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1.  Highlights from the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength or FAMuSS Study 
BioMed Research International  2013;2013:643575.
The purpose of the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength study or FAMuSS was to identify genetic factors that dictated the response of health-related fitness phenotypes to resistance exercise training (RT). The phenotypes examined were baseline muscle strength and muscle, fat, and bone volume and their response to RT. FAMuSS participants were 1300 young (24 years), healthy men (42%) and women (58%) that were primarily of European-American descent. They were genotyped for ~500 polymorphisms and completed the Paffenbarger Physical Activity Questionnaire to assess energy expenditure and time spent in light, moderate, and vigorous intensity habitual physical activity and sitting. Subjects then performed a 12-week progressive, unilateral RT program of the nondominant arm with the dominant arm used as a comparison. Before and after RT, muscle strength was measured with the maximum voluntary contraction and one repetition maximum, while MRI measured muscle, fat, and bone volume. We will discuss the history of how FAMuSS originated, provide a brief overview of the FAMuSS methods, and summarize our major findings regarding genotype associations with muscle strength and size, body composition, cardiometabolic biomarkers, and physical activity.
doi:10.1155/2013/643575
PMCID: PMC3885233  PMID: 24455711
2.  Microtubules Underlie Dysfunction in Duchenne Muscular Dystrophy 
Science signaling  2012;5(236):10.1126/scisignal.2002829.
Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca2+) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase–dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca2+ influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca2+ influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca2+ influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS–related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca2+ and ROS signaling in DMD and could be effective therapeutic targets for intervention.
doi:10.1126/scisignal.2002829
PMCID: PMC3835660  PMID: 22871609
3.  Identification of condition-specific regulatory modules through multi-level motif and mRNA expression analysis 
Many computational methods for identification of transcription regulatory modules often result in many false positives in practice due to noise sources of binding information and gene expression profiling data. In this paper, we propose a multi-level strategy for condition-specific gene regulatory module identification by integrating motif binding information and gene expression data through support vector regression and significant analysis. We have demonstrated the feasibility of the proposed method on a yeast cell cycle data set. The study on a breast cancer microarray data set shows that it can successfully identify the significant and reliable regulatory modules associated with breast cancer.
PMCID: PMC3749738  PMID: 20054984
transcription regulatory module; motif enrichment analysis; SVR; support vector regression; statistical significance analysis; multi-level regulator identification
4.  Status of LEPR Gene in PCB-exposed Population: A Quick Look 
Earlier, we have reported that Polychlorinated Biphenyls (PCBs) exposure in Slovak population has made differential gene expression that has linked to the possibilities of some diseases and disorder development in the studied population. Here we report that down-regulation of LEPR (Leptin receptor) gene in the 45-month children may have been following consequences in developing obesity later in life. A pilot high-throughput qRT-PCR [Taqman Low Density Array (TLDA)] study in a small population also corroborated the gene-expression results, and their pathways underlying the consequences of the diseases, amid further detailed large-scale population validation. The study shows the opportunity of predicting long-term effects of chemical exposures using selected genomic classifiers may reflect exposure effect and risk from environmental toxicants.
PMCID: PMC3670706  PMID: 23741107
PCBs; Gene Expression; Leptin Receptor; Obesity
5.  Novel Approaches to Corticosteroid Treatment in Duchenne Muscular Dystrophy 
doi:10.1016/j.pmr.2012.08.003
PMCID: PMC3606917  PMID: 23137739
Duchenne muscular dystrophy; Corticosteroids; Glucocorticoids; Dissociative steroids
6.  Differential gene expression and a functional analysis of PCB-exposed children: Understanding disease and disorder development 
Environment International  2011;40:143-154.
