Examining temporal trends in psychological distress and the co-occurrence of common substance use in a population-based sample of grade 7-12 students from 2013 to 2019.

PURPOSE: Characterizing trends and correlates of adolescent psychological distress is important due to observed global increases over the last 20 years. Substance use is a commonly discussed correlate, though we lack an understanding about how co-occurrence of these concerns has been changing over time. METHODS: Data came from repeated, representative, cross-sectional surveys of grade 7-12 students across Ontario, Canada conducted biennially from 2013 to 2019. Poisson regression with robust standard errors was used to examine changes in the joint association between psychological distress (operationalized as Kessler-6 [K6] scores ≥ 13) and substance use over time. Weighted prevalence ratios (PR) and their 99% confidence intervals were estimated, where p < 0.01 denotes statistical significance. RESULTS: The prevalence of psychological distress doubled between 2013 and 2019, with adjusted increases of about 1.2 times each survey year. This biennial increase did not differ based on sex, perceived social standing, school level, or any substance use. Students using substances consistently reported a higher prevalence of psychological distress (between 1.2 times and 2.7 times higher). There were similarly no differential temporal trends based on substance use for very high distress (K6 ≥ 19) or K6 items explored individually. CONCLUSION: Psychological distress steeply increased among adolescents and substance use remains important to assess and address alongside distress. However, the magnitude of temporal increases appears to be similar for adolescents reporting and not reporting substance use.

Playing for more than winning: Exploring sports participation, physical activity, and belongingness and their relationship with patterns of adolescent substance use and mental health.

BACKGROUND: Promoting adolescent sports participation and physical activity may be effective low-barrier prevention strategies for co-occurring adolescent substance use (SU) and mental health symptoms (MH). The objectives of this study were to: 1) explore associations between profiles of SU/MH and sports participation; and 2) determine whether physical activity and belongingness account for these associations. METHODS: Data came from a representative sample of 11,994 grade 9-12 Ontarian students (ages ~14-18) previously grouped into five SU/MH profiles based on patterns of use and symptoms. A series of multinomial logistic regressions, adjusted for socio-demographics and school clustering, were used to predict the risks of students belonging to SU/MH profiles based on: 1) school sports participation (>=weekly), 2) sports and physical activity (>=60minutes; 0-7 days), and 3) sports, physical activity, and school belongingness. RESULTS: Greater school sports participation, physical activity, and belongingness were each associated with reduced risks of belonging to most profiles with elevations in SU and/or MH symptoms relative to the low SU/MH profile (Relative Risk Ratios: sports=0.62-0.87, physical activity=0.78-0.98, belonging=0.75-0.83). Frequency of physical activity accounted for ~32-60% of the associations between sports and SU/MH profiles, while school belongingness accounted for the remaining associations. Physical activity and belongingness remained independently associated with SU/MH profiles. CONCLUSIONS: Findings suggest possible indirect associations between school sports participation and SU/MH profiles through physical activity and school belongingness, which may be promising prevention targets that have independent associations over and above sports. School sports participation may be one of a number of ways to achieve these goals.

Association of CYP2C9*2 with bosentan-induced liver injury.

Bosentan (Tracleer) is an endothelin receptor antagonist prescribed for the treatment of pulmonary arterial hypertension (PAH). Its use is limited by drug-induced liver injury (DILI). To identify genetic markers of DILI, association analyses were performed on 56 Caucasian PAH patients receiving bosentan. Twelve functional polymorphisms in five genes (ABCB11, ABCC2, CYP2C9, SLCO1B1, and SLCO1B3) implicated in bosentan pharmacokinetics were tested for associations with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and DILI. After adjusting for body mass index, CYP2C9*2 was the only polymorphism associated with ALT, AST, and DILI (ß = 2.16, P = 0.024; ß = 1.92, P = 0.016; odds ratio 95% CI = 2.29-∞, P = 0.003, respectively). Bosentan metabolism by CYP2C9*2 in vitro was significantly reduced compared with CYP2C9*1 and was comparable to that by CYP2C9*3. These results suggest that CYP2C9*2 is a potential genetic marker for prediction of bosentan-induced liver injury and warrants investigation for the optimization of bosentan treatment.

Preclinical assessment of the absorption, distribution, metabolism and excretion of GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide), an orally bioavailable systemic Hedgehog signalling pathway inhibitor.

GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers. The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3 ml min(-1) kg(-1) in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg(-1) in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096-0.649 ml min(-1) kg(-1) and the predicted volume of distribution was 0.766 l kg(-1). Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes. Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1-M3) and three sequential glucuronides (M4-M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3). GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC50 estimates greater than 20 microM. K(i)'s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24 microM, respectively. An evaluation with Simcyp suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15 microM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells. Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.

Metabolism and disposition of [(14)C]1-nitronaphthalene in male Sprague-Dawley rats.

