based on time post obesity-related intervention. Through this study, we can better understand and predict childhood obesity through metabolite analysis and monitoring.

Association between Physical Fitness and Cardiometabolic Risk of Children and Adolescents in Korea.

BACKGROUND: This study aimed to investigate the association between physical fitness and cardiometabolic health of Korean children and adolescents. METHODS: In total, 168 participants (89 boys and 79 girls) aged 10-16 years were recruited for the Intervention for Childhood and Adolescent Obesity via Activity and Nutrition Study in 2016. The subjects were categorized into two groups using the definition of metabolic syndrome by the International Diabetes Federation: metabolically unhealthy (with at least two of the five criteria) and healthy groups (with less than one criterion). Correlation analysis of the participants' general characteristics was performed. Odds ratios (ORs) of physical fitness for cardiometabolic risk were evaluated via logistic regression. RESULTS: Metabolically unhealthy children showed greater weight, height, and body mass index, higher Children's Depression Inventory score, and longer screen time than did the metabolically healthy children. Metabolically healthy children showed greater upper and lower extremity muscular strength than did the metabolically unhealthy children (P=0.04 and P<0.001, respectively). In the multiple logistic regression analysis, lower extremity muscle strength was inversely related to the clustered cardiometabolic risk of the children and adolescents with or without adjustment for confounders (OR, 4.32; 95% confidence interval [CI], 1.87-9.97; OR, 7.64; 95% CI, 1.55- 37.74, respectively). CONCLUSION: Physical fitness, especially lower extremity muscle strength, is significantly inversely associated with individual and clustered cardiometabolic risks in Korean children and adolescents.

Positive correlation of cg16672562 methylation with obesity-related traits in childhood obesity, and its independence with underlying HIF3A (hypoxia-inducible factor 3a) genetic background.

Differential methylations of the HIF3A (hypoxia-inducible factor 3a) gene have been linked to body mass index (BMI). To explore the association of these methylations to childhood obesity, we measured 5 CpG methylation sites (cg27146050, cg46801562, cg22891070, cg16672562 and cg46801675) in intron 1 of the HIF3A gene by pyrosequencing, in the Korean population (mean age: 13.9 yrs, 305 obese cases and 387 controls). Two CpG methylations, cg46801562 and cg16672562, had statistically significant association with childhood obesity (P = 2.09E-9 and 1.66E-7, respectively). Notably, in the case of cg16672562, all correlations were significantly positive with BMI (beta = 0.285, P = 1.652E-13), waist-hip ratio (beta = 0.0028, P = 1.42E-15) and fasting plasma glucose level (beta = 0.0645, P = 2.61E-4), when analyzed by linear regression, with age and sex as covariates. We investigated any genetic effect of cg16672562 methylation by using 14 single nucleotide polymorphisms (SNP) identified by exome sequencing of the HIF3A locus cg16672562 methylation showed no statistically significant changes due to the 14 polymorphisms. In this study, we show that cg16672562 is the most significant blood DNA methylation marker for childhood obesity in the Korean population, and might be independent of any underlying HIF3A genetic background.

The change of plasma ghrelin and leptin levels by the development of type 2 diabetes mellitus in patients with alcohol dependence.

BACKGROUND: There have been lots of studies about the relationship between chronic use of alcohol and the development of type 2 diabetes mellitus (T2DM). Chronic use of alcohol can be affected by the altered level of ghrelin and leptin which regulate food-seeking behavior having similar mechanism of controlling alcohol-craving behavior. Those peptides are known to be correlated with T2DM. Ghrelin and leptin also have been regarded as possible regulators of glucose metabolism and insulin function. Hence, there is the possibility that ghrelin and leptin can be related with deteriorated pathophysiology of T2DM in alcoholic patients. METHODS: Patients with alcohol dependence diagnosed by Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) underwent an 75 g oral glucose-tolerance test (OGTT), to classify them to normal glucose tolerance (NGT, n = 52), pre-diabetes including impaired glucose tolerance (IGT), impaired fasting glucose level (IFG) and combination of IGT and IFG (Pre-DM, n = 26) and T2DM (n = 24) groups. Fasting plasma ghrelin and leptin levels were compared among groups. RESULTS: There was no difference of ghrelin concentration among the groups but the leptin concentration was significantly different between NGT and T2DM group (p < 0.05). Increased leptin levels were significantly correlated with body mass index (BMI), insulin level, and insulin resistance. CONCLUSIONS: Chronic alcohol drinking might produce leptin resistance which makes leptin significantly correlated with fasting insulin concentration and insulin resistance. Therefore, we suppose that increased level of leptin by chronic alcohol use could be one of the main mechanisms that develop insulin resistance in alcoholic patients.

