Normalized Affymetrix expression data are biased by G-quadruplex formation

Probes with runs of four or more guanines (G-stacks) in their sequences can exhibit a level of hybridization that is unrelated to the expression levels of the mRNA that they are intended to measure. This is most likely caused by the formation of G-quadruplexes, where inter-probe guanines form Hoogsteen hydrogen bonds, which probes with G-stacks are capable of forming. We demonstrate that for a specific microarray data set using the Human HG-U133A Affymetrix GeneChip and RMA normalization there is significant bias in the expression levels, the fold change and the correlations between expression levels. These effects grow more pronounced as the number of G-stack probes in a probe set increases. Approximately 14 of the probe sets are directly affected. The analysis was repeated for a number of other normalization pipelines and two, FARMS and PLIER, minimized the bias to some extent. We estimate that ∼15 of the data sets deposited in the GEO database are susceptible to the effect. The inclusion of G-stack probes in the affected data sets can bias key parameters used in the selection and clustering of genes. The elimination of these probes from any analysis in such affected data sets outweighs the increase of noise in the signal. © 2011 The Author(s)

A close examination of double filtering with fold change and t test in microarray analysis

<p>Abstract</p> <p>Background</p> <p>Many researchers use the double filtering procedure with fold change and <it>t </it>test to identify differentially expressed genes, in the hope that the double filtering will provide extra confidence in the results. Due to its simplicity, the double filtering procedure has been popular with applied researchers despite the development of more sophisticated methods.</p> <p>Results</p> <p>This paper, for the first time to our knowledge, provides theoretical insight on the drawback of the double filtering procedure. We show that fold change assumes all genes to have a common variance while <it>t </it>statistic assumes gene-specific variances. The two statistics are based on contradicting assumptions. Under the assumption that gene variances arise from a mixture of a common variance and gene-specific variances, we develop the theoretically most powerful likelihood ratio test statistic. We further demonstrate that the posterior inference based on a Bayesian mixture model and the widely used significance analysis of microarrays (SAM) statistic are better approximations to the likelihood ratio test than the double filtering procedure.</p> <p>Conclusion</p> <p>We demonstrate through hypothesis testing theory, simulation studies and real data examples, that well constructed shrinkage testing methods, which can be united under the mixture gene variance assumption, can considerably outperform the double filtering procedure.</p

Time spent with cats is never wasted: Lessons learned from feline acromegalic cardiomyopathy, a naturally occurring animal model of the human disease

<div><p>Background</p><p>In humans, acromegaly due to a pituitary somatotrophic adenoma is a recognized cause of increased left ventricular (LV) mass. Acromegalic cardiomyopathy is incompletely understood, and represents a major cause of morbidity and mortality. We describe the clinical, echocardiographic and histopathologic features of naturally occurring feline acromegalic cardiomyopathy, an emerging disease among domestic cats.</p><p>Methods</p><p>Cats with confirmed hypersomatotropism (IGF-1>1000ng/ml and pituitary mass; n = 67) were prospectively recruited, as were two control groups: diabetics (IGF-1<800ng/ml; n = 24) and healthy cats without known endocrinopathy or cardiovascular disease (n = 16). Echocardiography was performed in all cases, including after hypersomatotropism treatment where applicable. Additionally, tissue samples from deceased cats with hypersomatotropism, hypertrophic cardiomyopathy and age-matched controls (n = 21 each) were collected and systematically histopathologically reviewed and compared.</p><p>Results</p><p>By echocardiography, cats with hypersomatotropism had a greater maximum LV wall thickness (6.5mm, 4.1–10.1mm) than diabetic (5.9mm, 4.2–9.1mm; Mann Whitney, p<0.001) or control cats (5.2mm, 4.1–6.5mm; Mann Whitney, p<0.001). Left atrial diameter was also greater in cats with hypersomatotropism (16.6mm, 13.0–29.5mm) than in diabetic (15.4mm, 11.2–20.3mm; Mann Whitney, p<0.001) and control cats (14.0mm, 12.6–17.4mm; Mann Whitney, p<0.001). After hypophysectomy and normalization of IGF-1 concentration (n = 20), echocardiographic changes proved mostly reversible. As in humans, histopathology of the feline acromegalic heart was dominated by myocyte hypertrophy with interstitial fibrosis and minimal myofiber disarray.</p><p>Conclusions</p><p>These results demonstrate cats could be considered a naturally occurring model of acromegalic cardiomyopathy, and as such help elucidate mechanisms driving cardiovascular remodeling in this disease.</p></div

