Electronic vapor product use and suicidal behavior in adolescents

Objective: To determine whether an association exists between electronic vapor product use (EVP) and suicidal behavior in middle and high school students Methods: Multilevel logistic regression analysis for suicidal behaviors using data from the 2017 Vermont Youth Risk Behavior Survey. Results: Middle school EVP users were significantly more likely to report suicidal behavior than non-users. No statistically significant association was found in high school students. Discussion: While further study is necessary to clarify the relationship between EVP use and suicidal behavior, these findings highlight the need for suicide prevention and smoking cessation efforts

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase.

The NEIL1 DNA glycosylase is one of eleven mammalian DNA glycosylases that partake in the first step of the base excision repair (BER) pathway. NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating (alanine) mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1 in vitro, and was able to phosphorylate the enzyme at residues S207, S306, and S61

Binding of NEIL1 and mutant enzymes to DNA.

<p>(A) Representative phosphor-autodiogram for binding of NEIL1-WT to a 35-mer furan-containing DNA where the substrate (10 pM) was incubated with increasing (0–600 nM) amounts of enzyme. Complex formation is indicated by the presence of shifted bands C1 and C2. (B) Graphical fitting of the EMSA data indicated above using GraphPad Prism 6. The data were fit to the one-site specific binding equation. The K_d values for WT and the NEIL1 mutants are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.t001" target="_blank">Table 1</a> and are representative of experiments performed in duplicate.</p

The JNK1 kinase interacts with NEIL1.

<p>(A) Western blot analysis of endogenous JNK1 present in HEK293T WCE and in NEIL1-FLAG immunoprecipitates. (B) Far-Western analysis to confirm the interaction between NEIL1 and JNK1. Top panel, Coomassie stained SDS-PAGE gel of NEIL1 and its phosphomimetic and ablating mutants (50 pmoles). Bottom panel, membrane after transfer, denaturation, slow refolding, binding to JNK1 from HEK293 WCE, and probing with an anti-JNK1 antibody. BSA was used as a negative control.</p

Phosphorylation of NEIL1 by JNK1 using an <i>in vitro</i> kinase assay.

<p>(A) <i>In vitro</i> kinase assays were performed with NEIL1 constructs expressed from E. coli cells and purified to homogeneity and active JNK1 kinase for 30 minutes at 32°C. γ-³²P incorporation was quantified using phosphor-autoradiography after SDS-PAGE analysis of the samples. Lane 1, no JNK1 control; lane 2, no NEIL1 control; lane 3–13 are WT, S207E, S207A, S306E, S306A, S61E, S61A, DE, DA, TE, TA, respectively. (B) Graphical representation of two experimental repeats of the <i>in vitro</i> kinase assay. Statistically significant values (at 95% confidence) were determined by a one-way Anova test where * denotes p-values <0.05, *** denotes p-values <0.0005, and **** denotes p values <0.0001. (C) Glycosylase activity assays were performed using Sp:C and AP:C substrates with increasing amounts of <i>in vitro</i> phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.</p

Sites of phosphorylation within the NEIL1 DNA glycosylase.

<p>(A) Domain map of NEIL1 indicating the position of known sites of phosphorylation. The residues S207, S306, and S61 identified in this study are shown in blue and the Y263 and S269 sites previously identified [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.ref039" target="_blank">39</a>] are indicated in black. (B) SDS-PAGE gel of SBP-tagged NEIL1 after affinity pull-down from HEK293T cell-extracts overexpressing NEIL1. The gel was stained with Coomassie blue and the NEIL1-SBP band was cut from the gel and digested with trypsin for identification of phosphorylated peptides via LC-MS/MS.</p

Identification of phosphorylation sites on NEIL1 by LC-MS/MS.

<p>(A) List of phosphorylated peptides identified from LC-MS/MS analysis of NEIL1-SBP with their corresponding monoisotopic mass m/z, charge-state (z), and Xcorr values. (B) Product-ion spectra (Scaffold software 4.3) for each phosphorylated peptide are displayed. The b- and y-ions are shown in red and blue, respectively, and the neutral loss peak resulting from the loss of a phosphogroup from the parent ion is indicated in green.</p

Glycosylase and lyase activity panel for human NEIL1-WT and the phosphomimetic/ablating mutants.

<p>Glycosylase assays were performed by incubating 20 nM of double-stranded DNA substrates (A) Sp:C and (B) AP:C and increasing amounts of enzyme with the following substrate to enzyme ratios: 1:0.5, 1:1, 1:4, and 1:16. “-” indicates a no enzyme negative control. Assays were performed at room temperature for 30 minutes. S and P indicate substrate and product, respectively. Data shown are representative of duplicate experiments.</p

Phosphomimetic/ phosphoablating mutants of NEIL1.

<p>Phosphomimetic/ phosphoablating mutants of NEIL1.</p