Stress distribution in a premolar 3D model with anisotropic and isotropic enamel.

The aim of this study was to compare the areas of stress concentration in a three-dimensional (3D) premolar tooth model with anisotropic or isotropic enamel using the finite element method. A computed tomography was imported to an image processing program to create the tooth model which was exported to a 3D modeling program. The mechanical properties and loading conditions were prescribed in Abaqus. In order to evaluate stresses, axial and oblique loads were applied simulating realistic conditions. Compression stress was observed on the side of load application, and tensile stress was observed on the opposite side. Tensile stress was concentrated mainly in the cervical region and in the alveolar insertion bone. Although stress concentration analyses of the isotropic 3D models produced similar stress distribution results when compared to the anisotropic models, tensile stress values shown by anisotropic models were smaller than the isotropic models. Oblique loads resulted in higher values of tensile stresses, which concentrate mainly in the cervical area of the tooth and in the alveolar bone insertion. Anisotropic properties must be utilized in enamel stress evaluation in non-carious cervical lesions.

Differential gene expression profiling of short and long term denervated muscle.

Skeletal muscle function and viability are dependent upon intact innervation. Peripheral nerve injury and muscle denervation cause muscle atrophy. Time to re-innervation is one of the most important determinants of functional outcome. While short-term denervation can result in nearly fully reversible changes in muscle mass, prolonged denervation leads to irreversible muscle impairment from profound atrophy, myocyte death and fibrosis. We performed transcriptional profiling to identify genes that were altered in expression in short-term (1 month) and long-term (3 month) denervated muscle and validated the microarray data by RT-PCR and Western blotting. Genes controlling cell death, metabolism, proteolysis, stress responses and protein synthesis/translation were altered in expression in the denervated muscle. A differential pattern of expression of genes encoding cell cycle regulators and extracellular matrix components was identified that correlated with the development of irreversible post-denervation changes. Genes encoding mediators of protein degradation were differentially expressed between 1 and 3 month denervated muscle suggesting different signaling networks are recruited over time to induce and maintain muscle atrophy. Understanding of the timing and type of pathological processes that are triggered by denervation may allow the design of interventions that delay or protect muscle from loss of nerve function.

Differential gene expression profile reveals deregulation of pregnancy specific beta1 glycoprotein 9 early during colorectal carcinogenesis.

BACKGROUND: APC (Adenomatous polyposis coli) plays an important role in the pathogenesis of both familial and sporadic colorectal cancer. Patients carrying germline APC mutations develop multiple colonic adenomas at younger age and higher frequency than non-carrier cases which indicates that silencing of one APC allele may be sufficient to initiate the transformation process. METHODS: To elucidate the biological dysregulation underlying adenoma formation we examined global gene expression profiles of adenomas and corresponding normal mucosa from an FAP patient. Differential expression of the most significant gene identified in this study was further validated by mRNA in situ hybridization, reverse transcriptase PCR and Northern blotting in different sets of adenomas, tumours and cancer cell lines. RESULTS: Eighty four genes were differentially expressed between all adenomas and corresponding normal mucosa, while only seven genes showed differential expression within the adenomas. The first group included pregnancy specific beta-1 glycoprotein 9 (PSG9) (p < 0.006). PSG9 is a member of the carcinoembryonic antigen (CEA)/PSG family and is produced at high levels during pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18) of FAP adenomas and 75% (45/60) of sporadic colorectal cancer cases tested. CONCLUSION: Detection of PSG9 expression in adenomas, and at higher levels in FAP cases, indicates that germline APC mutations and defects in Wnt signalling modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult except in association with cancer, and it appears to be an early molecular event associated with colorectal cancer monitoring of its expression may be useful as a biomarker for the early detection of this disease.

Cardioprotective stress response in the human fetal heart.

OBJECTIVE: We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype. METHODS: Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS: Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05). CONCLUSIONS: Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.

Gene expression profiles in children undergoing cardiac surgery for right heart obstructive lesions.

