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1.  Transcriptional profiling and biological pathway analysis of human equivalence PCB exposure in vitro: Indicator of Disease and disorder development in humans 
Environmental research  2015;138:202-216.
Background and Aims
Our earlier gene-expression studies with a Slovak PCBs-exposed population have revealed possible disease and disorder development in accordance with epidemiological studies. The present investigation aimed to develop an in vitro model system that can provide an indication of disrupted biological pathways associated with developing future diseases, well in advance of the clinical manifestations that may take years to appear in the actual human exposure scenario.
Methods
We used human PBMC (Primary Blood Mononuclear Cells) and exposed them to a mixture of human equivalence levels of PCBs (PCB-118,138,153,170,180) as found in the PCBs-exposed Slovak population. The microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR Taqman Low Density Array (TLDA) was done to further validate the selected 6 differentially expressed genes of our interest, viz., ARNT, CYP2D6, LEPR, LRP12, RRAD, TP53, with a small population validation sample (n=71).
Results
Overall, we revealed a discreet gene expression profile in the experimental model that resembled the diseases and disorders observed in PCBs-exposed population studies. The disease pathways included Endocrine System disorders, Genetic disorders, Metabolic diseases, Developmental disorders, and Cancers, strongly consistent with the evidence from epidemiological studies.
Interpretation
These gene finger prints could lead to the identification of populations and subgroups at high risk for disease, and can pose as early disease biomarkers well ahead of time, before the actual disease becomes visible.
doi:10.1016/j.envres.2014.12.031
PMCID: PMC4739739  PMID: 25725301
PCBs; Human PBMC; Gene expression; Taqman Low-density array (TLDA); Pathway Analysis; Disease and Disorders; Biomarkers
2.  TNFα-induced microRNAs control dystrophin expression in Becker muscular dystrophy 
Cell reports  2015;12(10):1678-1690.
Summary
The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45–47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven miRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31). microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNFα increases microRNA levels in vitro while NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.
Graphical abstract
doi:10.1016/j.celrep.2015.07.066
PMCID: PMC4757433  PMID: 26321630
3.  A ground truth based comparative study on clustering of gene expression data 
Given the variety of available clustering methods for gene expression data analysis, it is important to develop an appropriate and rigorous validation scheme to assess the performance and limitations of the most widely used clustering algorithms. In this paper, we present a ground truth based comparative study on the functionality, accuracy, and stability of five data clustering methods, namely hierarchical clustering, K-means clustering, self-organizing maps, standard finite normal mixture fitting, and a caBIG™ toolkit (VIsual Statistical Data Analyzer - VISDA), tested on sample clustering of seven published microarray gene expression datasets and one synthetic dataset. We examined the performance of these algorithms in both data-sufficient and data-insufficient cases using quantitative performance measures, including cluster number detection accuracy and mean and standard deviation of partition accuracy. The experimental results showed that VISDA, an interactive coarse-to-fine maximum likelihood fitting algorithm, is a solid performer on most of the datasets, while K-means clustering and self-organizing maps optimized by the mean squared compactness criterion generally produce more stable solutions than the other methods.
PMCID: PMC4737472  PMID: 18508478
Clustering Evaluation; Sample Clustering; Comparative Study; Gene Expression Data
4.  Parametric Response Mapping Monitors Temporal Changes on Lung CT Scans in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) 
Academic radiology  2014;22(2):186-194.
Rationale and Objectives
The longitudinal relationship between regional air trapping and emphysema remains unexplored. We have sought to demonstrate the utility of parametric response mapping (PRM), a computed tomography (CT) based biomarker, for monitoring regional disease progression in COPD patients, linking expiratory and inspiratory-based CT metrics over time.
Materials and Methods
Inspiratory and expiratory lung CT scans were acquired from 89 COPD subjects with varying GOLD status at 30 day (n=13) or one year (n=76) from baseline as part of the SPIROMICS clinical trial. PRMs of CT data were used to quantify the relative volumes of normal parenchyma (PRMNormal), emphysema (PRMEmph), and functional small airways disease (PRMfSAD). PRM measurement variability was assessed using the 30-day interval data. Changes in PRM metrics over a one-year period were correlated to pulmonary function (FEV1). A theoretical model that simulates PRM changes from COPD was compared to experimental findings.
