Physiological Genomics current issue

Translational informatics: enabling high-throughput research paradigms

Payne, P. R. O., Embi, P. J., Sen, C. K. — Fri, 06 Nov 2009 11:16:57 PST

A common thread throughout the clinical and translational research domains is the need to collect, manage, integrate, analyze, and disseminate large-scale, heterogeneous biomedical data sets. However, well-established and broadly adopted theoretical and practical frameworks and models intended to address such needs are conspicuously absent in the published literature or other reputable knowledge sources. Instead, the development and execution of multidisciplinary, clinical, or translational studies are significantly limited by the propagation of "silos" of both data and expertise. Motivated by this fundamental challenge, we report upon the current state and evolution of biomedical informatics as it pertains to the conduct of high-throughput clinical and translational research and will present both a conceptual and practical framework for the design and execution of informatics-enabled studies. The objective of presenting such findings and constructs is to provide the clinical and translational research community with a common frame of reference for discussing and expanding upon such models and methodologies.

Gene expression and muscle fiber function in a porcine ICU model

Fri, 06 Nov 2009 11:16:57 PST

Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation and immobilization in intensive care unit (ICU) patients are clinically challenging partly due to 1) the poorly understood intricate cellular and molecular networks and 2) the unavailability of an animal model mimicking this condition. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization, we have analyzed the expression profile of skeletal muscle biopsies taken at three time points during a 5-day period. Among the differentially regulated transcripts, extracellular matrix, energy metabolism, sarcomeric and LIM protein mRNA levels were downregulated, while ubiquitin proteasome system, cathepsins, oxidative stress responsive genes and heat shock proteins (HSP) mRNAs were upregulated. Despite 5 days of immobilization and mechanical ventilation single muscle fiber cross-sectional areas as well as the maximum force generating capacity at the single muscle fiber level were preserved. It is proposed that HSP induction in skeletal muscle is an inherent, primary, but temporary protective mechanism against protein degradation. To our knowledge, this is the first study that isolates the effect of immobilization and mechanical ventilation in an ICU condition from various other cofactors.

Lack of periostin leads to suppression of Notch1 signaling and calcific aortic valve disease

Fri, 06 Nov 2009 11:16:57 PST

The Postn gene encodes protein periostin. During embryonic development, it is highly expressed in the outflow tract (OFT) endocardial cushions of the developing heart, which give rise to several structures of the mature heart including the aortic valve. Periostin was previously implicated in osteoblast differentiation, cancer metastasis, and tooth and bone development, but its role in cardiac OFT development is unclear. To elucidate the role that periostin plays in the developing heart we analyzed cardiac OFT phenotype in mice after deletion of the Postn gene. We found that lack of periostin in the embryonic OFT leads to ectopic expression of the proosteogenic growth factor pleiotrophin (Ptn) and overexpression of delta-like 1 homolog (Dlk1), a negative regulator of Notch1, in the distal (prevalvular) cushions of the OFT. This resulted in suppression of Notch1 signaling, strong induction of the central transcriptional regulator of osteoblast cell fate Runx2, upregulation of osteopontin and osteocalcin expression, and subsequent calcification of the aortic valve. Our data suggest that periostin represses a default osteogenic program in the OFT cushion mesenchyme and promotes differentiation along a fibrogenic lineage. Lack of periostin causes derepression of the osteogenic potential of OFT mesenchymal cells, calcium deposition, and calcific aortic valve disease. These results establish periostin as a key regulator of OFT endocardial cushion mesenchymal cell fate during embryonic development.