In rats and mice, 1-nitronaphthalene (1-NN) produces both lung and liver toxicity. Even though these toxicities have been reported, the metabolism and disposition of 1-NN have not been elucidated. Therefore, studies were performed to characterize its fate after i.p. and i.v. administration to male Sprague-Dawley rats. After i.p. administration of [(14)C]1-NN (100 mg/kg; 60 microCi/kg), 84% of the dose was eliminated in the urine and feces by 48 h. At 96 h, 60% of the dose was recovered in the urine, 32% in the feces, and 1% collectively in the tissues, blood, and gastrointestinal contents. The terminal phase rate constant (k(term)) of 1-NN was 0.21 h(-1), the terminal phase half-life (T(1/2,term)) was 3.40 h, and the systemic bioavailability was 0.67. When administered i.v. (10 mg/kg; 120 microCi/kg), 85% of the dose was eliminated in the urine and feces by 24 h. At the end of the study (96 h), 56% of the dose was recovered in the urine, 36% in the feces, and 1% collectively in the tissues, blood, and gastrointestinal contents. Interestingly, 88% of the dose was secreted into bile by 8 h. The k(term) was 0.94 h(-1) and the T(1/2,term) was 0.77 h. The major urinary metabolite after both routes of administration was N-acetyl-S-(hydroxy-1-nitro-dihydronaphthalene)-L-cysteine. Other urinary metabolites identified include hydroxylated, dihydroxylated, glucuronidated, sulfated, and reduced metabolites, as well as dihydrodiol. The major biliary metabolite was hydroxy-glutathionyl-1-nitro-dihydronaphthalene. These data show that 1-NN undergoes extensive metabolism and enterohepatic recirculation, and the majority of the dose is eliminated in the urine.

SSB, encoding a ribosome-associated chaperone, is coordinately regulated with ribosomal protein genes.

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

The contemporary food supply of three northern Manitoba Cree communities.

A complex set of social, economic, cultural and environmental circumstances affecting native Canadians in northern regions has resulted in the dietary replacement of indigenous foods with marketed products not always of equivalent nutritional value. This article examines the current food supply in three northern Manitoba Cree communities by looking at the availability and preservation of traditional foods, the price of marketed foods and perceptions of the food supply. Data were obtained by questionnaire from older adults (over 55 years) and younger women (16-45 years) in each community. The food supply comprised a mix of traditional and marketed foods, with limited use of traditional methods of food preservation. Marketed food prices were high in communities without all-weather road access. Respondents expressed a desire for more traditional food. Promotion of traditional foods could increase nutrient intake, decrease food costs and contribute to a revival of interest in Cree culture.

Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

A heat shock transcription factor with reduced activity suppresses a yeast HSP70 mutant.

Strains carrying deletions in both the SSA1 and SSA2 HSP70 genes of Saccharomyces cerevisiae exhibit pleiotropic phenotypes, including the inability to grow at 37 degrees C or higher, reduced growth rate at permissive temperatures, increased HSP gene expression, and constitutive thermotolerance. A screen for extragenic suppressors of the ssa1 ssa2 slow-growth phenotype identified a spontaneous dominant suppressor mutation, EXA3-1 (R.J. Nelson, M. Heschl, and E.A. Craig, Genetics 131:277-285, 1992). Here we report that EXA3-1 is an allele of HSF1, which encodes the heat shock transcription factor (HSF). Strains containing the EXA3-1 allele in a wild-type background exhibit a 10- to 15-fold reduction in HSF activity during steady-state growth conditions as well as a delay in the accumulation of the SSA4, HSP26, and HSP104 mRNAs after a heat shock. EXA3-1-mediated suppression is the result of a single amino acid substitution of a highly conserved residue in the HSF DNA-binding domain which drastically reduces the ability of HSF to bind to heat shock elements as evaluated by band shift analysis. Together, these results indicate that the poor growth of ssa1 ssa2 strains is the result, at least in part, of the overproduction of a deleterious heat shock protein(s). This conclusion is supported by the fact that the levels of at least some heat shock proteins are reduced in ssa1 ssa2 cells containing the EXA3-1 allele. Surprisingly, strains containing the EXA3-1 allele in a wild-type HSP70 background grow early as well as the wild-type strain over a wide temperature range, displaying only a slight reduction in growth rate at 37 degrees Celsius, indicating that cells contain significantly more HSF activity than is require for growth under steady-state conditions.

The rightward gas vesicle operon in Halobacterium plasmid pNRC100: identification of the gvpA and gvpC gene products by use of antibody probes and genetic analysis of the region downstream of gvpC.