Results
PRM metrics varied by ~6.5% of total lung volume for PRMNormal and PRMfSAD and 1% for PRMEmph when testing 30-day repeatability. Over a one-year interval, only PRMEmph in severe COPD subjects produced significant change (19% to 21%). However, 11 of 76 subjects showed changes in PRMfSAD greater than variations observed from analysis of 30 day data. Mathematical model simulations agreed with experimental PRM results, suggesting fSAD is a transitional phase from normal parenchyma to emphysema.
Conclusions
PRM of lung CT scans in COPD patients provides an opportunity to more precisely characterize underlying disease phenotypes, with the potential to monitor disease status and therapy response.
doi:10.1016/j.acra.2014.08.015
PMCID: PMC4289437  PMID: 25442794
5.  Global Gene Expression Profiling in R155H Knock-In Murine Model of VCP Disease 
Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), which is characterized by progressive muscle weakness, dysfunction in bone remodeling, and frontotemporal dementia. More recently, VCP has been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. VCP plays a significant role in a plethora of cellular functions including membrane fusion, transcription activation, nuclear envelope reconstruction, post-mitotic organelle reassembly, cell cycle control. To elucidate the pathological mechanisms underlying the VCP disease progression, we have previously generated a VCPR155H/+ mouse model with the R155H mutation. Histological analyses of mutant muscle showed vacuolization of myofibrils, centrally located nuclei, and disorganized muscle fibers. Global expression profiling of VCPR155H/+ mice using gene annotations by DAVID identified key dysregulated signaling pathways including genes involved in the physiological system development and function, diseases and disorders, and molecular and cellular functions. There were a total of 212 significantly dysregulated genes, several of which are involved in the regulation of proteasomal function and NF-κB signaling cascade. Findings of the gene expression study were validated by using quantitative reverse transcriptase polymerase chain reaction analyses to test genes involved in various signaling cascades. This investigation reveals the importance of the VCPR155H/+ mouse model in the understanding of cellular and molecular mechanisms causing VCP-associated neurodegenerative diseases and in the discovery of novel therapeutic advancements and strategies for patients suffering with these debilitating disorders.
doi:10.1111/cts.12241
PMCID: PMC4329058  PMID: 25388089
6.  Intercellular Adhesion Molecule 1 and Progression of Percent Emphysema: The MESA Lung Study 
Respiratory medicine  2014;109(2):255-264.
Endothelial intercellular adhesion molecule (ICAM) 1 binds neutrophils and facilitates their transmigration into the lung; E-selectin facilitates leukocyte rolling. As neutrophils contribute to tissue destruction in emphysema and chronic obstructive pulmonary disease, we hypothesized that soluble ICAM-1 (sICAM-1) and E-selectin (sE-selectin) would be associated with longitudinal progression of emphysema and lung function decline.
The Multi-Ethnic Study of Atherosclerosis (MESA) enrolled participants 45-84 years old without clinical cardiovascular disease in 2000-02. The MESA Lung Study assessed percent emphysema (<-950 Hounsfield units) on cardiac (2000-07) and full-lung CT scans (2010-12), and spirometry was assessed twice over five years. sICAM-1 and sE-selectin were measured at baseline. Mixed-effect models adjusted for demographics, anthropometry, smoking, C-reactive protein, sphingomyelin and scanner factors.
Among 1,865 MESA Lung participants with measurement of sICAM-1 and percent emphysema the mean log-sICAM-1 was 5.5±0.3 ng/mL and percent emphysema increased 0.73 percentage points (95% CI: 0.34, 1.12; P<0.001) over ten years. A one SD increase in sICAM-1 was associated with an accelerated increase in percent emphysema of 0.23 percentage points over ten years (95% CI: 0.06, 0.39; P=0.007). No significant association was found for sE-selectin, or between any adhesion molecule and lung function.