Embrace diversity! Systems genetics-enabled discovery of disease networks

Voy, B. H., Aronow, B. J. — Fri, 06 Nov 2009 11:16:57 PST

Effects of atherogenic diet on hepatic gene expression across mouse strains

Fri, 06 Nov 2009 11:16:57 PST

Diets high in fat and cholesterol are associated with increased obesity and metabolic disease in mice and humans. To study the molecular basis of the metabolic response to dietary fat, 10 inbred strains of mice were fed atherogenic high-fat and control low-fat diets. Liver gene expression and whole animal phenotypes were measured and analyzed in both sexes. The effects of diet, strain, and sex on gene expression were determined irrespective of complex processes, such as feedback mechanisms, that could have mediated the genomic responses. Global gene expression analyses demonstrated that animals of the same strain and sex have similar transcriptional profiles on a low-fat diet, but strains may show considerable variability in response to high-fat diet. Functional profiling indicated that high-fat feeding induced genes in the immune response, indicating liver damage, and repressed cholesterol biosynthesis. The physiological significance of the transcriptional changes was confirmed by a correlation analysis of transcript levels with whole animal phenotypes. The results found here were used to confirm a previously identified quantitative trait locus on chromosome 17 identified in males fed a high-fat diet in two crosses, PERA x DBA/2 and PERA x I/Ln. The gene expression data and phenotype data have been made publicly available as an online tool for exploring the effects of atherogenic diet in inbred mouse strains (http://cgd-array.jax.org/DietStrainSurvey).

Genomic analyses reveal a conserved glutathione homeostasis pathway in the invertebrate chordate Ciona intestinalis

Nava, G. M., Lee, D. Y., Ospina, J. H., Cai, S.-Y., Gaskins, H. R. — Fri, 06 Nov 2009 11:16:57 PST

The major thiol redox buffer glutathione (l--glutamyl-l-cysteinylglycine, GSH) is central to cell fate determination, and thus, associated metabolic and regulatory pathways are exquisitely sensitive to a wide range of environmental cues. An imbalance of cellular redox homeostasis has emerged as a pathologic hallmark of a diverse range of human gene-environment disorders. Despite the central importance of GSH in cellular homeostasis, underlying genetic regulatory pathways remain poorly defined. This report describes the annotation and expression analysis of genes contributing to GSH homeostasis in the invertebrate chordate Ciona intestinalis. A core pathway comprising 19 genes contributing to the biosynthesis of GSH and its use as both a redox buffer and a conjugate in phase II detoxification as well as known transcriptional regulators were analyzed. These genes exhibit a high level of sequence conservation with corresponding human, rat, and mouse homologs and were expressed constitutively in tissues of adult animals. The GSH biosynthetic genes Gclc and Gclm were also responsive to the prototypical antioxidant tert-butylhydroquinone. The present evidence of a conserved GSH homeostasis pathway in C. intestinalis together with its phylogenetic position as a basal chordate and lifestyle as a filter feeder constantly exposed to natural marine toxins introduces this species as an important animal model for defining molecular mechanisms that potentially underlie genetic susceptibility to environmentally associated stress.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

Fri, 06 Nov 2009 11:16:57 PST

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens

Sim, C., Denlinger, D. L. — Fri, 06 Nov 2009 11:16:58 PST

Culex pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we examined, 4 were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause. In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic classes and genes involved in β-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late diapause and at diapause termination. RNA interference (RNAi) analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles in fat storage during early diapause. When expression of these genes is suppressed, female mosquitoes fail to sequester the lipids needed for overwintering.

miR-290 acts as a physiological effector of senescence in mouse embryo fibroblasts

Fri, 06 Nov 2009 11:16:58 PST

The culture-induced senescence of mouse embryo fibroblasts (MEF) correlates with reduction of cell proliferation. In this work we found that the accumulation of cells with 4C DNA content and the transcriptional change of several microRNAs (miRNAs or miRs) are relevant events in culture senescence. By comparing the miRNA expression profiles of physiologically senescent MEF and that of senescent MEF induced by the downregulation of leukemia-related factor, we identified miR-290 as a common upregulated miRNA. When miR-290 was transfected in presenescent MEF, SA-β-gal⁺ cells and p16, two markers of culture senescence, increased compared with control, indicating that miR-290 is causally involved in senescence. Interestingly, nocodazole (NCZ), which induces G2/M block, increased the percentage of senescent cells as well as the expression of miR-290 and of the tumor suppressor p16, thus mimicking culture senescence. As miR-290 was overexpressed in NCZ-treated cells and it was able to induce senescence in proliferating MEF, we investigated whether miR-290 and NCZ could share common mechanisms of culture senescence. Whereas the induction of SA-β-gal⁺ by miR-290 was not strengthened by coupling its transfection with NCZ treatment, the transfection of the antagomir 290 (d-290) plus NCZ treatment, while blocking cells at G2/M, suppressed SA-β-gal⁺ and p16 induction. On the basis of these findings we conclude that miR-290 might act as a physiological effector of NCZ induced as well as culture senescence via p16 regulation expanding the role of this miRNA from embryonic stem to differentiated cells.

