Browsing by Subject "gene expression"
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Item Accuracy of RNA-Seq and its dependence on sequencing depth(BMC Bioinformatics, 2012-08-24) Cai, Guoshuai; Li, Hua; Lu, Yue; Huang, Xuelin; Lee, Juhee; Muller, Peter; Ji, Yuan; Liang, ShoudanBackground: The cost of DNA sequencing has undergone a dramatical reduction in the past decade. As a result, sequencing technologies have been increasingly applied to genomic research. RNA-Seq is becoming a common technique for surveying gene expression based on DNA sequencing. As it is not clear how increased sequencing capacity has affected measurement accuracy of mRNA, we sought to investigate that relationship. Result: We empirically evaluate the accuracy of repeated gene expression measurements using RNA-Seq. We identify library preparation steps prior to DNA sequencing as the main source of error in this process. Studying three datasets, we show that the accuracy indeed improves with the sequencing depth. However, the rate of improvement as a function of sequence reads is generally slower than predicted by the binomial distribution. We therefore used the beta-binomial distribution to model the overdispersion. The overdispersion parameters we introduced depend explicitly on the number of reads so that the resulting statistical uncertainty is consistent with the empirical data that measurement accuracy increases with the sequencing depth. The overdispersion parameters were determined by maximizing the likelihood. We shown that our modified beta-binomial model had lower false discovery rate than the binomial or the pure beta-binomial models. Conclusion: We proposed a novel form of overdispersion guaranteeing that the accuracy improves with sequencing depth. We demonstrated that the new form provides a better fit to the data.Item Accuracy of RNA-Seq and its dependence on sequencing depth(BMC Bioinformatics, 2012-08-24) Cai, Guoshuai; Li, Hua; Lu, Yue; Huang, Xuelin; Lee, Juhee; Muller, Peter; Ji, Yuan; Liang, ShoudanBackground: The cost of DNA sequencing has undergone a dramatical reduction in the past decade. As a result, sequencing technologies have been increasingly applied to genomic research. RNA-Seq is becoming a common technique for surveying gene expression based on DNA sequencing. As it is not clear how increased sequencing capacity has affected measurement accuracy of mRNA, we sought to investigate that relationship. Result: We empirically evaluate the accuracy of repeated gene expression measurements using RNA-Seq. We identify library preparation steps prior to DNA sequencing as the main source of error in this process. Studying three datasets, we show that the accuracy indeed improves with the sequencing depth. However, the rate of improvement as a function of sequence reads is generally slower than predicted by the binomial distribution. We therefore used the beta-binomial distribution to model the overdispersion. The overdispersion parameters we introduced depend explicitly on the number of reads so that the resulting statistical uncertainty is consistent with the empirical data that measurement accuracy increases with the sequencing depth. The overdispersion parameters were determined by maximizing the likelihood. We shown that our modified beta-binomial model had lower false discovery rate than the binomial or the pure beta-binomial models. Conclusion: We proposed a novel form of overdispersion guaranteeing that the accuracy improves with sequencing depth. We demonstrated that the new form provides a better fit to the data.Item Alcohol-Induced Histone Acetylation Reveals a Gene Network Involved in Alcohol Tolerance(PLOS Genetics, 2013-12-12) Ghezzi, Alfredo; Krishnan, Harish R.; Lew, Linda; Prado III, Francisco J.; Ong, Darryl S.; Atkinson, Nigel S.Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.Item Bayesian Mixture Models for Assessment of Gene Differential Behaviour and Prediction of pCR through the Integration of Copy Number and Gene Expression Data(PLOS One, 2013-07-12) Trntini, Filippo; Ji, Yuan; Iwamoto, Takayuki; Qi, Yuan; Pusztai, Lajos; Muller, PeterWe consider modeling jointly microarray RNA expression and DNA copy number data. We propose Bayesian mixture models that define latent Gaussian probit scores for the DNA and RNA, and integrate between the two platforms via a regression of the RNA probit scores on the DNA probit scores. Such a regression conveniently allows us to include additional sample specific covariates such as biological conditions and clinical outcomes. The two developed methods are aimed respectively to make inference on differential behaviour of genes in patients showing different subtypes of breast cancer and to predict the pathological