Higher levels of sICAM-1 were independently associated with progression of percent emphysema in a general population sample.
doi:10.1016/j.rmed.2014.10.004
PMCID: PMC4331236  PMID: 25457724
emphysema; CT imaging; endothelium; intercellular adhesion molecule-1
7.  Exome Sequencing Identifies DYNC1H1 Variant Associated with Vertebral Abnormality and SMA-LED 
Pediatric neurology  2014;52(2):239-244.
Background
Molecular diagnosis of the distal spinal muscular atrophies or distal hereditary motor neuropathies remains challenging due to clinical and genetic heterogeneity. Next generation sequencing offers potential for identifying de novo mutations of causative genes in isolated cases.
Patient
We describe a 3.6 year old girl with congenital scoliosis, equinovarus and L5/S1 left hemivertebra who showed delayed walking and lower extremities atrophy. She was negative for SMN1 deletion testing and parents show no sign of disease.
Results
Whole exome sequencing of the affected girl showed a novel de novo heterozygous missense mutation c.1792C>T; (p.Arg598Cys) in the tail domain of the DYNC1H1 gene encoding for cytoplasmic dynein heavy chain 1. The mutation changed a highly conserved amino acid, and was absent from both parents.
Conclusion
De novo mutations of DYNC1H1 have been found in cases of autosomal dominant mental retardation with neuronal migration defects. Dominantly inherited mutations of DYNC1H1 have been reported to cause spinal muscular atrophy with predominance of lower extremity involvement (SMA-LED) and Charcot-Marie-Tooth type 2O (CMT2O). This is the first report of a de novo DYNC1H1 mutation in the SMA-LED phenotype with a spinal deformity (lumbar hemi vertebrae). This case also demonstrates the power of next generation sequencing to discover de novo mutations at a genome-wide scale.
doi:10.1016/j.pediatrneurol.2014.09.003
PMCID: PMC4351714  PMID: 25484024
spinal muscular atrophy with lower extremity predominance; SMALED; SMA-LED; distal spinal muscular atrophy; dSMA; whole exome sequencing; DYNC1H1; Charcot-Marie-Tooth
8.  Glucocorticoid Receptor (NR3C1) Variants Associate with the Muscle Strength and Size Response to Resistance Training 
PLoS ONE  2016;11(1):e0148112.
Glucocorticoid receptor (NR3C1) polymorphisms associate with obesity, muscle strength, and cortisol sensitivity. We examined associations among four NR3C1 polymorphisms and the muscle response to resistance training (RT). European-American adults (n = 602, 23.8±0.4yr) completed a 12 week unilateral arm RT program. Maximum voluntary contraction (MVC) assessed isometric strength (kg) and MRI assessed biceps size (cm2) pre- and post-resistance training. Subjects were genotyped for NR3C1 -2722G>A, -1887G>A, -1017T>C, and +363A>G. Men carrying the -2722G allele gained less relative MVC (17.3±1.2vs33.5±6.1%) (p = 0.010) than AA homozygotes; men with -1887GG gained greater relative MVC than A allele carriers (19.6±1.4vs13.2±2.3%) (p = 0.016). Women carrying the -1017T allele gained greater relative size (18.7±0.5vs16.1±0.9%) (p = 0.016) than CC homozygotes. We found sex-specific NR3C1 associations with the muscle strength and size response to RT. Future studies should investigate whether these associations are partially explained by cortisol’s actions in muscle tissue as they interact with sex differences in cortisol production.
doi:10.1371/journal.pone.0148112
PMCID: PMC4731199  PMID: 26821164
9.  KDDN: an open-source Cytoscape app for constructing differential dependency networks with significant rewiring 
Bioinformatics  2014;31(2):287-289.
