Generation of chromosome 1p/19q co-deletion by CRISPR/Cas9-guided genomic editing.

Background: Chromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis. Methods: We hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes. Results: After translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage. Conclusion: We demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.

Safest Roasting Times of Coffee To Reduce Carcinogenicity.

ABSTRACT: Roasting coffee results in not only the creation of carcinogens such as acrylamide, furan, and polycyclic aromatic hydrocarbons but also the elimination of carcinogens in raw coffee beans, such as endotoxins, preservatives, or pesticides, by burning off. However, it has not been determined whether the concentrations of these carcinogens are sufficient to make either light or dark roast coffee more carcinogenic in a living organism. An Ames test was conducted on light, medium, and dark roast coffee from three origins. We found that lighter roast coffee shows higher mutagenicity, which is reduced to the control level in dark roast coffee varieties, indicating that the roasting process is not increasing mutagenic potential but is beneficial to eliminating the existing carcinogens in raw coffee beans. This result suggests that dark roast coffee is safer and promotes further studies of the various carcinogens in raw coffee that have been burned off.

Smartphone Addiction and Anxiety in Adolescents - A Cross-sectional Study.

Objectives: The overuse of smartphones affects physical, social, and psychological well-being. However, research on smartphone addiction and anxiety disorders is scarce. Therefore, the objective of this study was to investigate the association between anxiety and smartphone addiction risk in Korean adolescents. Methods: This study used a cross-sectional survey method. We used the Revised Children's Manifest Anxiety Scale to assess anxiety symptoms and we used the Korean Smartphone Addiction Proneness Scale index to evaluate the degree of high-risk or at-risk for smartphone addiction. Results: Analyses were performed for 1733 adolescents, including 771 boys and 962 girls. The high-risk or at-risk group for smartphone addiction accounted for 20.1% (p < .0001). Total anxiety scale score, as well as physiological anxiety, oversensitivity, and social concern categories were statistically different among levels of smartphone addiction risk (all ps < .0001). Multivariate analysis showed that poor self-reported health level, higher risk of smartphone addiction, having fewer close friends, caffeine drink consumption, female sex, and alcohol use were associated with greater anxiety. Conclusions: Management of smartphone addiction seems to be essential for proper psychological health. There is an urgent need to develop a way to prevent smartphone addiction on a social level.

Association between attention deficit hyperactivity disorder and aggression subscales in adolescents.

INTRODUCTION: The aim of this study is to identify the association between Attention Deficit Hyperactivity Disorder (ADHD) proneness and aggressive propensity in adolescents. METHODS: A quantitative, large-scale, cross-sectional study was performed from April to May 2016 in Korea. The survey questionnaire included overall health behaviors, as well as scales for assessing ADHD proneness (revised short form of the Conners-Wells Adolescent Self-Report Scale; CASS[S]) and aggressive behavior (Buss-Perry Aggression Questionnaire; BPAQ) in adolescents. Area under the receiver operator characteristic (AUROC) curves was constructed to determine the cut-off value of total aggression score for discriminating ADHD proneness. RESULTS: A total of 2,432 students participated in the survey, and 1,872 of them completed the questionnaire, indicating a response rate of 77.0%. Based on CASS(S), 33 (1.8%) subjects were classified as the ADHD group. AUROC curve analysis showed that a score of 68.5 points had higher sensitivity (83.3%) and specificity (69.4%) to discriminate ADHD proneness. ADHD proneness was significantly associated with higher aggression subdomain scores (physical, verbal, anger, and hostility). Especially, anger and hostility had a stronger relationship with ADHD proneness than did physical and verbal aggression. A multivariable analysis demonstrated that ADHD proneness was significantly related to body mass index in the top 10% of the study population, alcohol consumption, gastrointestinal trouble, daytime sleepiness, and total aggression score of 68.5 points or higher. Adolescents who had total aggression scores of 68.5 points or higher showed a 9.8-fold (95% confidence interval [CI] 3.3-28.8) higher risk of ADHD compared with those who had scores less than 68.5 points. CONCLUSIONS: Our results demonstrated that ADHD proneness was significantly associated with aggression propensity. In particular, anger and hostility were more closely associated with ADHD proneness than were other aggression subdomains.

