MED: Genetics & Genomics Papers

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Evidence for a "Wattle and Daub" Model of the Cyst Wall of Entamoeba
(Public Library of Science, 2009-7-3) Chatterjee, Anirban; Ghosh, Sudip K.; Jang, Ken; Bullitt, Esther; Moore, Landon; Robbins, Phillips W.; Samuelson, John
The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a "wattle and daub" model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins). Author SummaryParasitic protists, which are spread by the fecal-oral route, have cyst walls that resist environmental insults (e.g. desiccation, stomach acids, bile, etc.). Entamoeba histolytica, the cause of amebic dysentery and liver abscess, is the only protist characterized to date that has chitin in its cyst wall. We have previously characterized Entamoeba chitin synthases, chitinases, and multivalent chitin-binding lectins called Jacob. Here we present evidence that the Entamoeba Jessie3 lectin contributes to the mortar or daub, which makes the cyst wall impenetrable to small molecules. First, the Jessie3 lectin was made after chitin and Jacob lectins had already been deposited onto the surface of encysting Entamoeba. Second, cysts became impenetrable to small molecules at the same time that Jessie3 was deposited into the wall. Third, recombinant Jessie3 lectins self-aggregated and caused transfected bacteria to agglutinate. These results suggest a "wattle and daub" model of the Ei cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).
Cofilin Activation in Peripheral CD4 T Cells of HIV-1 Infected Patients: A Pilot Study
(BioMed Central, 2008-10-17) Wu, Yuntao; Yoder, Alyson; Yu, Dongyang; Wang, Weifeng; Liu, Juan; Barrett, Tracey; Wheeler, David; Schlauch, Karen
Cofilin is an actin-depolymerizing factor that regulates actin dynamics critical for T cell migration and T cell activation. In unstimulated resting CD4 T cells, cofilin exists largely as a phosphorylated inactive form. Previously, we demonstrated that during HIV-1 infection of resting CD4 T cells, the viral envelope-CXCR4 signaling activates cofilin to overcome the static cortical actin restriction. In this pilot study, we have extended this in vitro observation and examined cofilin phosphorylation in resting CD4 T cells purified from the peripheral blood of HIV-1-infected patients. Here, we report that the resting T cells from infected patients carry significantly higher levels of active cofilin, suggesting that these resting cells have been primed in vivo in cofilin activity to facilitate HIV-1 infection. HIV-1-mediated aberrant activation of cofilin may also lead to abnormalities in T cell migration and activation that could contribute to viral pathogenesis.
Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes
(Public Library of Science, 2010-8-12) Stimpson, Kaitlin M.; Song, Ihn Young; Jauch, Anna; Holtgreve-Grez, Heidi; Hayden, Karen E.; Bridger, Joanna M.; Sullivan, Beth A.
Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment. Author Summary Endogenous human centromeres are defined by large arrays of α-satellite DNA. A portion of each α-satellite array is assembled into CENP-A chromatin, the structural and functional platform for kinetochore formation. Most chromosomes are monocentric, meaning they have a single centromere. However, genome rearrangement can produce chromosomes with two centromeres (dicentrics). In most organisms, dicentrics typically break during cell division; however, dicentric human chromosomes can be stable in mitosis and meiosis. This stability reflects centromere inactivation, a poorly understood phenomenon in which one centromere is functionally silenced. To explore molecular and genomic events that occur at the time of dicentric formation, we describe a cell-based system to create dicentric human chromosomes and monitor their behavior after formation. Such dicentrics can experience several fates, including centromere inactivation, breakage, or maintaining two functional centromeres. Unexpectedly, we also find that dicentrics with large (<20Mb) inter-centromeric distances are stable through at least 20 cell divisions. Our results highlight similarities and differences in dicentric behavior between humans and model organisms, and they provide evidence for one mechanism of centromere inactivation by centromeric deletion in some dicentrics. The ability to create dicentric human chromosomes provides a system to test other mechanisms of centromere disassembly and dicentric chromosome stability.