The goal of the present study is to understand the probable molecular mechanism of toxicities and the associated pathways related to observed pathophysiology in high PCB-exposed populations. We have performed a microarray-based differential gene expression analysis of children (mean age 46.1 months) of Central European descent from Slovak Republic in a well-defined study cohort. The subset of children having high blood PCB concentrations (>75 percentile) were compared against their low PCB counterparts (<25 percentile), with mean lipid-adjusted PCB values of 3.02±1.3 and 0.06±0.03 ng/mg of serum lipid, for the two groups, respectively (18.1±4.4 and 0.3±0.1 ng/ml of serum). The microarray was conducted with the total RNA from the peripheral blood mononuclear cells of the children using an Affymetrix platform (GeneChip Human genome U133 Plus 2.0 Array) and was analyzed by Gene Spring (GX 10.0). A highly significant set of 162 differentially expressed genes between high and low PCB groups (p value <0.00001) were identified and subsequently analyzed using the Ingenuity Pathway Analysis tool. The results indicate that Cell-To-Cell Signaling and Interaction, Cellular Movement, Cell Signaling, Molecular Transport, and Vitamin and Mineral Metabolism were the major molecular and cellular functions associated with the differentially altered gene set in high PCB-exposed children. The differential gene expressions appeared to play a pivotal role in the development of probable diseases and disorders, including cardiovascular disease and cancer, in the PCB-exposed population. The analyses also pointed out possible organ-specific effects, e.g., cardiotoxicity, hepatotoxicity and nephrotoxicity, in high PCB-exposed subjects. A few notable genes, such as BCL2, PON1, and ITGB1, were significantly altered in our study, and the related pathway analysis explained their plausible involvement in the respective disease processes, as mentioned. Our results provided insight into understanding the associated molecular mechanisms of complex gene-environment interactions in a PCB-exposed population. Future endeavors of supervised genotyping of pathway-specific molecular epidemiological studies and population biomarker validations are already underway to reveal individual risk factors in these PCB-exposed populations.
doi:10.1016/j.envint.2011.07.008
PMCID: PMC3247643  PMID: 21855147
PCB; Microarray; Gene expression; Environmental exposure; Functional analysis; PCB-exposed population
7.  The 1p13.3 LDL (C)-Associated Locus Shows Large Effect Sizes in Young Populations 
Pediatric research  2011;69(6):538-543.
Genome-wide association studies (GWAS) have identified polymorphic loci associated with coronary artery disease (CAD) risk factors (i. e. serum lipids) in adult populations (42–69 yrs). We hypothesized that younger populations would show a greater relative genetic component due to fewer confounding variables. We examined the influence of 20 GWAS loci associated with serum lipids and insulin metabolism, in a university student cohort (n=548; mean age= 24 yrs), and replicated statistically associated results in a second study cohort of primary school students (n=810, mean age= 11.5 yrs). 19 loci showed no relationship with studied risk factors in young adults. However, the ancestral allele of the rs646776 (SORT1) locus was strongly associated with increased low density lipoprotein cholesterol {LDL (C)} in young adults (TT: 97.6 ± 1.0 mg/dL {n=345}, vs. CT/CC: 87.3 ± 1.0 mg/dL {n=203}; p = 3 × 10−6) and children (TT: 94.0 ± 1.3 mg/dL {n=551}, vs. CT/CC: 84.7 ± 1.4 mg/dL {n=259}; p = 4 × 10−6). This locus is responsible for 3.6% of population variance in young adults and 2.5% of population variance in children. The effect size of the SORT1 locus is considerably higher in young populations (2.5%–4.1%) compared to older subjects (1%).
doi:10.1203/PDR.0b013e3182139227
PMCID: PMC3606915  PMID: 21297524
8.  Characterization of Transferrin Glycopeptide Structures in Human Cerebrospinal Fluid 
Transferrin in cerebrospinal fluid (CSF) exists as a mixture of silao and asialo glycoforms believed to originate from liver and brain respectively. We have previously shown that alteration in the asialo glycoform pattern could be an indication of certain anomalies in the central nervous system. Additionally, CSF asialo-transferrin has been shown to be a reliable marker to assess cerebrospinal leakage in head trauma. Therefore, the CSF transferrin glycoform pattern could be a useful diagnostic and prognostic tool. In this study we sought to characterize, in-depth, the transferrin glycovariants in cerebrospinal fluid using a combination of two-dimensional gel electrophoresis and high precision mass spectrometry analysis. Cerebrospinal fluid transferrin was detected as multiple spots (seven major spots) with different isoelectric points and slight shift in apparent molecular mass. High resolution (>60,000) and high accuracy (< 3 ppm error) mass spectrometry analysis revealed that each spot had a unique glycopeptide signature. MSn analysis enabled characterization of the glycan structure directly from the in-gel digested spots. The multiple spots detected for cerebrospinal fluid transferrin were mainly due to heterogeneity of di-antennary and tri-antennary glycans harboring a varying number of terminal N-acetylneuraminic acids and the existence of a high mannose and high N-acetylhexosamine glycosylated species.