Summary: We have developed an integrated molecular network learning method, within a well-grounded mathematical framework, to construct differential dependency networks with significant rewiring. This knowledge-fused differential dependency networks (KDDN) method, implemented as a Java Cytoscape app, can be used to optimally integrate prior biological knowledge with measured data to simultaneously construct both common and differential networks, to quantitatively assign model parameters and significant rewiring p-values and to provide user-friendly graphical results. The KDDN algorithm is computationally efficient and provides users with parallel computing capability using ubiquitous multi-core machines. We demonstrate the performance of KDDN on various simulations and real gene expression datasets, and further compare the results with those obtained by the most relevant peer methods. The acquired biologically plausible results provide new insights into network rewiring as a mechanistic principle and illustrate KDDN’s ability to detect them efficiently and correctly. Although the principal application here involves microarray gene expressions, our methodology can be readily applied to other types of quantitative molecular profiling data.
Availability: Source code and compiled package are freely available for download at http://apps.cytoscape.org/apps/kddn
Contact: yuewang@vt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu632
PMCID: PMC4287948  PMID: 25273109
10.  Mathematical modelling of transcriptional heterogeneity identifies novel markers and subpopulations in complex tissues 
Scientific Reports  2016;6:18909.
Tissue heterogeneity is both a major confounding factor and an underexploited information source. While a handful of reports have demonstrated the potential of supervised computational methods to deconvolute tissue heterogeneity, these approaches require a priori information on the marker genes or composition of known subpopulations. To address the critical problem of the absence of validated marker genes for many (including novel) subpopulations, we describe convex analysis of mixtures (CAM), a fully unsupervised in silico method, for identifying subpopulation marker genes directly from the original mixed gene expressions in scatter space that can improve molecular analyses in many biological contexts. Validated with predesigned mixtures, CAM on the gene expression data from peripheral leukocytes, brain tissue, and yeast cell cycle, revealed novel marker genes that were otherwise undetectable using existing methods. Importantly, CAM requires no a priori information on the number, identity, or composition of the subpopulations present in mixed samples, and does not require the presence of pure subpopulations in sample space. This advantage is significant in that CAM can achieve all of its goals using only a small number of heterogeneous samples, and is more powerful to distinguish between phenotypically similar subpopulations.
doi:10.1038/srep18909
PMCID: PMC4703969  PMID: 26739359
11.  The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse 
Toxicology reports  2015;2:838-849.
Phosphorodiamidate morpholino oligonucleotides (PMO) are used as a promising exon-skipping gene therapy for Duchenne Muscular Dystrophy (DMD). One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM) strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23). We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10) mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg). Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI). The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at day 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO.
Chemical compounds studied in this article: Phosphorodiamidate morpholino (PubChem CID: 22140692); isoflurane (PubChem CID: 3763); formic acid (PubChem CID: 284); acetonitrile (PubChem CID: 6342); acetone (PubChem CID: 180); methanol (PubChem CID: 887)
doi:10.1016/j.toxrep.2015.05.008
PMCID: PMC4512206  PMID: 26213685
PMO; urinary biomarkers; mdx-23; Duchenne Muscular Dystrophy; Clusterin; GGT1
12.  Nutrition and Health in Amphibian Husbandry 
Zoo biology  2014;33(6):485-501.
Amphibian biology is intricate, and there are many inter-related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists’ understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas.
doi:10.1002/zoo.21180
PMCID: PMC4685711  PMID: 25296396
aquarium; disease; ex situ; husbandry; life stage; nutrition; pathology; population; research; vitamin; water; zoo
13.  Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients 
Human Molecular Genetics  2014;23(24):6458-6469.
It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.
doi:10.1093/hmg/ddu366
PMCID: PMC4240201  PMID: 25027324
14.  Elusive sources of variability of dystrophin rescue by exon skipping 
Skeletal Muscle  2015;5:44.
Background
Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.
Methods
In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.
Results
The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.
Conclusions
While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13395-015-0070-6
PMCID: PMC4667482  PMID: 26634117
Duchenne muscular dystrophy; Dystrophin; Exon skipping; Variability; mdx-23
15.  Metabolite signatures of exercise training in human skeletal muscle relate to mitochondrial remodelling and cardiometabolic fitness 
Diabetologia  2014;57(11):2282-2295.