Health Behaviors Associated with Aggression in Korean Adolescents: Data from 2 Cross-sectional Studies.

Objectives: In this study, we identified the health-related behaviors associated with aggression and examined the trends in both aggression and health-related behaviors among adolescents in the Republic of Korea. Methods: We used 2 cross-sectional samples of adolescents collected from the same geographic region 10 years apart. We measured aggression using the Aggression Questionnaire. Subject characteristics of the questionnaire included age, sex, caffeine intake, alcohol consumption, smoking, regular exercise, use of medications, and unstable mood. Results: Data pertaining to 1316 and 894 students were extracted from 2006 and 2016 surveys, respectively. Based on the multivariate results, sex and smoking were associated with physical aggression in both surveys, whereas sex and unstable mood were linked to verbal aggression. Alcohol consumption, unstable mood, and use of pain medications were related to anger; indigestion and unstable mood were associated with hostility in both years. The total aggression score was significantly related to alcohol consumption and unstable mood in both years after adjusting for other confounders. Conclusions: Health-related behaviors such as alcohol consumption and unstable mood showed meaningful relations with aggression. Accordingly, we should increase public awareness of factors associated with aggression, and government agencies and schools should implement comprehensive prevention efforts.

Association between anxiety and aggression in adolescents: a cross-sectional study.

BACKGROUND: The purpose of this study was to investigate the relationship between anxiety proneness and aggressive behavior in adolescents. METHODS: A quantitative, large scale cross-sectional study was conducted in Korea. The survey questionnaire included general health behavior and scales for assessing anxiety (Revised Children's Manifest Anxiety Scale; RCMAS) and aggressive behavior (The Aggression Questionnaire; AQ) in adolescents. RESULTS: A total of 2432 students participated in the survey, and 1933 individuals completed the questionnaire, indicating a response rate of 79.5%. Based on RCMAS, 163 (8.4%) subjects were classified as the anxiety group. Aggressive behavior was significantly associated with higher anxiety scores. In particular, among four subdomains of aggression, anger and hostility had a stronger relationship with anxiety than did physical and verbal aggression. Multivariate analysis demonstrated that anxiety was independently associated with gender, age, headache, constipation, asthma, and aggression score. Adolescents with total aggression scores of 69 or higher showed a 9-fold (AOR = 9.00, CI = 6.33-13.51) higher risk of anxiety compared to those with under 69. CONCLUSION: Aggression and anxiety are important aspects of mental health in adolescents. Our results demonstrated that higher risk of anxiety was associated with total aggression scores. In particular, indirect aggression (i.e. anger and hostility) was more closely associated with anxiety than direct aggression.

Author Correction: ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors.

In the version of this Article originally published, Supplementary Fig. 6j showed incorrect values for the LS and AG4 glutathione samples, and Fig. 5c and Supplementary Fig. 6j did not include all n = 6 samples for the hESC, Y-hiPSC and AG4-ZSCAN10 groups as was stated in the legend. In addition, the bars for hESC, Y-hiPSC, AG4-ZCNAN10, AG4 and LS in Supplementary Fig. 6i and j have been reproduced from Fig. 5b and c, respectively. Fig. 6e was also reproduced in the lower panel of Supplementary Fig. 6h, to enable direct comparison of the data, however this was not explained in the original figure legends. The correct versions of these figures and their legends are shown below, and Supplementary Table 5 has been updated with the source data for all numerical data in the manuscript.

Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis.

INTRODUCTION: IDH1 mutation has been identified as an early genetic event driving low grade gliomas (LGGs) and it has been proven to exerts a powerful epigenetic effect. Cells containing IDH1 mutation are refractory to epigenetical reprogramming to iPSC induced by expression of Yamanaka transcription factors, a feature that we employed to study early genetic amplifications or deletions in gliomagenesis. METHODS: We made iPSC clones from freshly surgically resected IDH1 mutant LGGs by forced expression of Yamanaka transcription factors. We sequenced the IDH locus and analyzed the genetic composition of multiple iPSC clones by array-based comparative genomic hybridization (aCGH). RESULTS: We hypothesize that the primary cell pool isolated from LGG tumor contains a heterogeneous population consisting tumor cells at various stages of tumor progression including cells with early genetic lesions if any prior to acquisition of IDH1 mutation. Because cells containing IDH1 mutation are refractory to reprogramming, we predict that iPSC clones should originate only from LGG cells without IDH1 mutation, i.e. cells prior to acquisition of IDH1 mutation. As expected, we found that none of the iPSC clones contains IDH1 mutation. Further analysis by aCGH of the iPSC clones reveals that they contain regional chromosomal amplifications which are also present in the primary LGG cells. CONCLUSIONS: These results indicate that there exists a subpopulation of cells harboring gene amplification but without IDH1 mutation in the LGG primary cell pool. Further analysis of TCGA LGG database demonstrates that these regional chromosomal amplifications are also present in some cases of low grade gliomas indicating they are reoccurring lesions in glioma albeit at a low frequency. Taken together, these data suggest that regional chromosomal alterations may exist prior to the acquisition of IDH mutations in at least some cases of LGGs.