doi:10.1016/j.ijms.2011.06.021
PMCID: PMC3293479  PMID: 22408387
transferrin; cerebrospinal fluid; glycoforms; glycopeptides; glycans; LTQ-Orbitrap XL; high resolution; accurate mass
9.  Extensive and Prolonged Restoration of Dystrophin Expression with Vivo-Morpholino-Mediated Multiple Exon Skipping in Dystrophic Dogs 
Nucleic Acid Therapeutics  2012;22(5):306-315.
Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6–8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.
doi:10.1089/nat.2012.0368
PMCID: PMC3464409  PMID: 22888777
10.  Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction 
Skeletal Muscle  2012;2:19.
Background
Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs.
Methods
Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle.
Results
Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle.
Conclusion
EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.
doi:10.1186/2044-5040-2-19
PMCID: PMC3541266  PMID: 22974368
Neuromuscular junction; Eps homology domain containing protein; Endosomal transport; Endosomal recycling; Bungarotoxin; Endplate; Synapse; Skeletal muscle
11.  Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1G93A mouse model of amyotrophic lateral sclerosis 
Brain and Behavior  2012;2(5):563-575.
Galectins are pleiotropic carbohydrate-binding lectins involved in inflammation, growth/differentiation, and tissue remodeling. The functional role of galectins in amyotrophic lateral sclerosis (ALS) is unknown. Expression studies revealed increases in galectin-1 mRNA and protein in spinal cords from SOD1G93A mice, and in galectin-3 and -9 mRNAs and proteins in spinal cords of both SOD1G93A mice and sporadic ALS patients. As the increase in galectin-3 appeared in early presymptomatic stages and increased progressively through to end stage of disease in the mouse, it was selected for additional study, where it was found to be mainly expressed by microglia. Galectin-3 antagonists are not selective and do not readily cross the blood–brain barrier; therefore, we generated SOD1G93A/Gal-3−/− transgenic mice to evaluate galectin-3 deletion in a widely used mouse model of ALS. Disease progression, neurological symptoms, survival, and inflammation were assessed to determine the effect of galectin-3 deletion on the SOD1G93A disease phenotype. Galectin-3 deletion did not change disease onset, but resulted in more rapid progression through functionally defined disease stages, more severely impaired neurological symptoms at all stages of disease, and expiration, on average, 25 days earlier than SOD1G93A/Gal-3+/+ cohorts. In addition, microglial staining, as well as TNF-α, and oxidative injury were increased in SOD1G93A/Gal-3−/− mice compared with SOD1G93A/Gal-3+/+ cohorts. These data support an important functional role for microglial galectin-3 in neuroinflammation during chronic neurodegenerative disease. We suggest that elevations in galectin-3 by microglia as disease progresses may represent a protective, anti-inflammatory innate immune response to chronic motor neuron degeneration.
doi:10.1002/brb3.75
PMCID: PMC3489809  PMID: 23139902
Alternative activation; amyotrophic lateral sclerosis; microglia; motor neuron disease; SOD1
12.  Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model 
Skeletal Muscle  2012;2:16.
Background
Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice.
Methods
Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects.
Results
We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage.
Conclusions
The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.
doi:10.1186/2044-5040-2-16
PMCID: PMC3482394  PMID: 22908954
Duchenne muscular dystrophy; Muscle; AICAR; GW501516; Metabolism
13.  Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ 
Marine genomics  2011;4(1):33-40.
Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9,719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specializations using evolutionary diversity of organ function and cross-species mapping.
doi:10.1016/j.margen.2010.12.004
PMCID: PMC3412124  PMID: 21429463
Electric organ; Neuromuscular Junction (NMJ); Proteome; Torpedo californica; Novel Gene Discovery; Open Reading Frame (ORF)
14.  Genome-wide identification of significant aberrations in cancer genome 
BMC Genomics  2012;13:342.
Background
Somatic Copy Number Alterations (CNAs) in human genomes are present in almost all human cancers. Systematic efforts to characterize such structural variants must effectively distinguish significant consensus events from random background aberrations. Here we introduce Significant Aberration in Cancer (SAIC), a new method for characterizing and assessing the statistical significance of recurrent CNA units. Three main features of SAIC include: (1) exploiting the intrinsic correlation among consecutive probes to assign a score to each CNA unit instead of single probes; (2) performing permutations on CNA units that preserve correlations inherent in the copy number data; and (3) iteratively detecting Significant Copy Number Aberrations (SCAs) and estimating an unbiased null distribution by applying an SCA-exclusive permutation scheme.
Results
We test and compare the performance of SAIC against four peer methods (GISTIC, STAC, KC-SMART, CMDS) on a large number of simulation datasets. Experimental results show that SAIC outperforms peer methods in terms of larger area under the Receiver Operating Characteristics curve and increased detection power. We then apply SAIC to analyze structural genomic aberrations acquired in four real cancer genome-wide copy number data sets (ovarian cancer, metastatic prostate cancer, lung adenocarcinoma, glioblastoma). When compared with previously reported results, SAIC successfully identifies most SCAs known to be of biological significance and associated with oncogenes (e.g., KRAS, CCNE1, and MYC) or tumor suppressor genes (e.g., CDKN2A/B). Furthermore, SAIC identifies a number of novel SCAs in these copy number data that encompass tumor related genes and may warrant further studies.
Conclusions
Supported by a well-grounded theoretical framework, SAIC has been developed and used to identify SCAs in various cancer copy number data sets, providing useful information to study the landscape of cancer genomes. Open–source and platform-independent SAIC software is implemented using C++, together with R scripts for data formatting and Perl scripts for user interfacing, and it is easy to install and efficient to use. The source code and documentation are freely available at http://www.cbil.ece.vt.edu/software.htm.
doi:10.1186/1471-2164-13-342
PMCID: PMC3428679  PMID: 22839576
15.  Global gene expression and Ingenuity biological functions analysis on PCB 153 and 138 induced human PBMC in vitro reveals differential mode(s) of action in developing toxicities 
Environment international  2011;37(5):838-857.
Several reports have indicated that low level of polychlorinated biphenyl (PCB) exposure can adversely affect a multitude of physiological disorders and diseases in in vitro, in vivo, and as reported in epidemiological studies. This investigation is focused on the possible contribution of two most prevalent PCB congeners in vitro in developing toxicities. We used PCB 138 and 153 at the human equivalence level as model agents to test their specificity. We chose a global approach using oligonucleotide microarray technology to investigate modulated gene expression for biological effects, upon exposure of PCBs, followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We performed in vitro studies with human peripheral blood mononuclear cells (PBMC), where PBMC cells were exposed to respective PCBs for 48 hrs. Overall, our observation on gene expression indicated that PCB produces a unique signature affecting different pathways, specific for each congener. While analyzing these data through IPA, the prominent and interesting disease and disorders were Neurological disease, Cancer, Cardiovascular disease, respiratory disease, as well as endocrine system disorders Genetic disorders, and reproductive system disease. They showed strong resemblances with in vitro, in vivo, and in the epidemiological studies. A distinct difference was observed in renal and urological diseases, organisimal injury and abnormalities, dental disease, ophthalmic disease, and psychological disorders, which are only revealed by PCB 138 exposure, but not in PCB 153. The present study emphasizes the challenges of global gene expression in vitro and was correlated with the results of exposed human population. The microarray results give a molecular mechanistic insight and functional effects, following PCB exposure. The extent of changes in genes related to several possible mode(s) of action highlights the changes in cellular functions and signaling pathways that play major roles. In addition to understanding the pathways related to mode of action for chemicals, these data could lead to the identification of genomic signatures that could be used for screening of chemicals for their potential to cause disease and developmental disorders.