Transferability and Fine-Mapping of Genome-Wide Associated Loci for Adult Height across Human Populations
(Public Library of Science, 2009-12-22) Shriner, Daniel; Adeyemo, Adebowale; Gerry, Norman P.; Herbert, Alan; Chen, Guanjie; Doumatey, Ayo; Huang, Hanxia; Zhou, Jie; Christman, Michael F.; Rotimi, Charles N.
Human height is the prototypical polygenic quantitative trait. Recently, several genetic variants influencing adult height were identified, primarily in individuals of East Asian (Chinese Han or Korean) or European ancestry. Here, we examined 152 genetic variants representing 107 independent loci previously associated with adult height for transferability in a well-powered sample of 1,016 unrelated African Americans. When we tested just the reported variants originally identified as associated with adult height in individuals of East Asian or European ancestry, only 8.3% of these loci transferred (p-values=0.05 under an additive genetic model with directionally consistent effects) to our African American sample. However, when we comprehensively evaluated all HapMap variants in linkage disequilibrium (r2=0.3) with the reported variants, the transferability rate increased to 54.1%. The transferability rate was 70.8% for associations originally reported as genome-wide significant and 38.0% for associations originally reported as suggestive. An additional 23 loci were significantly associated but failed to transfer because of directionally inconsistent effects. Six loci were associated with adult height in all three groups. Using differences in linkage disequilibrium patterns between HapMap CEU or CHB reference data and our African American sample, we fine-mapped these six loci, improving both the localization and the annotation of these transferable associations.
A Genome-Wide Association Study of Hypertension and Blood Pressure in African Americans
(Public Library of Science, 2009-7-17) Adeyemo, Adebowale; Gerry, Norman; Chen, Guanjie; Herbert, Alan; Doumatey, Ayo; Huang, Hanxia; Zhou, Jie; Lashley, Kerrie; Chen, Yuanxiu; Christman, Michael; Rotimi, Charles
The evidence for the existence of genetic susceptibility variants for the common form of hypertension ("essential hypertension") remains weak and inconsistent. We sought genetic variants underlying blood pressure (BP) by conducting a genome-wide association study (GWAS) among African Americans, a population group in the United States that is disproportionately affected by hypertension and associated complications, including stroke and kidney diseases. Using a dense panel of over 800,000 SNPs in a discovery sample of 1,017 African Americans from the Washington, D.C., metropolitan region, we identified multiple SNPs reaching genome-wide significance for systolic BP in or near the genes: PMS1, SLC24A4, YWHA7, IPO7, and CACANA1H. Two of these genes, SLC24A4 (a sodium/potassium/calcium exchanger) and CACNA1H (a voltage-dependent calcium channel), are potential candidate genes for BP regulation and the latter is a drug target for a class of calcium channel blockers. No variant reached genome wide significance for association with diastolic BP (top scoring SNP rs1867226, p = 5.8×10−7) or with hypertension as a binary trait (top scoring SNP rs9791170, p = 5.1×10−7). We replicated some of the significant SNPs in a sample of West Africans. Pathway analysis revealed that genes harboring top-scoring variants cluster in pathways and networks of biologic relevance to hypertension and BP regulation. This is the first GWAS for hypertension and BP in an African American population. The findings suggests that, in addition to or in lieu of relying solely on replicated variants of moderate-to-large effect reaching genome-wide significance, pathway and network approaches may be useful in identifying and prioritizing candidate genes/loci for further experiments. Author Summary Despite intense research, the genetic risk factors for essential hypertension and blood pressure (BP) regulation have not been identified with consistency. We conducted a genome wide association scan using over 800,000 genetic markers in an African American sample of 1,017 adults in the Washington, D.C., area of the United States. We found evidence to suggest that genetic variants in several genes, including PMS1, SLC24A4, YWHA7, IPO7, and CACANA1H, are significantly associated with systolic BP levels. From our previous knowledge of human physiology, two of these genes have potential roles to play in BP regulation. The evidence for genetic variants influencing diastolic BP levels and hypertension status was weaker and inconclusive. To our knowledge, this is the first study that has used a genome-wide association approach to study hypertension and BP in an African American population, a minority group that experiences hypertension more frequently and more severely than other population groups in the United States. The findings will be useful to other researchers seeking to advance our understanding of the genetic factors that influence BP with the hope that these insights will eventually translate to new and better treatment options for hypertension in African Americans and other global populations.
