A systematic approach to reduce intraocular pressure for the treatment of glaucoma.

Glaucoma is the leading cause of irreversible blindness due to increased intraocular pressure (IOP) in the eye. We have developed a novel treatment option for glaucoma based on a real-time IOP-dependent nitric oxide synthase (NOS) and packed in a therapeutic contact lens to reduce the IOP. First, 1.6 nmole nitric oxide was produced from the genetic chassis, which was optimized for isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction in a T7 expression system. For biosafety concerns to human being, the csgAD genes responsible for curli biofilm formation in Escherichia coli were co-expressed with NOS in the designated NOSAD strain to strengthen the adherence of cells to the contact lens, thereby preventing the contamination into the eyes. Moreover, NOSAD is a diaminopimelic acid (DAP) auxotrophic strain, which cannot survive without supplementation of DAP and reached the critical consideration of biosafety to the environment. We also demonstrated that the nitric oxide diffusion was 3.6-times enhanced from penetration into the aqueous humor of porcine eyes. The deformation ratio of the contact lens was correlated to the change of IOP by using a digital image correlation (DIC) system in a porcine eye model. The novel systematic approach provides an alternative treatment for glaucoma patients in the future.

Shared genetic risk between eating disorder- and substance-use-related phenotypes: Evidence from genome-wide association studies.

Eating disorders and substance use disorders frequently co-occur. Twin studies reveal shared genetic variance between liabilities to eating disorders and substance use, with the strongest associations between symptoms of bulimia nervosa and problem alcohol use (genetic correlation [rg ], twin-based = 0.23-0.53). We estimated the genetic correlation between eating disorder and substance use and disorder phenotypes using data from genome-wide association studies (GWAS). Four eating disorder phenotypes (anorexia nervosa [AN], AN with binge eating, AN without binge eating, and a bulimia nervosa factor score), and eight substance-use-related phenotypes (drinks per week, alcohol use disorder [AUD], smoking initiation, current smoking, cigarettes per day, nicotine dependence, cannabis initiation, and cannabis use disorder) from eight studies were included. Significant genetic correlations were adjusted for variants associated with major depressive disorder and schizophrenia. Total study sample sizes per phenotype ranged from ~2400 to ~537 000 individuals. We used linkage disequilibrium score regression to calculate single nucleotide polymorphism-based genetic correlations between eating disorder- and substance-use-related phenotypes. Significant positive genetic associations emerged between AUD and AN (rg = 0.18; false discovery rate q = 0.0006), cannabis initiation and AN (rg = 0.23; q < 0.0001), and cannabis initiation and AN with binge eating (rg = 0.27; q = 0.0016). Conversely, significant negative genetic correlations were observed between three nondiagnostic smoking phenotypes (smoking initiation, current smoking, and cigarettes per day) and AN without binge eating (rgs = -0.19 to -0.23; qs < 0.04). The genetic correlation between AUD and AN was no longer significant after co-varying for major depressive disorder loci. The patterns of association between eating disorder- and substance-use-related phenotypes highlights the potentially complex and substance-specific relationships among these behaviors.

Meta-analysis of up to 622,409 individuals identifies 40 novel smoking behaviour associated genetic loci.

Smoking is a major heritable and modifiable risk factor for many diseases, including cancer, common respiratory disorders and cardiovascular diseases. Fourteen genetic loci have previously been associated with smoking behaviour-related traits. We tested up to 235,116 single nucleotide variants (SNVs) on the exome-array for association with smoking initiation, cigarettes per day, pack-years, and smoking cessation in a fixed effects meta-analysis of up to 61 studies (up to 346,813 participants). In a subset of 112,811 participants, a further one million SNVs were also genotyped and tested for association with the four smoking behaviour traits. SNV-trait associations with P < 5 × 10-8 in either analysis were taken forward for replication in up to 275,596 independent participants from UK Biobank. Lastly, a meta-analysis of the discovery and replication studies was performed. Sixteen SNVs were associated with at least one of the smoking behaviour traits (P < 5 × 10-8) in the discovery samples. Ten novel SNVs, including rs12616219 near TMEM182, were followed-up and five of them (rs462779 in REV3L, rs12780116 in CNNM2, rs1190736 in GPR101, rs11539157 in PJA1, and rs12616219 near TMEM182) replicated at a Bonferroni significance threshold (P < 4.5 × 10-3) with consistent direction of effect. A further 35 SNVs were associated with smoking behaviour traits in the discovery plus replication meta-analysis (up to 622,409 participants) including a rare SNV, rs150493199, in CCDC141 and two low-frequency SNVs in CEP350 and HDGFRP2. Functional follow-up implied that decreased expression of REV3L may lower the probability of smoking initiation. The novel loci will facilitate understanding the genetic aetiology of smoking behaviour and may lead to the identification of potential drug targets for smoking prevention and/or cessation.