doi:10.1016/j.envint.2011.02.010
PMCID: PMC3097535  PMID: 21470681
PCB 138; PCB 153; Human PBMC; Gene expression; IPA Analysis; Disease and Disorders
16.  Correction: Sphingosine-1-Phosphate Enhances Satellite Cell Activation in Dystrophic Muscles through a S1PR2/STAT3 Signaling Pathway 
PLoS ONE  2012;7(6):10.1371/annotation/7e7ac57d-30ae-4e49-9138-e3bdbe3491d2.
doi:10.1371/annotation/7e7ac57d-30ae-4e49-9138-e3bdbe3491d2
PMCID: PMC3368964
17.  Asthmatic Airway Epithelium Is Intrinsically Inflammatory and Mitotically Dyssynchronous 
Asthma is an inflammatory condition for which anti-inflammatory glucocorticoids are the standard of care. However, similar efficacy has not been shown for agents targeting inflammatory cells and pathways. This suggests a noninflammatory cell contributor (e.g., epithelium) to asthmatic inflammation. Herein, we sought to define the intrinsic and glucocorticoid-affected properties of asthmatic airway epithelium compared with normal epithelium. Human primary differentiated normal and asthmatic airway epithelia were cultured in glucocorticoid-free medium beginning at −48 hours. They were pulsed with dexamethasone (20 nM) or vehicle for 2 hours at −26, −2, +22, and +46 hours. Cultures were mechanically scrape-wounded at 0 hours and exposed continuously to bromodeoxyuridine (BrdU). Cytokine secretions were analyzed using cytometric bead assays. Wound regeneration/mitosis was analyzed by microscopy and flow cytometry. Quiescent normal (n = 3) and asthmatic (n = 6) epithelia showed similar minimal inflammatory cytokine secretion and mitotic indices. After wounding, asthmatic epithelia secreted more basolateral TGF-β1, IL-10, IL-13, and IL-1β (P < 0.05) and regenerated less efficiently than normal epithelia (+48 h wound area reduction = [mean ± SEM] 50.2 ± 7.5% versus 78.6 ± 7.7%; P = 0.02). Asthmatic epithelia showed 40% fewer BrdU+ cells at +48 hours (0.32 ± 0.05% versus 0.56 ± 0.07% of total cells; P = 0.03), and those cells were more dyssynchronously distributed along the cell cycle (52 ± 10, 25 ± 4, 23 ± 7% for G1/G0, S, and G2/M, respectively) than normal epithelia (71 ± 1, 12 ± 2, and 17 ± 2% for G1/G0, S, and G2/M, respectively). Dexamethasone pulses improved asthmatic epithelial inflammation and regeneration/mitosis. In summary, we show that inflammatory/fibrogenic cytokine secretions are correlated with dyssynchronous mitosis upon injury. Intermittent glucocorticoids simultaneously decreased epithelial cytokine secretions and resynchronized mitosis. These data, generated in an airway model lacking inflammatory cells, support the concept that epithelium contributes to asthmatic inflammation.
doi:10.1165/rcmb.2010-0029OC
PMCID: PMC3135846  PMID: 20705942
asthma; epithelial cells; mitosis; inflammation; glucocorticoids
18.  Delineation of a Gene Network Underlying the Pulmonary Response to Oxidative Stress in Asthma 
Journal of Investigative Medicine  2009;57(7):756-764.