BACKGROUND: The global myocardial stress response during cardiac surgery has not been systematically studied, nor is it known whether the response of the neonatal myocardium is intrinsically different from that of older children. To determine the age-related molecular basis of this response, we conducted microarray-based differential gene expression profiling on right ventricular tissue samples acquired in patients of varying ages with right ventricular outflow tract obstruction. METHODS: We studied gene expression profiles in 24 patients during operations for lesions involving right ventricular outflow tract obstruction age stratified into group I (7 patients, aged 5 to 66 days; mean, 30 days) and group II (17 patients, aged 4 months to 12.5 years; mean, 2.8 years). Myocardial samples were taken from the right ventricular outflow tract after aortic occlusion and archived in liquid nitrogen. RNA isolation, fluorescence labeling of complementary DNA, hybridization to spotted arrays containing 19,008 characterized or unknown human complementary DNAs, and quantitative fluorescence scanning of gene-expression intensity were performed at the University of Toronto Health Network Microarray Centre. Data were analyzed with the Significance Analysis for Microarrays program. Minimum Information About Microarray Experiments-compliant, log2-normalized data sets were compared to ascertain potential statistical differences in gene expression between patient groups. RESULTS: There were no hospital deaths or major postoperative morbid events. We identified 50 transcripts differentially expressed in the neonatal group (the predicted false discovery rate was <0.8 transcripts). The neonatal pattern of gene expression (group I) was dominated by genes with literature-validated cardioprotective, antihypertrophic, and antiproliferative properties, including increases in atrial natriuretic peptide, protein phosphatase 2A, small GTPase rap1, and protein inhibitor of activated STAT protein, PIASy. Several transcripts have not been previously reported in heart. CONCLUSIONS: Neonatal myocardium has a unique pattern of gene expression, which may result from developmental (age-related) differences or reflect a more severe disease phenotype independent of age effects per se. The neonatal transcript profile seems to reflect a stress-induced protective program composed of genes with functions diametrically opposed to those expected to be related to the pathogenesis of critical right ventricular outflow tract obstruction, thus revealing a novel and compensatory antidisease transcriptional response in the neonatal heart.

Design and implementation of microarray gene expression markup language (MAGE-ML).

BACKGROUND: Meaningful exchange of microarray data is currently difficult because it is rare that published data provide sufficient information depth or are even in the same format from one publication to another. Only when data can be easily exchanged will the entire biological community be able to derive the full benefit from such microarray studies. RESULTS: To this end we have developed three key ingredients towards standardizing the storage and exchange of microarray data. First, we have created a minimal information for the annotation of a microarray experiment (MIAME)-compliant conceptualization of microarray experiments modeled using the unified modeling language (UML) named MAGE-OM (microarray gene expression object model). Second, we have translated MAGE-OM into an XML-based data format, MAGE-ML, to facilitate the exchange of data. Third, some of us are now using MAGE (or its progenitors) in data production settings. Finally, we have developed a freely available software tool kit (MAGE-STK) that eases the integration of MAGE-ML into end users' systems. CONCLUSIONS: MAGE will help microarray data producers and users to exchange information by providing a common platform for data exchange, and MAGE-STK will make the adoption of MAGE easier.

A molecular compendium of genes expressed in multiple myeloma.

We have created a molecular resource of genes expressed in primary malignant plasma cells using a combination of cDNA library construction, 5' end single-pass sequencing, bioinformatics, and microarray analysis. In total, we identified 9732 nonredundant expressed genes. This dataset is available as the Myeloma Gene Index (www.uhnres.utoronto.ca/akstewart_lab).Predictably, the sequenced profile of myeloma cDNAs mirrored the known function of immunoglobulin-producing, high-respiratory rate, low-cycling, terminally differentiated plasma cells. Nevertheless, approximately 10% of myeloma-expressed sequences matched only entries in the database of Expressed Sequence Tags (dbEST) or the high-throughput genomic sequence (htgs) database. Numerous novel genes of potential biologic significance were identified. We therefore spotted 4300 sequenced cDNAs on glass slides creating a myeloma-enriched microarray. Several of the most highly expressed genes identified by sequencing, such as a novel putative disulfide isomerase (MGC3178), tumor rejection antigen TRA1, heat shock 70-kDa protein 5, and annexin A2, were also differentially expressed between myeloma and B lymphoma cell lines using this myeloma-enriched microarray. Furthermore, a defined subset of 34 up-regulated and 18 down-regulated genes on the array were able to differentiate myeloma from nonmyeloma cell lines. These not only include genes involved in B-cell biology such as syndecan, BCMA, PIM2, MUM1/IRF4, and XBP1, but also novel uncharacterized genes matching sequences only in the public databases. In summary, our expressed gene catalog and myeloma-enriched microarray contains numerous genes of unknown function and may complement other commercially available arrays in defining the molecular portrait of this hematopoietic malignancy. GenBank Accession numbers include BF169967-BF176369, BF185966-BF185969, and BF177280-BF177455.

DREAM is a critical transcriptional repressor for pain modulation.

Control and treatment of chronic pain remain major clinical challenges. Progress may be facilitated by a greater understanding of the mechanisms underlying pain processing. Here we show that the calcium-sensing protein DREAM is a transcriptional repressor involved in modulating pain. dream(-/-) mice displayed markedly reduced responses in models of acute thermal, mechanical, and visceral pain. dream(-/-) mice also exhibited reduced pain behaviors in models of chronic neuropathic and inflammatory pain. However, dream(-/-) mice showed no major defects in motor function or learning and memory. Mice lacking DREAM had elevated levels of prodynorphin mRNA and dynorphin A peptides in the spinal cord, and the reduction of pain behaviors in dream(-/-) mice was mediated through dynorphin-selective kappa (kappa)-opiate receptors. Thus, DREAM appears to be a critical transcriptional repressor in pain processing.