A regulatory variant of CHRM3 is associated with cannabis-induced hallucinations in European Americans.

Cannabis, the most widely used illicit drug, can induce hallucinations. Our understanding of the biology of cannabis-induced hallucinations (Ca-HL) is limited. We used the Semi-Structured Assessment for Drug Dependence and Alcoholism (SSADDA) to identify cannabis-induced hallucinations (Ca-HL) among long-term cannabis users (used cannabis ≥1 year and ≥100 times). A genome-wide association study (GWAS) was conducted by analyzing European Americans (EAs) and African Americans (AAs) in Yale-Penn 1 and 2 cohorts individually, then meta-analyzing the two cohorts within population. In the meta-analysis of Yale-Penn EAs (n = 1917), one genome-wide significant (GWS) signal emerged at the CHRM3 locus, represented by rs115455482 (P = 1.66 × 10-10), rs74722579 (P = 2.81 × 10-9), and rs1938228 (P = 1.57 × 10-8); signals were GWS in Yale-Penn 1 EAs (n = 1092) and nominally significant in Yale-Penn 2 EAs (n = 825). Two SNPs, rs115455482 and rs74722579, were available from the Collaborative Study on the Genetics of Alcoholism data (COGA; 3630 long-term cannabis users). The signals did not replicate, but when meta-analyzing Yale-Penn and COGA EAs, the two SNPs' association signals were increased (meta-P-values 1.32 × 10-10 and 2.60 × 10-9, respectively; n = 4291). There were no significant findings in AAs, but in the AA meta-analysis (n = 3624), nominal significance was seen for rs74722579. The rs115455482*T risk allele was associated with lower CHRM3 expression in the thalamus. CHRM3 was co-expressed with three psychosis risk genes (GABAG2, CHRNA4, and HRH3) in the thalamus and other human brain tissues and mouse GABAergic neurons. This work provides strong evidence for the association of CHRM3 with Ca-HL and provides insight into the potential involvement of thalamus for this trait.

The etiology of DSM-5 alcohol use disorder: Evidence of shared and non-shared additive genetic effects.

BACKGROUND: Alcoholism is a multifactorial disorder influenced by multiple gene loci, each with small effect. Studies suggest shared genetic influences across DSM-IV alcohol dependence symptoms, but shared effects across DSM-5 alcohol use disorder remains unknown. We aimed to test the assumption of genetic homogeneity across the 11 criteria of DSM-5 alcohol use disorder (AUD). METHODS: Data from 2596 alcohol using individuals of European ancestry from the Study of Addiction: Genetics and Environment were used to examine the genomewide SNP-heritability (h2SNP) and SNP-covariance (rGSNP) between 11 DSM-5 AUD symptoms. Phenotypic relationships between symptoms were examined to confirm an underlying liability of AUD and the SNP-heritability of the observed latent trait and the co-heritabilityamong AUD symptoms was assessed using Genomic-Relatedness-Matrix-Restricted-Maximum-Likelihood. Genetic covariance among symptoms was examined using factor analysis. RESULTS: Phenotypic relationships confirmed a unidimensional underlying liability to AUD. Factor and parallel analyses of the observed genetic variance/covariance provided evidence of genetic homogeneity. Additive genetic effects on DSM-5 AUD symptoms varied from 0.10 to 0.37 and largely overlapped (rG-SNP across symptoms ranged from 0.49 - 0.92). The additive genetic effect on the DSM-5 AUD factor was 0.36, 0.14 for DSM-5 AUD diagnosis, and was 0.22 for DSM-5 AUD severity. CONCLUSIONS: Common genetic variants influence DSM-5 AUD symptoms. Despite evidence for a common AUD factor, the evidence of only partially overlapping genetic effects across AUD symptoms further substantiates the need to simultaneously model common and symptom-specific genetic effects in molecular genetic studies in order to best characterize the genetic liability.