Molecular Weight Assessment of Proteins in Total Proteome Profiles using 1D-PAGE and LC/MS/MS
(BioMed Central, 2005-6-8) Ahmad, Q. Rushdy; Nguyen, Dat H.; Wingerd, Mark A.; Church, George M.; Steffen, Martin A.
BACKGROUND. The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures. RESULTS. We have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1×107 cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weight differences based on existing protein annotation. CONCLUSION. We demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification.
Integration of Relational and Hierarchical Network Information for Protein Function Prediction
(BioMed Central, 2008-8-22) Jiang, Xiaoyu; Nariai, Naoki; Steffen, Martin; Kasif, Simon; Kolaczyk, Eric D.
BACKGROUND. In the current climate of high-throughput computational biology, the inference of a protein's function from related measurements, such as protein-protein interaction relations, has become a canonical task. Most existing technologies pursue this task as a classification problem, on a term-by-term basis, for each term in a database, such as the Gene Ontology (GO) database, a popular rigorous vocabulary for biological functions. However, ontology structures are essentially hierarchies, with certain top to bottom annotation rules which protein function predictions should in principle follow. Currently, the most common approach to imposing these hierarchical constraints on network-based classifiers is through the use of transitive closure to predictions. RESULTS. We propose a probabilistic framework to integrate information in relational data, in the form of a protein-protein interaction network, and a hierarchically structured database of terms, in the form of the GO database, for the purpose of protein function prediction. At the heart of our framework is a factorization of local neighborhood information in the protein-protein interaction network across successive ancestral terms in the GO hierarchy. We introduce a classifier within this framework, with computationally efficient implementation, that produces GO-term predictions that naturally obey a hierarchical 'true-path' consistency from root to leaves, without the need for further post-processing. CONCLUSION. A cross-validation study, using data from the yeast Saccharomyces cerevisiae, shows our method offers substantial improvements over both standard 'guilt-by-association' (i.e., Nearest-Neighbor) and more refined Markov random field methods, whether in their original form or when post-processed to artificially impose 'true-path' consistency. Further analysis of the results indicates that these improvements are associated with increased predictive capabilities (i.e., increased positive predictive value), and that this increase is consistent uniformly with GO-term depth. Additional in silico validation on a collection of new annotations recently added to GO confirms the advantages suggested by the cross-validation study. Taken as a whole, our results show that a hierarchical approach to network-based protein function prediction, that exploits the ontological structure of protein annotation databases in a principled manner, can offer substantial advantages over the successive application of 'flat' network-based methods.
Lack of Association between Angiotensin-Converting Enzyme and Dementia of the Alzheimer's Type in an Elderly Arab Population in Wadi Ara, Israel
(Dove Medical Press, 2005) Bowirrat, Abdalla; Cui, Jing; Waraska, Kristin; Friedland, Robert P.; Oscar-Berman, Marlene; Farrer, Lindsay A.; Korczyn, Amos; Baldwin, Clinton T.
The angiotensin-converting enzyme (ACE), a protease involved in blood pressure regulation, has been implicated as an important candidate gene for Alzheimer's disease (AD). This study investigated whether the ACE gene insertion–deletion (ID) polymorphism is associated with risk of developing dementia of Alzheimer's type (DAT) in an Arab–Israeli community, a unique genetic isolate where there is a high prevalence of DAT. In contrast to several other studies, we found no evidence of an association between this polymorphism and either DAT or age-related cognitive decline (ARCD).