Cigarette smoke exposure induces a respiratory epithelial response that is mediated in part by oxidative stress. The contribution of oxidative stress to cigarette smoke-induced responses in asthmatic respiratory epithelium is not well understood. We sought to increase this understanding by employing data integration and systems biology approaches to publicly available microarray data deposited over the last several years. In this study, we analyzed 14 publicly available asthma- or tobacco-relevant data series and found four (two mouse and two human) that fulfilled adequate signal/noise thresholds using unsupervised clustering and F test statistics. Using significance filters and a four-way Venn diagram approach, we identified 26 overlapping genes in the epithelial transcriptional stress response to cigarette smoke and asthma. This test set corresponded to a 26-member gene/protein network containing 18 members that were highly-regulated in a fifth data series of direct lung oxidative stress. Of those network members, two stood out (i.e. tissue inhibitor of metalloproteinases 1 and thrombospondin 1) due to central location within the network and marked up-regulation sustained at later times in response to oxidative stress. These analyses identified key relationships and primary hypothetical targets for future studies of cigarette smoke-induced oxidative stress in asthma.
doi:10.231/JIM.0b013e3181b91a83
PMCID: PMC3328512  PMID: 19730131
Asthma; Microarray Analysis; Tobacco Smoke Pollution; Reactive Oxygen Species
19.  Glucocorticoid Efficacy in Asthma: Is Improved Tissue Remodeling Upstream of Anti-Inflammation 
Synthetic glucocorticoids, such as prednisone, are among the most widely prescribed drugs worldwide and are used to treat many acute and chronic inflammatory conditions. The current paradigm of glucocorticoid efficacy is that they are potent anti-inflammatory agents. Decreased inflammation in many disorders is thought to lead to decreased pathological tissue remodeling. However, this model has never been validated. In particular, improvements in inflammation have not been shown to improve the rate of lung function decline in asthma. Herein, we present an alternative paradigm, where GC efficacy is mediated through more successful tissue remodeling, with reduction in inflammation secondary to successful regeneration.
doi:10.231/JIM.0b013e3181b91654
PMCID: PMC3324850  PMID: 19730133
Glucocorticoid; Asthma; Tissue Remodeling; Lung; Inflammation
20.  DDN: a caBIG® analytical tool for differential network analysis 
Bioinformatics  2011;27(7):1036-1038.
Summary: Differential dependency network (DDN) is a caBIG® (cancer Biomedical Informatics Grid) analytical tool for detecting and visualizing statistically significant topological changes in transcriptional networks representing two biological conditions. Developed under caBIG® 's In Silico Research Centers of Excellence (ISRCE) Program, DDN enables differential network analysis and provides an alternative way for defining network biomarkers predictive of phenotypes. DDN also serves as a useful systems biology tool for users across biomedical research communities to infer how genetic, epigenetic or environment variables may affect biological networks and clinical phenotypes. Besides the standalone Java application, we have also developed a Cytoscape plug-in, CytoDDN, to integrate network analysis and visualization seamlessly.
Availability: The Java and MATLAB source code can be downloaded at the authors' web site http://www.cbil.ece.vt.edu/software.htm
Contact: yuewang@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr052
PMCID: PMC3065688  PMID: 21296752
21.  PUGSVM: a caBIGTM analytical tool for multiclass gene selection and predictive classification 
Bioinformatics  2010;27(5):736-738.
Summary: Phenotypic Up-regulated Gene Support Vector Machine (PUGSVM) is a cancer Biomedical Informatics Grid (caBIG™) analytical tool for multiclass gene selection and classification. PUGSVM addresses the problem of imbalanced class separability, small sample size and high gene space dimensionality, where multiclass gene markers are defined by the union of one-versus-everyone phenotypic upregulated genes, and used by a well-matched one-versus-rest support vector machine. PUGSVM provides a simple yet more accurate strategy to identify statistically reproducible mechanistic marker genes for characterization of heterogeneous diseases.
Availability: http://www.cbil.ece.vt.edu/caBIG-PUGSVM.htm.
Contact: yuewang@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq721
PMCID: PMC3042183  PMID: 21186245
22.  Multilevel support vector regression analysis to identify condition-specific regulatory networks 
Bioinformatics  2010;26(11):1416-1422.
Motivation: The identification of gene regulatory modules is an important yet challenging problem in computational biology. While many computational methods have been proposed to identify regulatory modules, their initial success is largely compromised by a high rate of false positives, especially when applied to human cancer studies. New strategies are needed for reliable regulatory module identification.
