Alternative-Splicing in the Exon-10 Region of GABAA Receptor β2 Subunit Gene: Relationships between Novel Isoforms and Psychotic Disorders

BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for beta(2)-subunit of gamma-aminobutyric acid type A (GABA(A)) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1) and beta(2S2), bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1) expression and decreased beta(2S2) expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression. Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD

GABA_A receptor subtype selectivity and structure-activity relationships of flavonoids

A flavonoid, 6-hydroxyflavone, was previously reported to bind to the benzodiazepine (BZ) site on GABA_A receptors with moderate binding affinity. In the present study, we showed that 6-hydroxyflavone partially potentiated GABA-induced currents in native GABA_A receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6-hydroxyflavone displayed significant preference for α2- and α3-containing subtypes, which were thought to mediate anxiolytic effect, compared to α1- and α5-containing subtypes expressed in HEK 293T cells. In mice, 6-hydroxyflavone exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6-hydroxyflavone as a promising drug candidate for the treatment of anxiety-like disorders. In addition, GABA_A receptor structure-efficacy relationships for flavones were also studied. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogues, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2’-hydroxyflavone analyzed in the present study, as well as 6,2’-dihydroxyflavone reported earlier. Whole-cell patch clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABA_A receptors acting through benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. Moreover, 2’-hydroxyflavone was shown to be a neutralizing modulator, different in efficacy from its structural analogue, 6,2’-dihydroxyflavone, a negative modulator of GABA_A receptors. The fact that flavone analogues differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2’-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure-efficacy relationships for flavones acting at GABA_A receptors, and hence facilitation of flavone-based drug development

GABA(A) receptor subtype selectivity underlying selective anxiolytic effect of baicalin

Baicalin, a naturally occurring flavonoid, was previously reported to induce anxiolytic-like effect devoid of sedation and myorelaxation in mice, acting through type A gamma-aminobutyric acid (GABA(A)) receptor benzodiazepine (BZ) site. The present Study further expanded the behavioral pharmacology profile of baicalin and subtype selectivity was explored as a possible mechanism underlying its in vivo effects on mice. Baicalin was characterized using convulsion, memory, and motor function related animal tests; and its selectivity towards recombinant GABA(A) receptor subtypes expressed in HEK 293T cells was determined by radioligand binding assay and electrophysiological studies. In the picrotoxin-induced seizure, step-through passive avoidance and rotarod tests, the anticonvulsant, amnesic and motor incoordination effects commonly associated with classical BZs were not observed when baicalin was administered at effective anxiolytic doses, demonstrating a separation of the anticonvulsant, amnesic and motor incoordination effects from the anxiolytic-like effect. Although baicalin exhibited higher binding affinity for the alpha(1)-containing GABAA subtype compared with alpha(2)-, alpha(3)-, and alpha(5)-containing subtypes, this was not statistically significant. In contrast to the classical BZ diazepam, baicalin showed significant preference for alpha(2)- and alpha(3)-containing subtypes compared to alpha(1)- and alpha(5)-containing subtypes in whole-cell patch clamp studies (P< 0.01). Its subtype selectivity suggested that baicalin exerted its in vivo anxiolytic-like effect mainly through the alpha(2)- and alpha(3)-containing subtypes. Therefore, the present study revealed an underlying mechanism for the selective anxiolytic profile of baicalin, suggesting alpha(2)- and alpha(3)-containing subtypes were important drug targets for flavonoid-based anxiolytics. (C) 2008 Elsevier Ltd. All rights reserved

GABA(A) receptor subtype selectivity underlying anxiolytic effect of 6-hydroxyflavone

6-Hydroxyflavone (6HF), a naturally occurring flavonoid, was previously reported to bind to type A gamma-aminobutyric acid (GABA(A)) receptors benzodiazepine (BZ) site with moderate binding affinity. In the present study, we showed that 6HF partially potentiated GABA-induced currents in native GABAA receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6HF displayed significant preference for alpha(2)- and alpha(3)-containing subtypes, which were thought to mediate anxiolytic effect, compared to of alpha(1)- and alpha(5)-containing subtypes expressed in HEK 293T cells. In mice, 6HF exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6HF as a promising drug candidate for the treatment of anxiety-like disorders. (C) 2010 Elsevier Inc. All rights reserved

Methodological reporting of randomized trials in five leading Chinese nursing journals