Previously Unidentified Changes in Renal Cell Carcinoma Gene Expression Identified by Parametric Analysis of Microarray Data
(BioMed Central, 2003-11-27) Lenburg, Marc E.; Liou, Louis S.; Gerry, Norman P.; Frampton, Garrett M.; Cohen, Herbert T.; Christman, Michael F.
BACKGROUND. Renal cell carcinoma is a common malignancy that often presents as a metastatic-disease for which there are no effective treatments. To gain insights into the mechanism of renal cell carcinogenesis, a number of genome-wide expression profiling studies have been performed. Surprisingly, there is very poor agreement among these studies as to which genes are differentially regulated. To better understand this lack of agreement we profiled renal cell tumor gene expression using genome-wide microarrays (45,000 probe sets) and compare our analysis to previous microarray studies. METHODS. We hybridized total RNA isolated from renal cell tumors and adjacent normal tissue to Affymetrix U133A and U133B arrays. We removed samples with technical defects and removed probesets that failed to exhibit sequence-specific hybridization in any of the samples. We detected differential gene expression in the resulting dataset with parametric methods and identified keywords that are overrepresented in the differentially expressed genes with the Fisher-exact test. RESULTS. We identify 1,234 genes that are more than three-fold changed in renal tumors by t-test, 800 of which have not been previously reported to be altered in renal cell tumors. Of the only 37 genes that have been identified as being differentially expressed in three or more of five previous microarray studies of renal tumor gene expression, our analysis finds 33 of these genes (89%). A key to the sensitivity and power of our analysis is filtering out defective samples and genes that are not reliably detected. CONCLUSIONS. The widespread use of sample-wise voting schemes for detecting differential expression that do not control for false positives likely account for the poor overlap among previous studies. Among the many genes we identified using parametric methods that were not previously reported as being differentially expressed in renal cell tumors are several oncogenes and tumor suppressor genes that likely play important roles in renal cell carcinogenesis. This highlights the need for rigorous statistical approaches in microarray studies.
Reversible and Permanent Effects of Tobacco Smoke Exposure on Airway Epithelial Gene Expression
(BioMed Central, 2007-09-25) Beane, Jennifer; Sebastiani, Paola; Liu, Gang; Brody, Jerome S.; Lenburg, Marc E.; Spira, Avrum
Oligonucleotide microarray analysis revealed 175 genes that are differentially expressed in large airway epithelial cells of people who currently smoke compared with those who never smoked, with 28 classified as irreversible, 6 as slowly reversible, and 139 as rapidly reversible. BACKGROUND. Tobacco use remains the leading preventable cause of death in the US. The risk of dying from smoking-related diseases remains elevated for former smokers years after quitting. The identification of irreversible effects of tobacco smoke on airway gene expression may provide insights into the causes of this elevated risk. RESULTS. Using oligonucleotide microarrays, we measured gene expression in large airway epithelial cells obtained via bronchoscopy from never, current, and former smokers (n = 104). Linear models identified 175 genes differentially expressed between current and never smokers, and classified these as irreversible (n = 28), slowly reversible (n = 6), or rapidly reversible (n = 139) based on their expression in former smokers. A greater percentage of irreversible and slowly reversible genes were down-regulated by smoking, suggesting possible mechanisms for persistent changes, such as allelic loss at 16q13. Similarities with airway epithelium gene expression changes caused by other environmental exposures suggest that common mechanisms are involved in the response to tobacco smoke. Finally, using irreversible genes, we built a biomarker of ever exposure to tobacco smoke capable of classifying an independent set of former and current smokers with 81% and 100% accuracy, respectively. CONCLUSION. We have categorized smoking-related changes in airway gene expression by their degree of reversibility upon smoking cessation. Our findings provide insights into the mechanisms leading to reversible and persistent effects of tobacco smoke that may explain former smokers increased risk for developing tobacco-induced lung disease and provide novel targets for chemoprophylaxis. Airway gene expression may also serve as a sensitive biomarker to identify individuals with past exposure to tobacco smoke.
