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Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

Background: The β2-adrenergic receptor (β2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions. Methodology/Principal Findings: We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5′UTR, coding, and 3′UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that β2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. “Whole-gene” transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface β2AR protein expression compared to the others. Agonist-promoted downregulation of β2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression. Conclusions/Significance: Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of β-agonist may be influenced by certain β2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where β2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

Using Near Infrared Reflectance Spectroscopy (NIRS) to predict the protein and energy digestibility of lupin kernel meals when fed to rainbow trout, Oncorhynchus mykiss

This study examined the potential of using near infrared spectroscopy (NIRS) to predict the nutrient composition, energy density and the digestible protein and digestible energy values of lupin kernel meals when fed to rainbow trout. A series of 136 lupin kernel meals were assessed for their protein and energy digestibilities using the diet-substitution approach in a series of 10 experiments over a 6-year period from 2002 to 2008. Two reference diets were also included in each experiment. Minimal variance in the digestibility parameters of both reference diets was observed among the experiments ensuring that there was a high degree of robustness in the across-experiment evaluations. The same lupin kernel meal samples were also scanned using a diode array near infrared spectrophotometer (DA-NIRS). The spectra obtained by the DA-NIRS were chemometrically calibrated against both the chemical composition and the digestible value data using multivariate analysis software. The cross validation tests used in this study provide a valid indication of the potential to predict the nutrient composition, energy value and digestible protein and energy values of the lupin kernel meals as used in diets for rainbow trout. That the standard errors of cross validation (SECV) of the parameters investigated were generally commensurate with the cross trial variation seen in the reference sample indicating robust calibrations for the two target parameters of digestible protein and digestible energy. Therefore this study demonstrates that within one raw material type that not only does significant variability in the digestible value of the raw materials exist, but that it is possible to use NIRS technology to provide rapid estimates of the digestible value of those raw materials in near real-time. © 2014 John Wiley & Sons Ltd

Switchgrass (\u3ci\u3ePanicum virgatum\u3c/i\u3e L.) polyubiquitin gene (\u3ci\u3ePvUbi1\u3c/i\u3e and \u3ci\u3ePvUbi2\u3c/i\u3e) promoters for use in plant transformation

Abstract Background The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5\u27 upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. Results PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5\u27 untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3\u27 UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5\u27 UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3\u27 UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. Conclusions The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots

Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches

Theory of Circle Maps and the Problem of One-Dimensional Optical Resonator with a Periodically Moving Wall

We consider the electromagnetic field in a cavity with a periodically oscillating perfectly reflecting boundary and show that the mathematical theory of circle maps leads to several physical predictions. Notably, well-known results in the theory of circle maps (which we review briefly) imply that there are intervals of parameters where the waves in the cavity get concentrated in wave packets whose energy grows exponentially. Even if these intervals are dense for typical motions of the reflecting boundary, in the complement there is a positive measure set of parameters where the energy remains bounded.Comment: 34 pages LaTeX (revtex) with eps figures, PACS: 02.30.Jr, 42.15.-i, 42.60.Da, 42.65.Y

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

The beta2-adrenergic receptor (beta2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

Is the socioeconomic gap in childhood exposure to secondhand smoke widening or narrowing?

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