complete response (pCR) of patients borrowing strength across the genomic platforms. Posterior inference is carried out via MCMC simulations. We demonstrate the proposed methodology using a published data set consisting of 121 breast cancer patients.Item Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles(PLOS One, 2013-06-07) Matsumoto, Yuiko; Buemio, Alvin; Chu, Randy; Vafaee, Mozhgon; Crews, DavidIn the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.Item Evidence for the down-regulation of ATP6V1H gene expression in type 2 diabetes(2012-05) Molina, Melanie; Field, LeanneAccording to the World Health Organization, there are approximately 346 million cases of diabetes worldwide, 90% of which are accredited specifically to type 2 diabetes. As type 2 diabetes continues to raise concern, researchers are beginning to turn their focus to the inherent factors that predispose individuals to the disease, one of which is genetics. Recently, a higher prevalence of type 2 diabetes was observed in a 2,500-member cohort of Mexican-Americans established on the US-Mexican border, specifically in Brownsville (Cameron County), Texas. The purpose of this research was threefold: 1) to study the demographic and physiological parameters of nine individuals in the CCHC who developed diabetes over the three years of the study period; 2) to investigate whether the gene encoding subunit H of V-ATPase (ATP6V1H) was down-regulated in the nine individuals as they progressed from pre-diabetes to type 2 diabetes; 3) to perform an extensive literature review of the ATP6V1H gene and its protein product, V-ATPase subunit H, to postulate what role it might play in the development of diabetes and its complications. Of the nine participants that developed type 2 diabetes, six were female and three were male (ages ranged from 38-81). Six different physiological parameters were measured in each individual at the time of their first and last visits to the clinic: triglyceride, cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, glucose and HbA1c. Higher levels of triglyceride, HDL cholesterol and glucose were found in the 9 individuals as they progressed from pre-diabetes to type 2 diabetes. These differences were statistically significant. There were no statistically significant differences in any of the other measured parameters. The gene expression of ATP6V1H was assayed using microarray techniques and a paired Z test revealed a statistically significant decrease (p-value = 7.18 x 10-11) in expression in all nine participants.Item Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator(BMC Microbiology, 2012-10-05) Caballero, Valeria C.; Toledo, Vivian P.; Maturana, Cristian; Fisher, Carolyn R.; Payne, Shelley M.; Salazar, Juan CarlosBackground: Glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. Results: The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. Conclusions: The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.Item Gene Expression Associated with White Syndromes in a Reef Building Coral, Acropora Hyacinthus(2015-05) Wright, Rachel M.; Aglyamova, Galina V.; Meyer, Eli; Matz, Mikhail V.; Wright, Rachel M.Corals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy. Results: Tag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies. Conclusions: Similarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.Item Gene expression profiling in hepatic tissue of newly weaned pigs fed pharmacological zinc and phytase supplemented diets(BMC Genomics, 2008-09-17) Martinez-Motemayor, Michelle M.; Hill, Gretchen M.; Raney, Nancy E.; Rilington, Valencia D.; Tempelman, Robert J.; Link, Jane E.; Wilkinson, Christopher P.; Ramos, Antonio M.; Ernst, Catherine W.Background: Zinc (Zn) is an essential trace element. However, Zn bioavailability from commonly consumed plants may be reduced due to phytic acid. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Moreover, adding supplemental phytase improves Zn bioavailability. Thus, we hypothesized that benefits of pharmacological Zn supplementation result from changes in gene expression that could be further affected by supplemental phytase. The goal of this study was to investigate the effects of feeding newly weaned pigs dietary Zn (150, 1,000, or 2,000 mg Zn/kg) as Zn oxide with or without phytase [500 phytase units (FTU)/kg] for 14 d on hepatic gene expression. Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg (n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray. -- Results: Expressed sequence tags for 61 putatively differentially expressed transcripts were cloned and sequenced. In addition, interrogation of a 13,297 element oligonucleotide microarray revealed 650 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation. Seven transcripts exhibiting differential expression in pigs fed pharmacological Zn with sequence similarities to genes encoding GLO1, PRDX4, ACY1, ORM1, CPB2, GSTM4, and HSP70.2 were selected for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and ACY1 (P < 0.01) mRNA abundances were confirmed to be greater in pigs fed 1,000 (n = 8) and 2,000 (n = 8) mg Zn/kg than in pigs fed 150 (n = 7) mg Zn/kg. Relative hepatic HSP70.2 (P < 0.002) mRNA abundance was confirmed to be lower in pigs fed 2,000 mg Zn/kg than in pigs fed 150 or 1,000 mg Zn/kg. -- Conclusion: Results suggest that feeding pharmacological Zn (1,000 or 2,000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.Item Identification of Novel sRNAs in Mycobacterial Species(PLOS One, 2013-11-14) Tsai, Chen-Hsun; Baranowski, Catherine; Livny, Jonathan; McDonough, Kathleen A.; Wade, Joseph T.; Contreras, Lydia M.Bacterial small RNAs (sRNAs) are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE) to map the 5′ and 3′ ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.Item The INIA Texas Gene Expression Database: An online tool for alcohol genomics(2012-04) Weyn-Vanhentenryck, Sebastien; Ponomarev, IgorAlcoholism is a serious condition that affects millions of people and costs billions of dollars each year in treatment, damages, and lost income. In addition, it carries a tremendous emotional burden. Alcoholism is caused by a combination of genetic and environmental factors, which have yet to be fully identified. Fortunately, alcoholism research, as well as research into other diseases with a genetic component, has greatly benefited from recent rapid developments in high-throughput genomic technologies and the development of relevant model organisms. This has been highly productive for progress in the field, but effective methods for identifying relevant data and for performing cross-dataset analyses have not been developed at the same pace. To help fulfill this need, I have developed the INIA (Integrative Neuroscience Initiative on Alcoholism) Texas Gene Expression Database (IT-GED), which is freely available at http://inia.icmb.utexas.edu. IT-GED is a web-based database which contains a compilation of the significantly expressed genes from each of several microarray datasets investigating the role of gene expression in the brain's regulation of alcohol consumption. The studies were performed both in model organisms (mouse and rat) and post-mortem humans. The data is presented via a user-friendly interface which provides advanced searching abilities for identifying genes of interest and tools for analysis of the data. These tools provide the ability to compare user data to every dataset in IT-GED in order to assess the significance of a group of genes across multiple datasets and the ability to generate visual networks of those genes in order to identify the ones that are likely the most functionally significant in the response to high alcohol consumption. IT-GED thus provides a means by which alcohol researchers can combine multiple sources of data to generate novel hypotheses concerning the genetic causes of alcoholism. The goal of IT-GED is to provide support for comparing and integrating results across gene expression studies of alcohol consumption and for generating novel hypotheses based on individual genes and gene-gene interactions by simplifying data access, providing various tools for analysis, and presenting users with an easy-to-use interface.Item Integrating Transcriptional, Metabolomic, and Physiological Responses to Drought Stress and Recovery in Switchgrass (Panicum Virgatum L.)(2014-06) Meyer, Eli; Aspinwall, Michael J.; Lowry, David B.; Palacio-Mejia, Juan Diego; Logan, Tierney L.; Fay, Phillip A.; Juenger, Thomas E.; Meyer, Eli; Aspinwall, Michael J.; Lowry, David B.; Palacio-Mejia, Juan Diego; Logan, Tierney L.; Juenger, Thomas E.In light of the changes in precipitation and soil water availability expected with climate change, understanding the mechanisms underlying plant responses to water deficit is essential. Toward that end we have conducted an integrative analysis of responses to drought stress in the perennial C-4 grass and biofuel crop, Panicum virgatum (switchgrass). Responses to soil drying and re-watering were measured at transcriptional, physiological, and metabolomic levels. To assess the interaction of soil moisture with diel light: dark cycles, we profiled gene expression in drought and control treatments under pre-dawn and mid-day conditions. Results: Soil drying resulted in reduced leaf water potential, gas exchange, and chlorophyll fluorescence along with differential expression of a large fraction of the transcriptome (37%). Many transcripts responded differently depending on