Genetic Analysis of the Spindle Checkpoint Genes san-1, mdf-2, bub-3 and the CENP-F Homologues hcp-1 and hcp-2 in Caenorhabditis Elegans
(BioMed Central, 2008-2-4) Hajeri, Vinita A.; Stewart, Anil M.; Moore, Landon L.; Padilla, Pamela A.
BACKGROUND: The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant. RESULTS: The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. CONCLUSION: Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.
Development of Admixture Mapping Panels for African Americans from Commercial High-Density SNP Arrays
(BioMed Central, 2010-7-5) Chen, Guanjie; Shriner, Daniel; Zhou, Jie; Doumatey, Ayo; Huang, Hanxia; Gerry, Norman P.; Herbert, Alan; Christman, Michael F.; Chen, Yuanxiu; Dunston, Georgia M.; Faruque, Mezbah U.; Rotimi, Charles N.; Adeyemo, Adebowale
BACKGROUND: Admixture mapping is a powerful approach for identifying genetic variants involved in human disease that exploits the unique genomic structure in recently admixed populations. To use existing published panels of ancestry-informative markers (AIMs) for admixture mapping, markers have to be genotyped de novo for each admixed study sample and samples representing the ancestral parental populations. The increased availability of dense marker data on commercial chips has made it feasible to develop panels wherein the markers need not be predetermined. RESULTS: We developed two panels of AIMs (~2,000 markers each) based on the Affymetrix Genome-Wide Human SNP Array 6.0 for admixture mapping with African American samples. These two AIM panels had good map power that was higher than that of a denser panel of ~20,000 random markers as well as other published panels of AIMs. As a test case, we applied the panels in an admixture mapping study of hypertension in African Americans in the Washington, D.C. metropolitan area. CONCLUSIONS" Developing marker panels for admixture mapping from existing genome-wide genotype data offers two major advantages: (1) no de novo genotyping needs to be done, thereby saving costs, and (2) markers can be filtered for various quality measures and replacement markers (to minimize gaps) can be selected at no additional cost. Panels of carefully selected AIMs have two major advantages over panels of random markers: (1) the map power from sparser panels of AIMs is higher than that of ~10-fold denser panels of random markers, and (2) clusters can be labeled based on information from the parental populations. With current technology, chip-based genome-wide genotyping is less expensive than genotyping ~20,000 random markers. The major advantage of using random markers is the absence of ascertainment effects resulting from the process of selecting markers. The ability to develop marker panels informative for ancestry from SNP chip genotype data provides a fresh opportunity to conduct admixture mapping for disease genes in admixed populations when genome-wide association data exist or are planned.
Transcriptomic and Metabolite Analyses of Cabernet Sauvignon Grape Berry Development
(BioMed Central, 2007-11-22) Deluc, Laurent G.; Grimplet, Jérôme; Wheatley, Matthew D.; Tillett, Richard L.; Quilici, David R.; Osborne, Craig; Schooley, David A.; Schlauch, Karen A.; Cushman, John C.; Cramer, Grant R.
BACKGROUND: Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. RESULTS: Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. CONCLUSION: These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.
Tissue-Specific mRNA Expression Profiling in Grape Berry Tissues
(BioMed Central, 2007-6-21) Grimplet, Jerome; Deluc, Laurent G.; Tillett, Richard L.; Wheatley, Matthew D.; Schlauch, Karen A.; Cramer, Grant R.; Cushman, John C.
BACKGROUND: Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. RESULTS: Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. CONCLUSION: These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality.