Biological Significance of the Suppression of Oxidative Phosphorylation in Induced Pluripotent Stem Cells.

We discovered that induced pluripotent stem cell (iPSC) clones generated from aged tissue donors (A-iPSCs) fail to suppress oxidative phosphorylation. Compared to embryonic stem cells (ESCs) and iPSCs generated from young donors (Y-iPSCs), A-iPSCs show poor expression of the pluripotent stem cell-specific glucose transporter 3 (GLUT3) and impaired glucose uptake, making them unable to support the high glucose demands of glycolysis. Persistent oxidative phosphorylation in A-iPSCs generates higher levels of reactive oxygen species (ROS), which leads to excessive elevation of glutathione (a ROS-scavenging metabolite) and a blunted DNA damage response. These phenotypes were recapitulated in Y-iPSCs by inhibiting pyruvate dehydrogenase kinase (PDK) or supplying citrate to activate oxidative phosphorylation. In addition, oxidative phosphorylation in A-iPSC clones depletes citrate, a nuclear source of acetyl group donors for histone acetylation; this consequently alters histone acetylation status. Expression of GLUT3 in A-iPSCs recovers the metabolic defect, DNA damage response, and histone acetylation status.

Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8-/- or Dicer-/- embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8-/- ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8-/- ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8-/- PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs.

RNA Exosome Complex-Mediated Control of Redox Status in Pluripotent Stem Cells.

The RNA exosome complex targets AU-rich element (ARE)-containing mRNAs in eukaryotic cells. We identified a transcription factor, ZSCAN10, which binds to the promoters of multiple RNA exosome complex subunits in pluripotent stem cells to maintain subunit gene expression. We discovered that induced pluripotent stem cell clones generated from aged tissue donors (A-iPSC) show poor expression of ZSCAN10, leading to poor RNA exosome complex expression, and a subsequent elevation in ARE-containing RNAs, including glutathione peroxidase 2 (Gpx2). Excess GPX2 leads to excess glutathione-mediated reactive oxygen species scavenging activity that blunts the DNA damage response and apoptosis. Expression of ZSCAN10 in A-iPSC recovers RNA exosome gene expression, the DNA damage response, and apoptosis. These findings reveal the central role of ZSCAN10 and the RNA exosome complex in maintaining pluripotent stem cell redox status to support a normal DNA damage response.

ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors.

Induced pluripotent stem cells (iPSCs), which are used to produce transplantable tissues, may particularly benefit older patients, who are more likely to suffer from degenerative diseases. However, iPSCs generated from aged donors (A-iPSCs) exhibit higher genomic instability, defects in apoptosis and a blunted DNA damage response compared with iPSCs generated from younger donors. We demonstrated that A-iPSCs exhibit excessive glutathione-mediated reactive oxygen species (ROS) scavenging activity, which blocks the DNA damage response and apoptosis and permits survival of cells with genomic instability. We found that the pluripotency factor ZSCAN10 is poorly expressed in A-iPSCs and addition of ZSCAN10 to the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) during A-iPSC reprogramming normalizes ROS-glutathione homeostasis and the DNA damage response, and recovers genomic stability. Correcting the genomic instability of A-iPSCs will ultimately enhance our ability to produce histocompatible functional tissues from older patients' own cells that are safe for transplantation.

Canonical microRNAs Enable Differentiation, Protect Against DNA Damage, and Promote Cholesterol Biosynthesis in Neural Stem Cells.

Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, and therefore represent a promising donor tissue source for treating neurodegenerative diseases and repairing injuries of the nervous system. However, it remains unclear how canonical microRNAs (miRNAs), the subset of miRNAs requiring the Drosha-Dgcr8 microprocessor and the type III RNase Dicer for biogenesis, regulate NSCs. In this study, we established and characterized Dgcr8-/- NSCs from conditionally Dgcr8-disrupted mouse embryonic brain. RNA-seq analysis demonstrated that disruption of Dgcr8 in NSCs causes a complete loss of canonical miRNAs and an accumulation of pri-miRNAs. Dgcr8-/- NSCs can be stably propagated in vitro, but progress through the cell cycle at reduced rates. When induced for differentiation, Dgcr8-/- NSCs failed to differentiate into neurons, astrocytes, or oligodendrocytes under permissive conditions. Compared to Dgcr8+/- NSCs, Dgcr8-/- NSCs exhibit significantly increased DNA damage. Comparative RNA-seq analysis and gene set enrichment analysis (GSEA) revealed that Dgcr8-/- NSCs significantly downregulate genes associated with neuronal differentiation, cell cycle progression, DNA replication, protein translation, and DNA damage repair. Furthermore, we discovered that Dgcr8-/- NSCs significantly downregulate genes responsible for cholesterol biosynthesis and demonstrated that Dgcr8-/- NSCs contain lower levels of cholesterol. Together, our data demonstrate that canonical miRNAs play essential roles in enabling lineage specification, protecting DNA against damage, and promoting cholesterol biosynthesis in NSCs.

Canonical MicroRNA Activity Facilitates but May Be Dispensable for Transcription Factor-Mediated Reprogramming.

MicroRNAs (miRNAs) are important regulators of reprogramming of somatic cells into induced pluripotent stem cells (iPSCs); however, it is unclear whether miRNAs are required for reprogramming and whether miRNA activity as a whole facilitates reprogramming. Here we report on successful reprogramming of mouse fibroblasts and neural stem cells (NSCs) lacking Dgcr8, a factor required for the biogenesis of canonical miRNAs, by Yamanaka factors, albeit at decreased efficiencies. Though iPSCs derived from Dgcr8-deficient mouse fibroblasts or NSCs were able to self-renew and expressed pluripotency-associated markers, they exhibited poor differentiation potential into mature somatic tissues, similar to Dgcr8(-/-) embryonic stem cells. The differentiation defects could be rescued with expression of DGCR8 cDNA. Our data demonstrate that while miRNA activity as a whole facilitates reprogramming, canonical miRNA may be dispensable in the derivation of iPSCs.

The epithelial-mesenchymal transition factor SNAIL paradoxically enhances reprogramming.

Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.

Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

Multifaceted regulation of somatic cell reprogramming by mRNA translational control.

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.

Association between Internet overuse and aggression in Korean adolescents.

BACKGROUND: The purpose of this study was to examine the association between Internet overuse and aggression. METHODS: A total of 2336 high school students (boys, 57.5%; girls, 42.5%) in South Korea completed the structured questionnaire. The severity of Internet overuse was evaluated using Young's Internet Addiction Test. Aggression was measured using the Aggression Questionnaire, a modified hostility inventory by Buss and Perry. RESULTS: The proportions of boys who were classified as severe addicts and moderate addicts were 2.5% and 53.7%, respectively. For girls, the corresponding proportions were 1.9% and 38.9%, respectively. manova results for univariate analysis showed that sex, duration of Internet use, most-frequently used Internet activities, level of Internet addiction, smoking, alcohol, and caffeine were significantly associated with aggression scores. From multivariate analysis, it was found that smoking, alcohol, and level of Internet addiction were independently associated with all aggressive characteristics. Internet addiction scores were also significantly associated with all aggression scores from simple and multiple linear regression analyses (parameter estimate = 0.54-0.58 for total aggression). Pearson correlation results showed that Internet addiction scores revealed the highest correlation coefficients with aggression among Internet-related factors, age, and sex. Severe Internet-addicted boys showed higher scores in all aggression characteristics than severe Internet-addicted girls, even though it was not statistically significant in every characteristic. However, there was no sex effect on the association between Internet overuse and aggression. CONCLUSION: This study shows that Internet overuse is strongly associated with aggression in adolescents.