Aims/hypothesis
Targeted metabolomic and transcriptomic approaches were used to evaluate the relationship between skeletal muscle metabolite signatures, gene expression profiles and clinical outcomes in response to various exercise training interventions. We hypothesised that changes in mitochondrial metabolic intermediates would predict improvements in clinical risk factors, thereby offering novel insights into potential mechanisms.
Methods
Subjects at risk of metabolic disease were randomised to six months of inactivity or one of five aerobic and/or resistance training programmes (n = 112). Pre/post-intervention assessments included cardiorespiratory fitness (V̇O2peak), serum triacylglycerols (TGs) and insulin sensitivity (SI). In this secondary analysis, muscle biopsy specimens were used for targeted mass spectrometry-based analysis of metabolic intermediates and measurement of mRNA expression of genes involved in metabolism.
Results
Exercise regimens with the largest energy expenditure produced robust increases in muscle concentrations of even-chain acylcarnitines (median 37–488%), which correlated positively with increased expression of genes involved in muscle uptake and oxidation of fatty acids. Along with free carnitine, the aforementioned acylcarnitine metabolites were related to improvements in V̇O2peak, TGs and SI (R = 0.20–0.31, p < 0.05). Muscle concentrations of the tricarboxylic acid cycle intermediates succinate and succinylcarnitine (R = 0.39 and 0.24, p < 0.05) emerged as the strongest correlates of SI.
Conclusions/interpretation
The metabolic signatures of exercise-trained skeletal muscle reflected reprogramming of mitochondrial function and intermediary metabolism and correlated with changes in cardiometabolic fitness. Succinate metabolism and the succinate dehydrogenase complex emerged as a potential regulatory node that intersects with whole-body insulin sensitivity. This study identifies new avenues for mechanistic research aimed at understanding the health benefits of physical activity.
Trial registration
ClinicalTrials.gov NCT00200993 and NCT00275145
doi:10.1007/s00125-014-3343-4
PMCID: PMC4182127  PMID: 25091629
Acylcarnitines; Branched-chain amino acids; Metabolomics; Physical activity; Skeletal muscle; Succinate
16.  ET-64PRE-CLINICAL ASSESSMENT OF DISSOCIATIVE STEROIDS AS DEXAMETHASONE SUBSTITUTE FOR TREATMENT IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) 
Neuro-Oncology  2014;16(Suppl 5):v93.
BACKGROUND: Although corticosteroids are widely used to treat cancer, cerebral edema, and other inflammatory disorders, their exact mechanism of action is poorly understood and less toxic alternatives are needed. The purpose of this study was to use a genetically engineered DIPG murine model to conduct pre-clinical testing of a new orally-administered steroid derivative, VBP15, which promises to reduce side effects through dissociating GRE-mediated transcription and NFkB-mediated modulatory actions. METHODS: First, to compare VPB15 to dexamethasone in reducing pro-inflammatory signals in vitro, BSG D10 murine and Control D10 cells were expanded in culture for 24 hours, serum starved and treated at 12 hours with dexamethasone, VBP15 or DMSO (control) and no drug in complete media for two hours. Using extracted RNA from the cells, NanoString evaluated specific inflammatory pathways. Second, in vivo efficacy of VBP15 was assessed. CSPG4+/+ neurospheres were into the brainstem of 18 mice at P2, 7 were treated with oral dexamethasone, 7 with VBP15 and 4 with cherry syrup (control) for up to 45 days. RESULTS: (In Vitro) Compared to DMSO, both dexamethasone and VBP15 given 12 hours into serum starvation decreased cytokine expression of IL10, IL6 and Tnf, which are cytokines in the NfKB pathway. (In Vivo) Post-mortem H&E staining revealed that 17 of 18 mice had developed tumor. Kaplan-Meir survival curves indicated a survival advantage for mice treated with VBP15 compared to dexamethasone or control. CONCLUSIONS: VPB15 has similar or better efficacy vs. dexamethasone (DEX) in reducing pro-inflammatory signals in brainstem glioma cells in vitro, and VBP15-treated DIPG mice show better survival compared to dexamethasone in vivo. These pilot results warrant more extensive pre-clinical testing and possibly development of a clinical trial.
doi:10.1093/neuonc/nou255.61
PMCID: PMC4218161
17.  Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy 
PLoS ONE  2015;10(10):e0141240.