Evidence of MyomiR network regulation of {beta}-myosin heavy chain gene expression during skeletal muscle atrophy

McCarthy, J. J., Esser, K. A., Peterson, C. A., Dupont-Versteegden, E. E. — Fri, 06 Nov 2009 11:16:58 PST

There is a growing recognition that noncoding RNAs (ncRNA) play an important role in the regulation of gene expression. A class of small (19–22 nt) ncRNAs, known as microRNAs (miRs), have received a great deal of attention lately because of their ability to repress gene expression through a unique posttranscriptional 3'-untranslated region (UTR) mechanism. The objectives of the current study were to identify miRs expressed in the rat soleus muscle and determine if their expression was changed in response to hindlimb suspension. Comprehensive profiling revealed 151 miRs were expressed in the soleus muscle and expression of 18 miRs were significantly (P < 0.01) changed after 2 and/or 7 days of hindlimb suspension. The significant decrease (16%) in expression of muscle-specific miR-499 in response to hindlimb suspension was confirmed by RT-PCR and suggested activation of the recently proposed miR encoded by myosin gene (MyomiR) network during atrophy. Further analysis of soleus muscle subjected to hindlimb suspension for 28 days provided evidence consistent with MyomiR network repression of β-myosin heavy chain gene (β-MHC) expression. The significant downregulation of network components miR-499 and miR-208b by 40 and 60%, respectively, was associated with increased expression of Sox6 (2.2-fold) and Purβ (23%), predicted target genes of miR-499 and known repressors of β-MHC expression. A Sox6 3'-UTR reporter gene confirmed Sox6 is a target gene of miR-499. These results further expand the role of miRs in adult skeletal muscle and are consistent with a model in which the MyomiR network regulates slow myosin expression during muscle atrophy.

Changes in behavior and gene expression induced by caloric restriction in C57BL/6 mice

Fri, 06 Nov 2009 11:16:58 PST

Caloric restriction (CR) is an effective method for prevention of age-associated diseases as well as overweight and obesity; however, there is controversy regarding the effects of dieting regimens on behavior. In this study, we investigated two different dieting regimens: repeated fasting and refeeding (RFR) and daily feeding of half the amount of food consumed by RFR mice (CR). CR and RFR mice had an approximate 20% reduction in food intake compared with control mice. Open field, light-dark transition, elevated plus maze, and forced swimming tests indicated that CR, but not RFR, reduced anxiety- and depressive-like behaviors, with a reduction peak on day 8. Using a mouse whole genome microarray, we analyzed gene expression in the prefrontal cortex, amygdala, and hypothalamus. In addition to the CR-responsive genes commonly modified by RFR and CR, each regimen differentially changed the expression of distinct genes in each region. The most profound change was observed in the amygdalas of CR mice: 884 genes were specifically upregulated. Ingenuity pathway analysis revealed that these 884 genes significantly modified nine canonical pathways in the amygdala. -Adrenergic and dopamine receptor signalings were the two top-scoring pathways. Quantitative RT-PCR confirmed the upregulation of six genes in these pathways. Western blotting confirmed that CR specifically increased dopamine- and cAMP-regulated phosphoprotein (Darpp-32), a key regulator of dopamine receptor signaling, in the amygdala. Our results suggest that CR may change behavior through altered gene expression.