time of day (e.g. up-regulation pre-dawn and down-regulation mid-day). Genes associated with C-4 photosynthesis were down-regulated during drought, while C-4 metabolic intermediates accumulated. Rapid changes in gene expression were observed during recovery from drought, along with increased water use efficiency and chlorophyll fluorescence. Conclusions: Our findings demonstrate that drought responsive gene expression depends strongly on time of day and that gene expression is extensively modified during the first few hours of drought recovery. Analysis of covariation in gene expression, metabolite abundance, and physiology among plants revealed non-linear relationships that suggest critical thresholds in drought stress responses. Future studies may benefit from evaluating these thresholds among diverse accessions of switchgrass and other C-4 grasses.Item Integration of miRNA and Protein Profiling Reveals Coordinated Neuroadaptations in the Alcohol-Dependent Mouse Brain(PLOS One, 2013-12-16) Gorini, Giorgio; Nunez. Yuury O.; Mayfield, R. DayneThe molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE) procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.Item International plant molecular biology: a bright future for green science(Genome Biology, 2012-11-19) Sung, Sibum; Huq, Enamul; Chan, Jeffery Z.A report on the 10th International Congress of Plant Molecular Biology, Jeju, South Korea, October 21-26, 2012.Item The investigation of gene expression in FIBP, GATA-2, SNP136, SNP hOGGI in human prostate cancer(2007) Amini, Andrew; George B. KittoItem No Control Genes Required: Bayesiian Analysis of qRT-PCR Data(PLOS One, 2013-08-19) Matz, Mikhail V.; Wright, Rachel M.; Scott, James G.Background: Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR) is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. Results: In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC) algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts). Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the “classic” analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization) but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. Conclusions: Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.Item Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung(PLOS One, 2013-12-06) Huse, Holy K.; Kwon, Taejoon; Zlosnik, James E.A.; Speert, David P.; Marcotte, Edward M.; Whiteley, MarvinThe gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.Item Syndecan-1 Regulates Vasculat Smooth Muscle Cell Phenotype(PLOS One, 2014-02-25) Chaterji, Somali; Lam, Christoffer H.; Ho, Derek S.; Proske, Daniel C.; Baker, Aaron B.Objective: We examined the role of syndecan-1 in modulating the phenotype of vascular smooth muscle cells in the context of endogenous inflammatory factors and altered microenvironments that occur in disease or injury-induced vascular remodeling. Methods and Results: Vascular smooth muscle cells (vSMCs) display a continuum of phenotypes that can be altered during vascular remodeling. While the syndecans have emerged as powerful and complex regulators of cell function, their role in controlling vSMC phenotype is unknown. Here, we isolated vSMCs from wild type (WT) and syndecan-1 knockout (S1KO) mice. Gene expression and western blotting studies indicated decreased levels of α-smooth muscle actin (α-SMA), calponin, and other vSMC-specific differentiation markers in S1KO relative to WT cells. The spread area of the S1KO cells was found to be greater than WT cells, with a corresponding increase in focal adhesion formation, Src phosphorylation, and alterations in actin cytoskeletal arrangement. In addition, S1KO led to increased S6RP phosphorylation and decreased AKT and PKC-α phosphorylation. To examine whether these changes were present in vivo, isolated aortae from aged WT and S1KO mice were stained for calponin. Consistent with our in-vitro findings, the WT mice aortae stained higher for calponin relative to S1KO. When exposed to the inflammatory cytokine TNF-α, WT vSMCs had an 80% reduction in syndecan-1 expression. Further, with TNF-α, S1KO vSMCs produced increased pro-inflammatory cytokines relative to WT. Finally, inhibition of interactions between syndecan-1 and integrins αvβ3 and αvβ5 using the inhibitory peptide synstatin appeared to have similar effects on vSMCs as knocking out syndecan-1, with decreased expression of vSMC differentiation markers and increased expression of inflammatory cytokines, receptors, and osteopontin. Conclusions: Taken together, our results support that syndecan-1 promotes vSMC differentiation and quiescence. Thus, the presence of syndecan-1 would have a protective effect against vSMC dedifferentiation and this activity is linked to interactions