Objective
Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.
Methods
A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m2 as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.
Results
Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027).
Conclusions
We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.
doi:10.1371/journal.pone.0141240
PMCID: PMC4626372  PMID: 26513582
18.  Obesity-Related Genetic Variants and their Associations with Physical Activity 
Sports Medicine - Open  2015;1(1):34.
Background
Meta-analysis of genome-wide association studies identified obesity-related genetic variants. Due to the pleiotropic effects of related phenotypes, we tested six of these obesity-related genetic variants for their association with physical activity: fat mass and obesity-associated (FTO)(rs9939609)T>A, potassium channel tetramerization domain containing (KCTD15) (rs11084753)G>A, melanocortin receptor4 (MC4R)(rs17782313)T>C, neuronal growth regulator 1 (NEGR1)(rs2815752)A>G, SH2B adapter protein 1 (SH2B1)(rs7498665)A>G, and transmembrane protein18 (TMEM18)(rs6548238)C>T.
Method
European-American women (n = 263) and men (n = 229) (23.5 ± 0.3 years, 24.6 ± 0.2 kg/m2) were genotyped and completed the Paffenbarger physical activity Questionnaire. Physical activity volume in metabolic energy equivalents [MET]-hour/week was derived from the summed time spent (hour/week) times the given MET value for vigorous, moderate, and light intensity physical activity, and sitting and sleeping, respectively. Multivariable adjusted [(age, sex, and body mass index (BMI)] linear regression tested associations among genotype (dominant/recessive model) and the log of physical activity volume.
Result
MC4R (rs17782313)T>C explained 1.1 % (p = 0.02), TMEM18(rs6548238)C>T 1.2 % (p = 0.01), and SH2B1 (rs7498665)A>G 0.6 % (p = 0.08) of the variability in physical activity volume. Subjects with the MC4R C allele spent 3.5 % less MET-hour/week than those with the TT genotype (p = 0.02). Subjects with the TMEM18 T allele spent 4.1 % less MET-hour/week than those with the CC genotype (p = 0.01). Finally, subjects with the SH2B1 GG genotype spent 3.6 % less MET-hour/week than A allele carriers (p = 0.08).
Conclusion
Our findings suggest a shared genetic influence among some obesity-related gene loci and physical activity phenotypes that should be explored further. Physical activity volume differences by genotype have public health importance equating to 11–13 lb weight difference annually.
doi:10.1186/s40798-015-0036-6
PMCID: PMC4607705  PMID: 26495240
19.  A Numerical Study of Heat and Water Vapor Transfer in MDCT-Based Human Airway Models 
Annals of biomedical engineering  2014;42(10):2117-2131.
A three-dimensional (3D) thermo-fluid model is developed to study regional distributions of temperature and water vapor in three multi-detector row computed-tomography (MDCT)-basedhuman airwayswith minute ventilations of 6, 15 and 30 L/min. A one-dimensional (1D) model is also solved to provide necessary initial and boundary conditionsforthe 3D model. Both 3D and 1D predicted temperature distributions agree well with available in vivo measurement data. On inspiration, the 3D cold high-speed air stream is split at the bifurcation to form secondary flows, with its cold regions biased toward the inner wall. The cold air flowing along the wall is warmed up more rapidly than the air in the lumen center. The repeated splitting pattern of air streams caused by bifurcations acts as an effective mechanism for rapid heat and mass transfer in 3D. This provides a key difference from the 1D model, where heating relies largely on diffusion in the radial direction, thus significantly affecting gradient-dependent variables, such as energy flux and water loss rate. We then propose the correlations for respective heat and mass transfer in the airways of up to 6 generations: Nu=3.504(ReDaDt)0.277, R = 0.841 and Sh=3.652(ReDaDt)0.268, R = 0.825, where Nu is the Nusselt number, Sh is the Sherwood number, Re is the branch Reynolds number, Da is the airway equivalent diameter, and Dt is the tracheal equivalentdiameter.