Preoperative toxoplasma gondii serostatus does not affect long-term survival of cardiac transplant recipients: analysis of the Spanish Heart Transplantation Registry

[Abstract] Background. It's unclear whether pre-transplant T. gondii seropositivity is associated with impaired survival in heart transplant recipients. Objectives. To test the above-mentioned hypothesis in the Spanish Heart Transplantation Registry. Methods. Post-transplant outcomes of 4048 patients aged > 16 years who underwent first, single-organ heart transplantation in 17 Spanish institutions from 1984 to 2014 were studied. Long-term post-transplant survival and survival free of cardiac death or retransplantation of 2434 (60%) T. gondii seropositive recipients and 1614 (40%) T. gondii seronegative recipients were compared. Results. T. gondii seropositive recipients were older, had higher body mass index, and presented higher prevalence of hypertension, hypercholesterolemia, COPD and Cytomegalovirus seropositivity than T. gondii seronegative recipients. In univariable analysis, pre-transplant T. gondii seropositivity was associated with increased post-transplant all-cause mortality (non-adjusted HR 1.15; 95% CI 1.04–1.26). However, this effect was no longer statistically significant after multivariable adjustment by recipient's age and sex (adjusted HR 1.01, 95% CI 0.92–1.11). Extended multivariable adjustment by other potential confounders showed similar results (adjusted HR 0.99, 95% CI 0.89–1.11). T. gondii seropositivity had no significant effect on the composite outcome cardiac death or retransplantation (non-adjusted HR 1.08, 95% CI 0.95–1.24, p = 0.235). The distribution of the causes of death was comparable in T. gondii seropositive and T. gondii seronegative recipients. No statistically significant impact of donor's T. gondii serostatus or donor-recipient T. gondii serostatus matching on post-transplant survival was observed. Conclusions. Our analysis did not show a significant independent effect of preoperative T. gondii serostatus on long-term outcomes after heart transplantation

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10[superscript 54] circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

The Heart Is an Early Target of Anthrax Lethal Toxin in Mice: A Protective Role for Neuronal Nitric Oxide Synthase (nNOS)

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS−/− mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS−/− mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS−/− and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS−/− mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS−/− and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

Array data extractor (ADE): a LabVIEW program to extract and merge gene array data

BACKGROUND: Large data sets from gene expression array studies are publicly available offering information highly valuable for research across many disciplines ranging from fundamental to clinical research. Highly advanced bioinformatics tools have been made available to researchers, but a demand for user-friendly software allowing researchers to quickly extract expression information for multiple genes from multiple studies persists. FINDINGS: Here, we present a user-friendly LabVIEW program to automatically extract gene expression data for a list of genes from multiple normalized microarray datasets. Functionality was tested for 288 class A G protein-coupled receptors (GPCRs) and expression data from 12 studies comparing normal and diseased human hearts. Results confirmed known regulation of a beta 1 adrenergic receptor and further indicate novel research targets. CONCLUSIONS: Although existing software allows for complex data analyses, the LabVIEW based program presented here, “Array Data Extractor (ADE)”, provides users with a tool to retrieve meaningful information from multiple normalized gene expression datasets in a fast and easy way. Further, the graphical programming language used in LabVIEW allows applying changes to the program without the need of advanced programming knowledge