BACKGROUND: Randomized controlled trials (RCTs) are not always well reported, especially in terms of their methodological descriptions. This study aimed to investigate the adherence of methodological reporting complying with CONSORT and explore associated trial level variables in the Chinese nursing care field. METHODS: In June 2012, we identified RCTs published in five leading Chinese nursing journals and included trials with details of randomized methods. The quality of methodological reporting was measured through the methods section of the CONSORT checklist and the overall CONSORT methodological items score was calculated and expressed as a percentage. Meanwhile, we hypothesized that some general and methodological characteristics were associated with reporting quality and conducted a regression with these data to explore the correlation. The descriptive and regression statistics were calculated via SPSS 13.0. RESULTS: In total, 680 RCTs were included. The overall CONSORT methodological items score was 6.34 ± 0.97 (Mean ± SD). No RCT reported descriptions and changes in "trial design," changes in "outcomes" and "implementation," or descriptions of the similarity of interventions for "blinding." Poor reporting was found in detailing the "settings of participants" (13.1%), "type of randomization sequence generation" (1.8%), calculation methods of "sample size" (0.4%), explanation of any interim analyses and stopping guidelines for "sample size" (0.3%), "allocation concealment mechanism" (0.3%), additional analyses in "statistical methods" (2.1%), and targeted subjects and methods of "blinding" (5.9%). More than 50% of trials described randomization sequence generation, the eligibility criteria of "participants," "interventions," and definitions of the "outcomes" and "statistical methods." The regression analysis found that publication year and ITT analysis were weakly associated with CONSORT score. CONCLUSIONS: The completeness of methodological reporting of RCTs in the Chinese nursing care field is poor, especially with regard to the reporting of trial design, changes in outcomes, sample size calculation, allocation concealment, blinding, and statistical methods

Collapse Mechanism of Transmission Tower Subjected to Strong Wind Load and Dynamic Response of Tower-Line System

Transmission towers are prone to collapse under strong wind load, resulting in significant economic losses. In order to investigate the collapse mechanism and failure modes of the transmission tower under strong wind load and whether the wind vibration factor can greatly reflect the increasing effect of the fluctuating wind, the finite element method (FEM) is utilized to analyze the ultimate bearing capacity of a typical 220 kV transmission tower. The results show that the collapse of the tower under strong wind loads is usually due to the buckling of the leg members. When the reference wind speed is equal to 27 m/s, a small part of the main leg members reaches their yield strength, while the diagonal members are still in the elastic range, and the deformation of the transmission tower is unapparent at this wind speed. When reference wind speed is equal or greater than 30 m/s, the growing variety of main legs is totally into the plastic yield stage, and the overall deformation of this tower is visible. Therefore, the transmission tower is collapsed due to the large deformation caused by the elastic-plastic buckling of leg members. Based on the aforementioned study, a finite element model involving three transmission towers and four span transmission lines is established to analyze the dynamic response of the tower-line system below fluctuating wind. Results show that the wind-induced coefficients designed by current code not only notably satisfy the stress response of tower components subjected to fluctuating wind loads in the elastic phase but also accurately assess the collapse displacement of the transmission tower. The increasing effect of displacement on the top tower under fluctuating wind, unfortunately, could not considerably reply with the investigated factor, and the load-carrying capacity of the transmission tower in the plastic phase can be overestimated by static calculation results

Collapse Mechanism of Transmission Tower Subjected to Strong Wind Load and Dynamic Response of Tower-Line System

Transmission towers are prone to collapse under strong wind load, resulting in significant economic losses. In order to investigate the collapse mechanism and failure modes of the transmission tower under strong wind load and whether the wind vibration factor can greatly reflect the increasing effect of the fluctuating wind, the finite element method (FEM) is utilized to analyze the ultimate bearing capacity of a typical 220 kV transmission tower. The results show that the collapse of the tower under strong wind loads is usually due to the buckling of the leg members. When the reference wind speed is equal to 27 m/s, a small part of the main leg members reaches their yield strength, while the diagonal members are still in the elastic range, and the deformation of the transmission tower is unapparent at this wind speed. When reference wind speed is equal or greater than 30 m/s, the growing variety of main legs is totally into the plastic yield stage, and the overall deformation of this tower is visible. Therefore, the transmission tower is collapsed due to the large deformation caused by the elastic-plastic buckling of leg members. Based on the aforementioned study, a finite element model involving three transmission towers and four span transmission lines is established to analyze the dynamic response of the tower-line system below fluctuating wind. Results show that the wind-induced coefficients designed by current code not only notably satisfy the stress response of tower components subjected to fluctuating wind loads in the elastic phase but also accurately assess the collapse displacement of the transmission tower. The increasing effect of displacement on the top tower under fluctuating wind, unfortunately, could not considerably reply with the investigated factor, and the load-carrying capacity of the transmission tower in the plastic phase can be overestimated by static calculation results