Sensitization of human K562 leukemic cells to TRAIL-induced apoptosis by inhibiting the DNA-PKcs/Akt-mediated cell survival pathway.

Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, human K562 leukemic cells showed resistance to TRAIL-induced apoptosis. Interestingly, K562/R3 cells, a stable TRAIL-sensitive variant isolated from K562 cells, showed down-regulation of DNA-PK/Akt pathway and a high responsiveness to TRAIL-mediated growth inhibition and apoptosis. We revealed that siRNA-mediated suppression of DNA-PKcs led to decreased phosphorylation of Akt and Bad, a target molecule of Akt, and increased expression of DR4/DR5. Also, we found that suppression of DNA-PKcs using siRNA down-regulated c-FLIP and sensitized K562 cells to TRAIL-induced apoptosis through activation of caspase-8, -9 and -3. In addition, we revealed that treatment with DMNB, a specific inhibitor of DNA-PK, resulted in an increase of DR4/DR5 mRNA levels and their surface expression and a decrease of c-FLIP mRNA levels in K562 cells. DMNB potentiated TRAIL-induced cytotoxicity and apoptosis through inhibition of DNA-PK/Akt pathway and activation of caspase-8, -9 and -3 in K562 cells. This study is the first to show that a protective role of DNA-PK/Akt pathway against TRAIL-induced apoptosis and thus TRAIL in combination with agents that inhibit DNA-PK/Akt pathway would have clinical applicability in treating TRAIL-insensitive human leukemic cells. This model may provide a novel framework for overcoming TRAIL resistance of other cancer cells with agents that inhibit DNA-PK/Akt pathway.

p19(ras) amplifies p73beta-induced apoptosis through mitochondrial pathway.

p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

Internalization and trafficking mechanisms of coxsackievirus B3 in HeLa cells.

Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3 internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes.

Immunogenicity of a DNA vaccine for coxsackievirus B3 in mice: protective effects of capsid proteins against viral challenge.

Coxsackievirus (CVB) 3 induces viral myocarditis and ultimately dilated cardiomyopathy (DCM). However, there is no vaccine in clinical use. We constructed recombinant CVB3 plasmids using a highly effective mammalian expression vector and evaluated their immunogenicity in vivo on the basis of survival rate. Four recombinant plasmids were constructed, which encode CVB3 capsid proteins (VP1 or VP3) or VP1 partial proteins (VP1-1 or VP1-2), and used to immunize BALB/c mice by electroporation. Although VP1, VP3, VP1-1, and VP1-2 induced specific antibodies against the corresponding proteins in mice, neutralizing antibodies were not present in the sera. These recombinant plasmids, except VP1-1 (12.5%), dramatically increased the survival rate in mice at 46 days after challenge (42.9-75.0%, p<0.05). VP3 (75.0%) protected mice against viral infection and the middle regions of VP1 (VP1-2, 50.5%) conferred a protective effect like that conferred by VP1 (42.9%), suggesting that the epitopes in VP3 as well as in the middle of VP1 protect against CVB3 infection in vivo. In conclusion, some recombinant CVB3 plasmids used in this study reduced the destruction of myocytes and improved the survival rates in mice immunized and challenged compared with the control. Thus, pCA-VP3 as well as pCA-VP1 are good candidates for a CVB3 DNA vaccine.

Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death.

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.

Modification of serine 392 is a critical event in the regulation of p53 nuclear export and stability.

Although it has been shown that phosphorylations of p53 serine its residues are critical events for the regulation of their function, the specific biological effects of each of these phosphorylations, especially at serine 392, remain to be elucidated. Serine 392 has been proposed to play a role in the tetramerization of p53 and in the enhancement of its DNA-binding affinity. However, this is not consistent with other reports showing that substitution of serine 392 does not disrupt p53 function. These discrepancies suggest that modification of serine 392 may contribute to p53 activity through other transactivating pathways. In this study, we demonstrate that this C-terminal serine residue (p53-392S) in fact plays a critical role in the regulation of p53 stability such that substitution with alanine (p53-392A) strongly enhances p53 stability without disrupting mouse double minute 2 binding. Additionally, the p53-392A mutant is localized mainly in the nucleus, whereas both wild-type p53 and a glutamic acid mutant, p53-392E, are evenly distributed throughout the cytoplasm and nucleus. However, each of these p53 species had similar effects on both cell cycle inhibition and apoptosis, in response to either UV or adriamycin treatment. Moreover, p53-392A protein was resistant to E6-mediated degradation. Our results suggest that although serine 392 is not essential for the transactivation and nuclear import of p53, it exerts important effects upon p53 stability via the inhibition of its nuclear export mechanism.