Cisplatin exposure impairs ionocytes and hair cells in the skin of zebrafish embryos.

This study aimed to assess the sublethal effects of a platinum-based compound, cisplatin, using a zebrafish model. Zebrafish embryos were incubated in different concentrations of cisplatin at 0-96 h post-fertilization. Using a non-invasive, scanning ion-selective electrode technique (SIET), we measured the functions of hair cells (Ca2+ influx) and ionocytes ([H+] gradients). The survival rate, hatching rate, phenotype, body length, whole-body ion (Na+, Cl-, and Ca2+) and Pt contents were also determined. The effects of cisplatin on zebrafish embryos were demonstrated as first impairing hair cell function (at 1 µM of cisplatin), the hair cell number, and body ion content of Cl- (at 10 µM of cisplatin), then decreasing ionocyte acid secretion and overall body ion contents of Na+ and Ca2+ (at 50 µM of cisplatin). The body length and ionocyte density decreased at 100 µM of cisplatin, and survival decreased at 500 µM of cisplatin. As the cisplatin concentration increased, the accumulation of Pt in fish embryos also increased. These results revealed that hair cells are significantly more susceptible to cisplatin toxicity than ionocytes. By determining the lowest observed effective concentration of cisplatin that caused in vivo functional alterations of zebrafish hair cells and skin ionocytes, this model demonstrated 500-fold greater sensitivity than by detecting changes in survival, for early assessment of the effects of platinum-based chemotherapeutic drugs on fish.

Exome Chip Meta-analysis Fine Maps Causal Variants and Elucidates the Genetic Architecture of Rare Coding Variants in Smoking and Alcohol Use.

BACKGROUND: Smoking and alcohol use have been associated with common genetic variants in multiple loci. Rare variants within these loci hold promise in the identification of biological mechanisms in substance use. Exome arrays and genotype imputation can now efficiently genotype rare nonsynonymous and loss of function variants. Such variants are expected to have deleterious functional consequences and to contribute to disease risk. METHODS: We analyzed ∼250,000 rare variants from 16 independent studies genotyped with exome arrays and augmented this dataset with imputed data from the UK Biobank. Associations were tested for five phenotypes: cigarettes per day, pack-years, smoking initiation, age of smoking initiation, and alcoholic drinks per week. We conducted stratified heritability analyses, single-variant tests, and gene-based burden tests of nonsynonymous/loss-of-function coding variants. We performed a novel fine-mapping analysis to winnow the number of putative causal variants within associated loci. RESULTS: Meta-analytic sample sizes ranged from 152,348 to 433,216, depending on the phenotype. Rare coding variation explained 1.1% to 2.2% of phenotypic variance, reflecting 11% to 18% of the total single nucleotide polymorphism heritability of these phenotypes. We identified 171 genome-wide associated loci across all phenotypes. Fine mapping identified putative causal variants with double base-pair resolution at 24 of these loci, and between three and 10 variants for 65 loci. Twenty loci contained rare coding variants in the 95% credible intervals. CONCLUSIONS: Rare coding variation significantly contributes to the heritability of smoking and alcohol use. Fine-mapping genome-wide association study loci identifies specific variants contributing to the biological etiology of substance use behavior.

Transancestral GWAS of alcohol dependence reveals common genetic underpinnings with psychiatric disorders.

