Trinucleotide expansions cause disease by both protein-and RNAmediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.T ranslation of mRNA into protein is an exquisitely regulated, almost error-free process. Well-established rules of translational initiation have been used as a cornerstone in biology to understand gene expression and to predict the consequences of disease-causing mutations (1). For microsatellite expansion disorders, mutations within or outside ATG-initiated ORFs are thought to cause disease either by protein gain-of-function, protein loss-of-function, or RNA gain-of-function mechanisms (2, 3).Microsatellite expansion mutations that express polyglutamine (polyGln) expansion proteins include Huntington disease (Huntingtin, HD), spinal bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17. Since the discovery of these CAG·CTG expansion mutations, efforts to understand disease mechanisms have focused on elucidating the molecular effects of the polyGln proteins expressed from these loci. Although these polyGln expansion proteins bear no similarity to each other apart from the polyGln tract, a hallmark of these diseases is protein accumulation and aggregation in nuclear or cytoplasmic inclusions. Surprisingly, although the polyGln expansion proteins are widely expressed in the CNS and other tissues, only restricted populations of neurons are vulnerable in each disease (3).Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the best-characterized examples of RNA-mediated expansion disorders (2). The mutation causing DM1 is a CTG-repeat expansion located in the 3′ UTR of the dystrophia myotonica-protein kinase (DMPK) gene. Although DM1 can be clinically more severe than DM2, the discovery of the DM2 mutation and several mouse models provide strong support that many features of these diseases result from RNA gain-of-function effects in which the dysregulation of RNA-binding proteins is mediated by the expression of CUG and CCUG transcripts (4). Additionally, RNA gain-of-function effects have been reported for CGG and CAG expansion RNAs (5, 6).Both RNA and protein mechanisms appear to operate...

IMPORTANCE Exposure to nicotine in electronic cigarettes (e-cigarettes) is becoming increasingly common among adolescents who report never having smoked combustible tobacco.OBJECTIVE To evaluate whether e-cigarette use among 14-year-old adolescents who have never tried combustible tobacco is associated with risk of initiating use of 3 combustible tobacco products (ie, cigarettes, cigars, and hookah). DESIGN, SETTING, AND PARTICIPANTSLongitudinal repeated assessment of a school-based cohort at baseline (fall 2013, 9th grade, mean age = 14.1 years) and at a 6-month follow-up (spring 2014, 9th grade) and a 12-month follow-up (fall 2014, 10th grade). Ten public high schools in Los Angeles, California, were recruited through convenience sampling. Participants were students who reported never using combustible tobacco at baseline and completed follow-up assessments at 6 or 12 months (N = 2530). At each time point, students completed self-report surveys during in-classroom data collections. EXPOSURE Student self-report of whether he or she ever used e-cigarettes (yes or no) at baseline. MAIN OUTCOMES AND MEASURESSix-and 12-month follow-up reports on use of any of the following tobacco products within the prior 6 months: (1) any combustible tobacco product (yes or no); (2) combustible cigarettes (yes or no), (3) cigars (yes or no); (4) hookah (yes or no); and (5) number of combustible tobacco products (range: 0-3).RESULTS Past 6-month use of any combustible tobacco product was more frequent in baseline e-cigarette ever users (n = 222) than never users (n = 2308) at the 6-month follow-up (30.7% vs 8.1%, respectively; difference between groups in prevalence rates, 22.7% [95% CI, 16.4%-28.9%]) and at the 12-month follow-up (25.2% vs 9.3%, respectively; difference between groups, 15.9% [95% CI, 10.0%-21.8%]). Baseline e-cigarette use was associated with greater likelihood of use of any combustible tobacco product averaged across the 2 follow-up periods in the unadjusted analyses (odds ratio [OR], 4.27 [95% CI,) and in the analyses adjusted for sociodemographic, environmental, and intrapersonal risk factors for smoking (OR, 2.73 [95% CI, 2.00-3.73]). Product-specific analyses showed that baseline e-cigarette use was positively associated with combustible cigarette (OR, 2.65 [95% CI, 1.73-4.05]), cigar (OR, 4.85 [95% CI,), and hookah (OR, 3.25 [95% CI, 2.29-4.62]) use and with the number of different combustible products used (OR, 4.26 [95% CI,) averaged across the 2 follow-up periods.CONCLUSIONS AND RELEVANCE Among high school students in Los Angeles, those who had ever used e-cigarettes at baseline compared with nonusers were more likely to report initiation of combustible tobacco use over the next year. Further research is needed to understand whether this association may be causal.

Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within this fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. This approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three dimensional peptide fractionation involving sequential steps of preparative IEF, strong cation exchange, and capillary reversed phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.