doi:10.1007/s10439-014-1074-9
PMCID: PMC4162841  PMID: 25081386
Thermodynamics; human airway temperature; water vapor; computed tomography
20.  Mitotic Asynchrony Induces Transforming Growth Factor-β1 Secretion from Airway Epithelium 
We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-β1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-β1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-β1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-β1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony.
doi:10.1165/rcmb.2013-0396OC
PMCID: PMC4189490  PMID: 24669775
asthma; mitosis; transforming growth factor-β1; fibrosis
21.  miRTarVis: an interactive visual analysis tool for microRNA-mRNA expression profile data 
BMC Proceedings  2015;9(Suppl 6):S2.
Background
MicroRNAs (miRNA) are short nucleotides that down-regulate its target genes. Various miRNA target prediction algorithms have used sequence complementarity between miRNA and its targets. Recently, other algorithms tried to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. Some web-based tools are also introduced to help researchers predict targets of miRNAs from miRNA-mRNA expression profile data. A demand for a miRNA-mRNA visual analysis tool that features novel miRNA prediction algorithms and more interactive visualization techniques exists.
Results
We designed and implemented miRTarVis, which is an interactive visual analysis tool that predicts targets of miRNAs from miRNA-mRNA expression profile data and visualizes the resulting miRNA-target interaction network. miRTarVis has intuitive interface design in accordance with the analysis procedure of load, filter, predict, and visualize. It predicts targets of miRNA by adopting Bayesian inference and MINE analyses, as well as conventional correlation and mutual information analyses. It visualizes a resulting miRNA-mRNA network in an interactive Treemap, as well as a conventional node-link diagram. miRTarVis is available at http://hcil.snu.ac.kr/~rati/miRTarVis/index.html.
Conclusions
We reported findings from miRNA-mRNA expression profile data of asthma patients using miRTarVis in a case study. miRTarVis helps to predict and understand targets of miRNA from miRNA-mRNA expression profile data.
doi:10.1186/1753-6561-9-S6-S2
PMCID: PMC4547149  PMID: 26361498
MicroRNA; mRNA; Visualization; Expression profile; Target prediction
22.  Upregulated IL-1β in dysferlin-deficient muscle attenuates regeneration by blunting the response to pro-inflammatory macrophages 
Skeletal Muscle  2015;5:24.
Background
Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study.
Methods
To test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts.
Results
Dysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1β and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1β, which inhibited it. Importantly, blockade of IL-1β signaling significantly improved differentiation of dysferlin-deficient cells.
Conclusions
We propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1β signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle.
doi:10.1186/s13395-015-0048-4
PMCID: PMC4527226  PMID: 26251696
Skeletal muscle; Myoblasts; Macrophages; Cell-cell interactions; Muscular dystrophy; Dysferlin; LGMD2B
23.  Longitudinal changes in airway remodeling and air trapping in severe asthma 
Academic radiology  2014;21(8):986-993.
Rationale and Objectives
Previous cross-sectional studies have demonstrated that airway wall thickness and air trapping are greater in subjects with severe asthma than in those with mild-to-moderate asthma. However, a better understanding of how airway remodeling and lung density change over time is needed. This study aims to evaluate predictors of airway wall remodeling and change in lung function and lung density over time in severe asthma.
Materials and Methods
Phenotypic characterization and quantitative multidetector computed tomography (MDCT) of the chest was performed at baseline and ∼2.6 years later in 38 participants with asthma (severe n=24, mild-moderate n=14) and 9 normal controls from the Severe Asthma Research Program.