Liability to alcohol dependence (AD) is heritable, but little is known about its complex polygenic architecture or its genetic relationship with other disorders. To discover loci associated with AD and characterize the relationship between AD and other psychiatric and behavioral outcomes, we carried out the largest genome-wide association study to date of DSM-IV-diagnosed AD. Genome-wide data on 14,904 individuals with AD and 37,944 controls from 28 case-control and family-based studies were meta-analyzed, stratified by genetic ancestry (European, n = 46,568; African, n = 6,280). Independent, genome-wide significant effects of different ADH1B variants were identified in European (rs1229984; P = 9.8 × 10-13) and African ancestries (rs2066702; P = 2.2 × 10-9). Significant genetic correlations were observed with 17 phenotypes, including schizophrenia, attention deficit-hyperactivity disorder, depression, and use of cigarettes and cannabis. The genetic underpinnings of AD only partially overlap with those for alcohol consumption, underscoring the genetic distinction between pathological and nonpathological drinking behaviors.

Nutrient intake determines post-maturity molting in the golden orb-web spider Nephila pilipes (Araneae: Araneidae).

While molting occurs in the development of many animals, especially arthropods, post-maturity molting (PMM, organisms continue to molt after sexual maturity) has received little attention. The mechanism of molting has been studied intensively; however, the mechanism of PMM remains unknown although it is suggested to be crucial for the development of body size. In this study, we investigated factors that potentially induce PMM in the golden orb-web spider Nephila pilipes, which has the greatest degree of sexual dimorphism among terrestrial animals. We manipulated the mating history and the nutrient consumption of the females to examine whether they affect PMM. The results showed that female spiders under low nutrition were more likely to molt as adults, and mating had no significant influence on the occurrence of PMM. Moreover, spiders that underwent PMM lived longer than those that did not and their body sizes were significantly increased. Therefore, we concluded that nutritional condition rather than mating history affect PMM.

Instantaneous responses to high-frequency chest wall oscillation in patients with acute pneumonic respiratory failure receiving mechanical ventilation: A randomized controlled study.

BACKGROUND: Endotracheal intubation and prolonged immobilization of patients receiving mechanical ventilation may reduce expectoration function. High-frequency chest wall oscillation (HFCWO) may ameliorate airway secretion movement; however, the instantaneous changes in patients' cardiopulmonary responses are unknown. Moreover, HFCWO may influence ventilator settings by the vigorous oscillation. The aim of this study was to investigate these issues. METHODS: Seventy-three patients (52 men) aged 71.5 ±â€Š13.4 years who were intubated with mechanical ventilation for pneumonic respiratory failure were recruited and randomly classified into 2 groups (HFCWO group, n = 36; and control group who received conventional chest physical therapy (CCPT, n = 37). HFCWO was applied with a fixed protocol, whereas CCPT was conducted using standard protocols. Both groups received sputum suction after the procedure. Changes in ventilator settings and the subjects' responses were measured at preset intervals and compared within groups and between groups. RESULTS: Oscillation did not affect the ventilator settings (all P > 0.05). The mean airway pressure, breathing frequency, and rapid shallow breathing index increased, and the tidal volume and SpO2 decreased (all P < 0.05). After sputum suction, the peak airway pressure (Ppeak) and minute ventilation decreased (all P < 0.05). The HFCWO group had a lower tidal volume and SpO2 at the end of oscillation, and lower Ppeak and tidal volume after sputum suction than the CCPT group. CONCLUSIONS: HFCWO affects breathing pattern and SpO2 but not ventilator settings, whereas CCPT maintains a steadier condition. After sputum suction, HFCWO slightly improved Ppeak compared to CCPT, suggesting that the study extends the indications of HFCWO for these patients in intensive care unit. (ClinicalTrials.gov number NCT02758106, retrospectively registered.).

Genome-wide time-to-event analysis on smoking progression stages in a family-based study.

