The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly.

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

Cloning, expression, and evolutionary analysis of α-gliadin genes from Triticum and Aegilops genomes.

Fifteen novel α-gliadin genes were cloned and sequenced from Triticum and related Aegilops genomes by allele-specific polymerase chain reaction (AS-PCR). Sequence comparison displayed high diversities in the α-gliadin gene family. Four toxic epitopes and glutamine residues in the two polyglutamine domains facilitated these α-gliadins to be assigned to specific chromosomes. Five representative α-gliadin genes were successfully expressed in Escherichia coli, and their amount reached a maximum after 4 h induced by isopropyl-ß-D-thiogalactoside (IPTG), indicating a high level of expression under the control of T7 promoter. The transcriptional expression of α-gliadin genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR) showed a similar up-down regulation pattern in different genotypes. A neighbor-joining tree constructed with both full-open reading frame (ORF) α-gliadin genes and pseudogenes further revealed the origin and phylogenetic relationships among Triticum and related Aegilops genomes. The evolutionary analysis demonstrated that α-gliadin genes evolved mainly by synonymous substitutions under strong purifying selection during the evolutionary process.

Identification and molecular characterisation of HMW glutenin subunit 1By16* in wild emmer.

In this study, a novel y-type high molecular weight glutenin subunit (HMW-GS) in wild emmer wheat Triticum turgidum L. var. dicoccoides (Körn.) accession KU1952 was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE) and matrix-assisted laser desorption ionisation/time-of-flight/mass spectrometry (MALDI-TOF-MS). Its electrophoretic mobility and molecular weight were similar to those of 1By16 and was designated as 1By16*. The complete coding sequence of the 1By16* gene isolated by allelic-specific polymerase chain reaction (AS-PCR) consists of 2,157 bp, encoding 729 amino acid residues. The real presence and authenticity of the 1By16* gene in KU1952 were further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), heterologous expression and Western blotting. The molecular structure as well as phylogenetic analysis revealed that 1By16* had 21 single-nucleotide polymorphism (SNP) variations and possessed greater similarity with superior quality subunits 1By15 and 1By16 of common wheat. Secondary structure prediction displayed higher α-helix and ß-strand contents in the 1By16* subunit, which could form a superior gluten structure and, consequently, might have positive effects on dough quality. Our results suggest that 1By16* is expected to be a new potential gene for wheat quality improvement.

Application and validation of a computerized cough acquisition system for objective monitoring of acute cough: a meta-analysis.

STUDY OBJECTIVE: The purpose of the meta-analysis was to understand the antitussive effect of treatment with dextromethorphan hydrobromide, 30 mg, vs placebo over a 3-h treatment period in patients with cough due to uncomplicated upper respiratory tract infection (URTI), and to show that the computerized system for acquisition and analysis of cough sound was consistent and reproducible across the individual studies. STUDY DESIGN: The six studies used for the meta-analysis were randomized, double-blind, parallel-group, single-dose, placebo-controlled studies with a 3-h postdose cough evaluation period. SETTING: One study was conducted in Durban, South Africa, and five studies were conducted in Bombay, India. Four studies took place in clinics, and two studies were in-home studies. PATIENTS: Seven hundred ten adult patients with cough due to uncomplicated URTI who were otherwise healthy and who satisfied the inclusion/exclusion criteria for the meta-analysis. MEASUREMENTS AND RESULTS: For each patient, a standard baseline was calculated pretreatment, then a 3-h continuous cough recording was made after treatment was initiated. Five efficacy variables were measured in 30-min intervals: cough bouts, cough components, cough effort, cough intensity, and cough latency. The meta-analysis showed consistent results across most of the studies for each of the efficacy variables. It demonstrated significantly greater overall reductions in cough bouts, cough components, and cough effort, and an increase in cough latency for patients treated with dextromethorphan hydrobromide, 30 mg, vs those treated with placebo. CONCLUSION: The results of a meta-analysis of the six clinical studies show that the antitussive effect of a single dose of dextromethorphan hydrobromide, 30 mg, has been established. The consistent nature of the results shows that the computerized cough acquisition and analysis system is a valid and reproducible methodology for evaluating cough associated with URTI.

Methods of recording and analysing cough sounds.

Efforts have been directed to evolve a computerized system for acquisition and multi-dimensional analysis of the cough sound. The system consists of a PC-AT486 computer with an ADC board having 12 bit resolution. The audio cough sound is acquired using a sensitive miniature microphone at a sampling rate of 8 kHz in the computer and simultaneously recorded in real time using a digital audio tape recorder which also serves as a back up. Analysis of the cough sound is done in time and frequency domains using the digitized data which provide numerical values for key parameters like cough counts, bouts, their intensity and latency. In addition, the duration of each event and cough patterns provide a unique tool which allows objective evaluation of antitussive and expectorant drugs. Both on-line and off-line checks ensure error-free performance over long periods of time. The entire system has been evaluated for sensitivity, accuracy, precision and reliability. Successful use of this system in clinical studies has established what perhaps is the first integrated approach for the objective evaluation of cough.

Evaluation of antitussive agents in man.

Methodology to evaluate the efficacy of antitussive drugs rely largely on subjective methods and cough counts. There are few studies in cough due to natural disease especially using objective techniques. This paper presents data from a series of randomized, double blind, placebo controlled clinical trials in cough due to both chronic bronchopulmonary disease and acute upper respiratory tract infections. In these studies, cough was quantified using a standardized and validated computerized system for the acquisition and multidimensional analysis of the cough sound. Key objective parameters like cough counts, intensity, latency and total effort expended were studied. Guaiphenesin and bromhexine showed significant expectorant effects in patients with productive cough due to chronic bronchopulmonary disease. Differences were observed in speed of action, and objective and subjective measures, that probably indicate differences in drug action. More recently, three studies evaluated the antitussive drug dextromethorphan in non-productive cough due to uncomplicated upper respiratory tract infections. Reproducible cough suppressant effects were demonstrated after a single 30 mg dose using objective measures of cough counts, latency and total effort. These results establish the sensitivity and robustness of the cough quantitation methodology in the objective evaluation of cough treatments.