Smoking-Induced Gene Expression Changes in the Bronchial Airway Are Reflected in Nasal and Buccal Epithelium
(BioMed Central, 2008-5-30) Sridhar, Sriram; Schembri, Frank; Zeskind, Julie; Shah, Vishal; Gustafson, Adam M.; Steiling, Katrina; Liu, Gang; Dumas, Yves-Martine; Zhang, Xiaohui; Brody, Jerome S.; Lenburg, Marc E.; Spira, Avrum
BACKGROUND: Cigarette smoking is a leading cause of preventable death and a significant cause of lung cancer and chronic obstructive pulmonary disease. Prior studies have demonstrated that smoking creates a field of molecular injury throughout the airway epithelium exposed to cigarette smoke. We have previously characterized gene expression in the bronchial epithelium of never smokers and identified the gene expression changes that occur in the mainstem bronchus in response to smoking. In this study, we explored relationships in whole-genome gene expression between extrathorcic (buccal and nasal) and intrathoracic (bronchial) epithelium in healthy current and never smokers. RESULTS: Using genes that have been previously defined as being expressed in the bronchial airway of never smokers (the "normal airway transcriptome"), we found that bronchial and nasal epithelium from non-smokers were most similar in gene expression whencompared to other epithelial and nonepithelial tissues, with several antioxidant, detoxification, and structural genes being highly expressed in both the bronchus and nose. Principle component analysis of previously defined smoking-induced genes from the bronchus suggested that smoking had a similar effect on gene expression in nasal epithelium. Gene set enrichment analysis demonstrated that this set of genes was also highly enriched among the genes most altered by smoking in both nasal and buccal epithelial samples. The expression of several detoxification genes was commonly altered by smoking in all three respiratory epithelial tissues, suggesting a common airway-wide response to tobacco exposure. CONCLUSION: Our findings support a relationship between gene expression in extra- and intrathoracic airway epithelial cells and extend the concept of a smoking-induced field of injury to epithelial cells that line the mouth and nose. This relationship could potentially be utilized to develop a non-invasive biomarker for tobacco exposure as well as a non-invasive screening or diagnostic tool providing information about individual susceptibility to smoking-induced lung diseases.
Genetic mModifiers of Hb E/β0 Thalassemia Identified by a Two-Stage Genome-Wide Association Study
(BioMed Central, 2010-3-30) Sherva, Richard; Sripichai, Orapan; Abel, Kenneth; Ma, Qianli; Whitacre, Johanna; Angkachatchai, Vach; Makarasara, Wattanan; Winichagoon, Pranee; Svasti, Saovaros; Fucharoen, Suthat; Braun, Andreas; Farrer, Lindsay A.
BACKGROUND: Patients with Hb E/β0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/β0 thalassemia and normal α-globin genes. METHODS: First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping. RESULTS: After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the β-globin gene cluster had major alleles associated with severe disease. The most significant was bthal_bg200 (odds ratio (OR) = 5.56, P = 2.6 × 10-13). Seven SNPs in two distinct LD blocks within a region centromeric to the β-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 × 10-11). Several previously unreported SNPs were also significantly associated with disease severity. CONCLUSIONS: These results suggest that there may be an additional regulatory region centromeric to the β-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression.
Nonsteroidal Anti-Inflammatory Drug Use and Alzheimer's Disease Risk: The MIRAGE Study
(BioMed Central, 2005-1-12) Yip, Agustín G.; Green, Robert C.; Huyck, Matthew; Cupples, L. Adrienne; Farrer, Lindsay A.
BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAID) use may protect against Alzheimer's disease (AD) risk. We sought examine the association between NSAID use and risk of AD, and potential effect modification by APOE-ε4 carrier status and ethnicity. METHODS: The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD. Subjects comprised 691 AD patients (probands) and 973 family members enrolled at 15 research centers between 1996 and 2002. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. Ethnicity was similarly examined as an effect modifier. RESULTS: NSAID use was less frequent in cases compared to controls in the overall sample (adjusted OR = 0.64; 95% CI = 0.38–1.05). The benefit of NSAID use appeared more pronounced among APOE-ε4 carriers (adjusted OR = 0.49; 95% CI = 0.24–0.98) compared to non-carriers, although this association was not statistically significant. The pattern of association was similar in Caucasian and African Americans. CONCLUSIONS: NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.

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