BACKGROUND: Various pivotal stages in smoking behavior can be identified, including initiation, conversion from experimenting to established use, development of tolerance, and cessation. Previous studies have shown high heritability for age of smoking initiation and cessation; however, time-to-event genome-wide association studies aiming to identify underpinning genes that accelerate or delay these transitions are missing to date. METHODS: We investigated which single nucleotide polymorphisms (SNPs) across the whole genome contribute to the hazard ratio of transition between different stages of smoking behavior by performing time-to-event analyses within a large Finnish twin family cohort (N = 1962), and further conducted mediation analyses of plausible intermediate traits for significant SNPs. RESULTS: Genome-wide significant signals were detected for three of the four transitions: (1) for smoking cessation on 10p14 (P = 4.47e-08 for rs72779075 flanked by RP11-575N15 and GATA3), (2) for tolerance on 11p13 (P = 1.29e-08 for rs11031684 in RP1-65P5.1), mediated by smoking quantity, and on 9q34.12 (P = 3.81e-08 for rs2304808 in FUBP3), independent of smoking quantity, and (3) for smoking initiation on 19q13.33 (P = 3.37e-08 for rs73050610 flanked by TRPM4 and SLC6A16) in analysis adjusted for first time sensations. Although our top SNPs did not replicate, another SNP in the TRPM4-SLC6A16 gene region showed statistically significant association after region-based multiple testing correction in an independent Australian twin family sample. CONCLUSION: Our results suggest that the functional effect of the TRPM4-SLC6A16 gene region deserves further investigation, and that complex neurotransmitter networks including dopamine and glutamate may play a critical role in smoking initiation. Moreover, comparison of these results implies that genetic contributions to the complex smoking behavioral phenotypes vary among the transitions.

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes.

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

A 3p26-3p25 genetic linkage finding for DSM-IV major depression in heavy smoking families.

OBJECTIVE: The authors tested for genetic linkage of DSM-IV-diagnosed major depressive disorder in families that were ascertained for cigarette smoking. METHOD: Within a study that targeted families characterized by a history of smoking, analyses derived a subset of 91 Australian families with two or more offspring with a history of DSM-IV major depressive disorder (affected sibling pairs, N=187) and 25 Finnish families (affected sibling pairs, N=33). Within this affected sibling pairs design, the authors conducted nonparametric linkage analysis. RESULTS: In the Australian heavy smoking families, the authors found a genome-wide significant multipoint LOD score of 4.14 for major depressive disorder on chromosome 3 at 24.9 cM (3p26-3p25). CONCLUSIONS: Genome-wide significant linkage was detected for major depressive disorder on chromosome 3p in a sample ascertained for smoking. A linkage peak at this location was also observed in an independent study of major depressive disorder.

A quantitative-trait genome-wide association study of alcoholism risk in the community: findings and implications.

BACKGROUND: Given moderately strong genetic contributions to variation in alcoholism and heaviness of drinking (50% to 60% heritability) with high correlation of genetic influences, we have conducted a quantitative trait genome-wide association study (GWAS) for phenotypes related to alcohol use and dependence. METHODS: Diagnostic interview and blood/buccal samples were obtained from sibships ascertained through the Australian Twin Registry. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed with 8754 individuals (2062 alcohol-dependent cases) selected for informativeness for alcohol use disorder and associated quantitative traits. Family-based association tests were performed for alcohol dependence, dependence factor score, and heaviness of drinking factor score, with confirmatory case-population control comparisons using an unassessed population control series of 3393 Australians with genome-wide SNP data. RESULTS: No findings reached genome-wide significance (p = 8.4 × 10(-8) for this study), with lowest p value for primary phenotypes of 1.2 × 10(-7). Convergent findings for quantitative consumption and diagnostic and quantitative dependence measures suggest possible roles for a transmembrane protein gene (TMEM108) and for ANKS1A. The major finding, however, was small effect sizes estimated for individual SNPs, suggesting that hundreds of genetic variants make modest contributions (1/4% of variance or less) to alcohol dependence risk. CONCLUSIONS: We conclude that 1) meta-analyses of consumption data may contribute usefully to gene discovery; 2) translation of human alcoholism GWAS results to drug discovery or clinically useful prediction of risk will be challenging; and 3) through accumulation across studies, GWAS data may become valuable for improved genetic risk differentiation in research in biological psychiatry (e.g., prospective high-risk or resilience studies).