Results
Subjects with severe asthma had a significant decline in post-bronchodilator FEV1% predicted over time (p = <0.001). Airway wall thickness measured by MDCT was increased at multiple airway generations in severe asthma compared to mild-to-moderate asthma (wall area percent (WA%): p <0.05) and normals (p <0.05) at baseline and year 2. Over time, there was an increase in WA% and wall thickness (WT%) in all subjects (p = 0.030 and 0.009 respectively) with no change in emphysema-like lung or air trapping. Baseline pre-bronchodilator FEV1% inversely correlated with WA% and WT% (both p = <0.05). In a multivariable regression model, baseline WA%, race and healthcare utilization were predictors of subsequent airway remodeling.
Conclusions
Severe asthma subjects have a greater decline in lung function over time than normal subjects or those with mild-to-moderate asthma. MDCT provides a noninvasive measure of airway wall thickness that may predict subsequent airway remodeling.
doi:10.1016/j.acra.2014.05.001
PMCID: PMC4100072  PMID: 25018070
Severe asthma; computed tomography; airway remodeling
24.  Topomorphologic Separation of Fused Isointensity Objects via Multiscale Opening: Separating Arteries and Veins in 3-D Pulmonary CT 
A novel multiscale topomorphologic approach for opening of two isointensity objects fused at different locations and scales is presented and applied to separating arterial and venous trees in 3-D pulmonary multidetector X-ray computed tomography (CT) images. Initialized with seeds, the two isointensity objects (arteries and veins) grow iteratively while maintaining their spatial exclusiveness and eventually form two mutually disjoint objects at convergence. The method is intended to solve the following two fundamental challenges: how to find local size of morphological operators and how to trace continuity of locally separated regions. These challenges are met by combining fuzzy distance transform (FDT), a morphologic feature with a topologic fuzzy connectivity, and a new morphological reconstruction step to iteratively open finer and finer details starting at large scales and progressing toward smaller scales. The method employs efficient user intervention at locations where local morphological separability assumption does not hold due to imaging ambiguities or any other reason. The approach has been validated on mathematically generated tubular objects and applied to clinical pulmonary noncontrast CT data for separating arteries and veins. The tradeoff between accuracy and the required user intervention for the method has been quantitatively examined by comparing with manual outlining. The experimental study, based on a blind seed selection strategy, has demonstrated that above 95% accuracy may be achieved using 25–40 seeds for each of arteries and veins. Our method is very promising for semiautomated separation of arteries and veins in pulmonary CT images even when there is no object-specific intensity variation at conjoining locations.
doi:10.1109/TMI.2009.2038224
PMCID: PMC4517589  PMID: 20199919
Artery; computed tomography (CT); fuzzy connectivity; fuzzy distance transform (FDT); morphology; pulmonary imaging; scale; vascular tree; vein
25.  Antisense Oligo-Mediated Multiple Exon Skipping in a Dog Model of Duchenne Muscular Dystrophy 
Exon skipping is currently one of the most promising molecular therapies for Duchenne muscular dystrophy (DMD). We have recently developed multiple exon skipping targeting exons 6 and 8 in dystrophin mRNA of canine X-linked muscular dystrophy (CXMD), an animal model of DMD, which exhibits severe dystrophic phenotype in skeletal muscles and cardiac muscle. We have induced efficient exon skipping both in vitro and in vivo by using cocktail antisense 2’ O-methyl oligonucleotides (2’OMePS) and cocktail phosphorodiamidate morpholino oligomers (morpholinos, or PMOs) and ameliorated phenotype of dystrophic dogs by systemic injections. The multiple exon skipping (double exon skipping) shown here provides the prospect of choosing deletions that optimize the functionality of the truncated dystrophin protein for DMD patients by using a common cocktail that could be validated as a single drug and also potentially applicable for more than 90% of DMD patients.
doi:10.1007/978-1-61737-982-6_20
PMCID: PMC4491489  PMID: 21194037
Multiple exon skipping; Morpholinos (phosphorodiamidate morpholino oligomers); 2′ O-methylated antisense oligomers (phosphorothioate); Dystrophic dogs (canine X-linked muscular dystrophy); Duchenne/Becker muscular dystrophies

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