Results: We present a new approach, namely multilevel support vector regression (ml-SVR), to systematically identify condition-specific regulatory modules. The approach is built upon a multilevel analysis strategy designed for suppressing false positive predictions. With this strategy, a regulatory module becomes ever more significant as more relevant gene sets are formed at finer levels. At each level, a two-stage support vector regression (SVR) method is utilized to help reduce false positive predictions by integrating binding motif information and gene expression data; a significant analysis procedure is followed to assess the significance of each regulatory module. To evaluate the effectiveness of the proposed strategy, we first compared the ml-SVR approach with other existing methods on simulation data and yeast cell cycle data. The resulting performance shows that the ml-SVR approach outperforms other methods in the identification of both regulators and their target genes. We then applied our method to breast cancer cell line data to identify condition-specific regulatory modules associated with estrogen treatment. Experimental results show that our method can identify biologically meaningful regulatory modules related to estrogen signaling and action in breast cancer.
Availability and implementation: The ml-SVR MATLAB package can be downloaded at http://www.cbil.ece.vt.edu/software.htm
Contact: xuan@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq144
PMCID: PMC2872001  PMID: 20375112
23.  Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice 
Background
Cardiomyopathy in Duchenne muscular dystrophy (DMD) is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy.
Methods
Three month old female mdx mice were exposed to the β1 receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188) (460 mg/kg/dose i.p. daily). Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD) and picrosirius red staining.
Results
BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p < 0.02) and cardiac fibrosis (p < 0.01) over 4 weeks compared to unexposed controls. P188 treatment of mdx mice significantly increased heart rate (median 593 vs. 667 bpm; p < 0.001) after 2 weeks and prevented a decrease in cardiac function in isoproterenol exposed mice (Shortening Fraction = 46 ± 6% vs. 35 ± 6%; p = 0.007) after 4 weeks. P188 treated mdx mice did not show significant differences in cardiac fibrosis, but demonstrated significantly increased EBD positive fibers.
Conclusions
This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice.
doi:10.1186/1471-2261-11-20
PMCID: PMC3123649  PMID: 21575230
Duchenne muscular dystrophy; mdx; poloxamer; cardiomyopathy; isoproterenol
24.  Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction 
Skeletal Muscle  2011;1:20.
Background
During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins.
Results
Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome.
Conclusions
Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ.
doi:10.1186/2044-5040-1-20
PMCID: PMC3156643  PMID: 21798097
25.  Motif-guided sparse decomposition of gene expression data for regulatory module identification 
BMC Bioinformatics  2011;12:82.
Background
Genes work coordinately as gene modules or gene networks. Various computational approaches have been proposed to find gene modules based on gene expression data; for example, gene clustering is a popular method for grouping genes with similar gene expression patterns. However, traditional gene clustering often yields unsatisfactory results for regulatory module identification because the resulting gene clusters are co-expressed but not necessarily co-regulated.
Results
We propose a novel approach, motif-guided sparse decomposition (mSD), to identify gene regulatory modules by integrating gene expression data and DNA sequence motif information. The mSD approach is implemented as a two-step algorithm comprising estimates of (1) transcription factor activity and (2) the strength of the predicted gene regulation event(s). Specifically, a motif-guided clustering method is first developed to estimate the transcription factor activity of a gene module; sparse component analysis is then applied to estimate the regulation strength, and so predict the target genes of the transcription factors. The mSD approach was first tested for its improved performance in finding regulatory modules using simulated and real yeast data, revealing functionally distinct gene modules enriched with biologically validated transcription factors. We then demonstrated the efficacy of the mSD approach on breast cancer cell line data and uncovered several important gene regulatory modules related to endocrine therapy of breast cancer.
Conclusion
We have developed a new integrated strategy, namely motif-guided sparse decomposition (mSD) of gene expression data, for regulatory module identification. The mSD method features a novel motif-guided clustering method for transcription factor activity estimation by finding a balance between co-regulation and co-expression. The mSD method further utilizes a sparse decomposition method for regulation strength estimation. The experimental results show that such a motif-guided strategy can provide context-specific regulatory modules in both yeast and breast cancer studies.
doi:10.1186/1471-2105-12-82
PMCID: PMC3072956  PMID: 21426557

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