The extreme halophile Halobacterium halobium synthesizes intracellular gas-filled vesicles that confer buoyancy. A cluster of 13 genes on the 200-kb endogenous plasmid pNRC100 has been implicated in the biosynthesis of gas vesicles. Here, we show that two gas vesicle proteins are encoded by genes in the rightward operon, gvpA and gvpC, by Western blotting (immunoblotting) analysis with antibodies directed against LacZ-GvpA and LacZ-GvpC fusion proteins. Our results are consistent with previous data showing that the gvpA gene product is the major gas vesicle protein and demonstrate for the first time that the gvpC gene product is also present in H. halobium gas vesicles. Northern (RNA) blotting analysis showed two RNA species, an abundant 0.35-kb transcript of gvpA and a minor 2.5-kb transcript of gvpAC, and a third gene 3' to gvpAC, named gvpN. The gvpN gene encodes a hypothetical acidic protein with a molecular weight of 39,000 and a nucleotide binding motif. We used a site-directed mutagenesis method involving double recombination in Escherichia coli to insert a kanamycin resistance cassette just beyond the stop codon of gvpN. Introduction of the mutated gene cluster into an H. halobium mutant with a deletion of the entire gas vesicle gene cluster resulted in gas vesicle-positive transformants; this result suggests that gvpN is the last gene of the rightward gas vesicle transcription unit. We discuss the design and utility of the kanamycin resistance cassette for the mutagenesis of other genes in large operons.

Genetic transformation of a halophilic archaebacterium with a gas vesicle gene cluster restores its ability to float.

The halophilic archaebacterium, Halobacterium halobium, and many other aquatic bacteria synthesize gas-filled vesicles for flotation. We recently identified a cluster of 13 genes (gvpMLKJIHGFEDACN) on a 200-kb H. halobium plasmid, pNRC100, involved in gas vesicle synthesis. We have cloned and reconstructed the gvp gene cluster on an H. halobium-E. coli shuttle plasmid. Transformation of H. halobium Vac- mutants lacking the entire gas vesicle gene region with the gvp gene cluster results in restoration of their ability to float. These results open the way toward further genetic analysis of gas vesicle gene functions and directed flotation of other microorganisms with potential biotechnological applications.

Analysis of insertion mutants reveals two new genes in the pNRC100 gas vesicle gene cluster of Halobacterium halobium.

The archaebacterium, Halobacterium halobium, achieves buoyancy through synthesis of intracellular gas-filled vesicles. The plasmid-encoded gene (gvpA) specifying the major structural gas vesicle protein has previously been cloned and sequenced allowing the analysis of high-frequency mutations to the vesicle negative phenotype. Among eighteen gas vesicle mutants analyzed, four were observed to contain insertion elements 0.2 to 2 kb upstream of the structural gene. To explain the phenotype of these mutants, the upstream area was analyzed by DNA sequencing and transcriptional mapping. This analysis showed the presence of two open reading frames, gvpD and gvpE, which are of opposite transcriptional orientation to gvpA (gene order gvpA-D-E). gvpD begins 201 nucleotides from the gvpA structural gene and is 1608 nucleotides long while gvpE begins two nucleotides from the 3'-end of gvpD and is 573 nucleotides long. Primer extension analysis showed the occurrence of divergent promoters in the gvpA-gvpD intergenic region with the transcription start sites separated by 109 nucleotides. The sites of three insertion sequences in gas vesicle mutants mapped within gvpE while the fourth insertion site mapped near the N-terminal coding region of gvpD. Homology between the gvpDE gene region and a chromosomal site in a H. halobium NRC-1 derivative and in several other Halobacterium strains was identified by Southern hybridization.

High-frequency mutations in a plasmid-encoded gas vesicle gene in Halobacterium halobium.

Gas vesicle-deficient mutants of Halobacterium halobium arise spontaneously at high frequency (about 1%). The mutants are readily detected, forming translucent colonies on agar plates in contrast to opaque wild-type colonies. To investigate the mechanism of this mutation, we recently cloned a plasmid-encoded gas vesicle protein gene, gvpA, from H. halobium. In the wild-type NRC-1 strain the gvpA gene is encoded by a multicopy plasmid of approximately 150 kilobase pairs (kb). We have now characterized 18 gas vesicle-deficient mutants and 4 revertants by phenotypic and Southern hybridization analyses. Our results indicate that the mutants fall into three major classes. Class I mutants are partially gas vesicle-deficient (Vac(delta-)) and unstable, giving rise to completely gas vesicle-deficient (Vac(-)) derivatives and Vac(+) revertants at frequencies of 1-5%. The restriction map of the gvpA gene region in class I mutants is unchanged but the gene copy number is reduced compared to the Vac(+) strains. Class II mutants can be either Vac(delta-) or completely Vac(-) but are relatively stable. They contain insertion sequences within or upstream of the gvpA gene. A Vac(-) class II mutant, R1, contains the 1.3-kb insertion sequence, ISH3, within the gvpA gene, whereas four Vac(delta-) class II mutants contain other insertion sequences upstream of the gene. Class III mutants are stable Vac(-) derivatives of either the wild-type or class I mutants and have no detectable copies of the gvpA gene. Based on these results, we discuss the mechanisms of gas vesicle mutations in H. halobium.