IMPORTANCE Modern digital platforms are easily accessible and intensely stimulating; it is unknown whether frequent use of digital media may be associated with symptoms of attention-deficit/hyperactivity disorder (ADHD).OBJECTIVE To determine whether the frequency of using digital media among 15-and 16-year-olds without significant ADHD symptoms is associated with subsequent occurrence of ADHD symptoms during a 24-month follow-up.DESIGN, SETTING, AND PARTICIPANTS Longitudinal cohort of students in 10 Los Angeles County, California, high schools recruited through convenience sampling. Baseline and 6-, 12-, 18-, and 24-month follow-up surveys were administered from September 2014 (10th grade) to December 2016 (12th grade). Of 4100 eligible students, 3051 10th-graders (74%) were surveyed at the baseline assessment.EXPOSURES Self-reported use of 14 different modern digital media activities at a high-frequency rate over the preceding week was defined as many times a day (yes/no) and was summed in a cumulative index (range, 0-14).MAIN OUTCOMES AND MEASURES Self-rated frequency of 18 ADHD symptoms (never/rare, sometimes, often, very often) in the 6 months preceding the survey. The total numbers of 9 inattentive symptoms (range, 0-9) and 9 hyperactive-impulsive symptoms (range, 0-9) that students rated as experiencing often or very often were calculated. Students who had reported experiencing often or very often 6 or more symptoms in either category were classified as being ADHD symptom-positive. RESULTS Among the 2587 adolescents (63% eligible students; 54.4% girls; mean [SD] age 15.5 years [0.5 years]) who did not have significant symptoms of ADHD at baseline, the median follow-up was 22.6 months (interquartile range [IQR], months). The mean (SD) number of baseline digital media activities used at a high-frequency rate was 3.62 (3.30); 1398 students (54.1%) indicated high frequency of checking social media (95% CI, 52.1%-56.0%), which was the most common media activity. High-frequency engagement in each additional digital media activity at baseline was associated with a significantly higher odds of having symptoms of ADHD across follow-ups (OR, 1.11; 95% CI, 1.06-1.16). This association persisted after covariate adjustment (OR, 1.10; 95% CI, 1.05-1.15). The 495 students who reported no high-frequency media use at baseline had a 4.6% mean rate of having ADHD symptoms across follow-ups vs 9.5% among the 114 who reported 7 high-frequency activities (difference; 4.9%; 95% CI, 2.5%-7.3%) and vs 10.5% among the 51 students who reported 14 high-frequency activities (difference, 5.9%; 95% CI, 2.6%-9.2%).CONCLUSIONS AND RELEVANCE Among adolescents followed up over 2 years, there was a statistically significant but modest association between higher frequency of digital media use and subsequent symptoms of ADHD. Further research is needed to determine whether this association is causal.

Importance-Research indicates that e-cigarette use (vaping) among adolescents is associated with the initiation and progression of combustible cigarette smoking, yet the reasons for this association are unknown.Objective-To evaluate whether use of e-cigarettes with higher nicotine concentrations is associated with subsequent increases in the frequency and intensity of combustible cigarette smoking and vaping.Design/Setting/Participants-Prospective school-based cohort; surveys were administered in Spring (baseline, 10 th grade) and Fall (6-month follow-up, 11 th grade) 2015 among past 30-day vapers with available nicotine concentration data (N=181) from 10 high schools in the Los Angeles, CA metropolitan area. Exposure-Self-report of baseline e-cigarette nicotine concentration (i.e., none [0 mg/mL], low [1-5 mg/mL], medium [6-17 mg/mL] or high [18 mg/mL or greater]) typically used during the past 30-days. Main Outcome(s) and Measure(s)-Frequency of past 30-day combustible cigarette smoking and e-cigarette use (0 days [none], 1-2 days [infrequent], ≥ 3 days [frequent]) and daily smoking and vaping intensity (number of cigarettes smoked per day, number of vaping episodes per day and number of puffs per vaping episode) at 6-month follow-up.

Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.

E-cigarette vaping is reported by 37% of US 10th-grade adolescents 1 and is associated with subsequent initiation of combustible cigarette smoking. 2 Whether individuals who vape and transition to combustible cigarettes are experimenting or progress to more frequent and heavy smoking is unknown. In addition, because some adolescents use e-cigarettes as a smoking cessation aid, 3 adolescent smokers who vape could be more likely to reduce their smoking levels over time. Therefore, associations of vaping with subsequent smoking frequency and heaviness pattern among adolescents were examined.Methods | Respondents were students in 10 public high schools in Los Angeles County, California, enrolled in a longitudinal study approved by the University of Southern California institutional review board and detailed elsewhere. 2 This analysis used data from surveys administered during fall (baseline for this report) and spring (6-month follow-up) of 10th grade (2014)(2015).Surveys included e-cigarette and combustible cigarette use questions from prior research, 1,2 which were used to create variables for baseline vaping (never, prior [ever-vaper with no past 30-day vaping], infrequent [vaped 1-2 days during past 30 days], or frequent [vaped ≥3 days]), and baseline and follow-up past 30-day smoking frequency (nonsmoker, infrequent smoker [1-2 days], frequent smoker [≥3 days]) and heaviness (0, <1, 1, or ≥2 cigarettes per day on smoking days).Generalized estimating equation ordinal (cumulative logit) logistic regression models were used to assess the association between baseline vaping and follow-up frequency or heaviness of smoking, with adjustment for baseline smoking frequency or heaviness using SAS (SAS Institute), version 9.3. The baseline vaping × baseline smoking interaction term was then added to test differential associations of baseline vaping with follow-up smoking by baseline smoking status. Each model was retested after adjusting for age, sex, ethnicity, highest parental education, whether the student lived with both parents, ever use of alcohol or drugs, ever use of any combustible tobacco product, family history of smoking, depressive symptoms (Cronbach α = .94), UPPS Impulsive Behavior Scale lack of premeditation (α = .94) and sensationseeking (α = .91) subscales, delinquent behavior (α = .81), peer smoking, smoking susceptibility (α = .87), and smoking expectancies (α = .46). Details on covariate measures are reported elsewhere. 2 Significance was .05 (2-tailed). See modeling details in Table 1.

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.