with integrins αvβ3 and αvβ5.Item Synthesis and biologic properties of hydrophilic sapphyrins, a new class of tumor-selective inhibitors of gene expression(Molecular Cancer, 2007-01-19) Wang, Zhong; Lecane, Philip S.; Thiemann, Patrica; Fan, Qing; Cortez, Cecilia; Ma, Xuan; Tonev, Danielle; Miles, Dale; Naumovski, Louie; Miller, Richard A.; Magda, Darren; Cho, Dong-Gyu; Sessler, Jonathan L.; Pike, Brian L.; Yeligar, Samantha M.; Karaman, Mazen W.; Hacia, Joseph G.Background: Sapphyrin analogues and related porphyrin-like species have attracted attention as anticancer agents due to their selective localization in various cancers, including hematologic malignancies, relative to surrounding tissues. Sapphyrins are electron affinic compounds that generate high yields of singlet oxygen formation. Although initially explored in the context of photodynamic therapy, sapphyrins have intrinsic anticancer activity that is independent of their photosensitizing properties. However, the mechanisms for their anticancer activity have not been fully elucidated. -- Results: We have prepared a series of hydrophilic sapphyrins and evaluated their effect on proliferation, uptake, and cell death in adherent human lung (A549) and prostate (PC3) cancer cell lines and in an A549 xenograft tumor model. PCI-2050, the sapphyrin derivative with the highest in vitro growth inhibitory activity, significantly lowered 5-bromo-2'-deoxyuridine incorporation in S-phase A549 cells by 60% within eight hours and increased levels of reactive oxygen species within four hours. The growth inhibition pattern of PCI-2050 in the National Cancer Institute 60 cell line screen correlated most closely using the COMPARE algorithm with known transcriptional or translational inhibitors. Gene expression analyses conducted on A549 plateau phase cultures treated with PCI-2050 uncovered wide-spread decreases in mRNA levels, which especially affected short-lived transcripts. Intriguingly, PCI-2050 increased the levels of transcripts involved in RNA processing and trafficking, transcriptional regulation, and chromatin remodeling. We propose that these changes reflect the activation of cellular processes aimed at countering the observed wide-spread reductions in transcript levels. In our A549 xenograft model, the two lead compounds, PCI-2050 and PCI-2022, showed similar tumor distributions despite differences in plasma and kidney level profiles. This provides a possible explanation for the better tolerance of PCI-2022 relative to PCI-2050. -- Conclusion: Hydrophilic sapphyrins were found to display promise as novel agents that localize to tumors, generate oxidative stress, and inhibit gene expression.Item Widespread Misinterpretable ChIP-seq Bias in Yeast(PLOS One, 2013-12-09) Park, Daechan; Lee, Yaelim; Bhupindersingh, Gurvani; Lyer, Vishwanath R.Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to detect genome-wide interactions between a protein of interest and DNA in vivo. Loci showing strong enrichment over adjacent background regions are typically considered to be sites of binding. Insufficient attention has been given to systematic artifacts inherent to the ChIP-seq procedure that might generate a misleading picture of protein binding to certain loci. We show here that unrelated transcription factors appear to consistently bind to the gene bodies of highly transcribed genes in yeast. Strikingly, several types of negative control experiments, including a protein that is not expected to bind chromatin, also showed similar patterns of strong binding within gene bodies. These false positive signals were evident across sequencing platforms and immunoprecipitation protocols, as well as in previously published datasets from other labs. We show that these false positive signals derive from high rates of transcription, and are inherent to the ChIP procedure, although they are exacerbated by sequencing library construction procedures. This expression bias is strong enough that a known transcriptional repressor like Tup1 can erroneously appear to be an activator. Another type of background bias stems from the inherent nucleosomal structure of chromatin, and can potentially make it seem like certain factors bind nucleosomes even when they don't. Our analysis suggests that a mock ChIP sample offers a better normalization control for the expression bias, whereas the ChIP input is more appropriate for the nucleosomal periodicity bias. While these controls alleviate the effect of the biases to some extent, they are unable to eliminate it completely. Caution is therefore warranted regarding the interpretation of data that seemingly show the association of various transcription and chromatin factors with highly transcribed genes in yeast.