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1.  Microbial hitchhikers on intercontinental dust: catching a lift in Chad 
The ISME Journal  2012;7(4):850-867.
Ancient mariners knew that dust whipped up from deserts by strong winds travelled long distances, including over oceans. Satellite remote sensing revealed major dust sources across the Sahara. Indeed, the Bodélé Depression in the Republic of Chad has been called the dustiest place on earth. We analysed desert sand from various locations in Chad and dust that had blown to the Cape Verde Islands. High throughput sequencing techniques combined with classical microbiological methods showed that the samples contained a large variety of microbes well adapted to the harsh desert conditions. The most abundant bacterial groupings in four different phyla included: (a) Firmicutes—Bacillaceae, (b) Actinobacteria—Geodermatophilaceae, Nocardiodaceae and Solirubrobacteraceae, (c) Proteobacteria—Oxalobacteraceae, Rhizobiales and Sphingomonadaceae, and (d) Bacteroidetes—Cytophagaceae. Ascomycota was the overwhelmingly dominant fungal group followed by Basidiomycota and traces of Chytridiomycota, Microsporidia and Glomeromycota. Two freshwater algae (Trebouxiophyceae) were isolated. Most predominant taxa are widely distributed land inhabitants that are common in soil and on the surfaces of plants. Examples include Bradyrhizobium spp. that nodulate and fix nitrogen in Acacia species, the predominant trees of the Sahara as well as Herbaspirillum (Oxalobacteraceae), a group of chemoorganotrophic free-living soil inhabitants that fix nitrogen in association with Gramineae roots. Few pathogenic strains were found, suggesting that African dust is not a large threat to public health.
PMCID: PMC3603401  PMID: 23254516
aeolian; high throughput sequencing; Bodélé Depression; Republic of Chad; wind erosion
2.  Spread of Virulent Group A Streptococcus Type emm59 from Montana to Wyoming, USA 
Emerging Infectious Diseases  2014;20(4):679-681.
Full-genome sequencing showed that a recently emerged and hypervirulent clone of group A Streptococcus type emm59 active in Canada and parts of the United States has now caused severe invasive infections in rural northeastern Wyoming. Phylogenetic analysis of genome data indicated that the strain was likely introduced from Montana.
PMCID: PMC3966365
group A Streptococcus; GAS; bacteria; emm59; streptococci; invasive disease; virulence; genome sequencing; epidemiology; necrotizing fasciitis; Montana; Wyoming; United States
3.  Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6 
PLoS ONE  2014;9(3):e92608.
Brown adipose tissue (BAT) plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1) that differentiates BAT from its energy storing white adipose tissue (WAT) counterpart. The clinical implication of “classical” BAT (originates from Myf5 positive myoblastic lineage) or the “beige” fat (originates through trans-differentiation of WAT) activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6) induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn) and Cyclooxygenase-2 (Cox2). Furthermore, pathway analyses using the Causal Reasoning Engine (CRE) identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R). Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.
PMCID: PMC3962431  PMID: 24658703
4.  Genomic View of Bipolar Disorder Revealed by Whole Genome Sequencing in a Genetic Isolate 
PLoS Genetics  2014;10(3):e1004229.
Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.
Author Summary
Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable efforts genetic studies have yet to reveal the precise genetic underpinnings of the disorder. In this study we have analyzed a large extended pedigree of Old Order Amish that segregates bipolar disorder. Our study design integrates both dense genotype and whole-genome sequence data. In a combined linkage and association analysis we identify five chromosomal regions with nominally significant or suggestive evidence for linkage, several of which constitute replication of earlier linkage findings for bipolar disorder in non-Amish families. Association analysis of genetic variants in each of the linkage regions yielded a number of plausible candidate genes for bipolar disorder. The striking genetic heterogeneity we observed in this genetic isolate has profound implications for the study of bipolar disorder in the general population.
PMCID: PMC3953017  PMID: 24625924
5.  Validation of nigrostriatal PET measures: critical limits 
Annals of neurology  2013;73(3):390-396.
Molecular imaging and clinical endpoints are frequently discordant in Parkinson disease (PD) clinical trials raising questions about validity of these imaging measures to reflect disease severity.
We compared striatal uptake for 3 PET tracers with in vitro measures of nigral cell counts and striatal dopamine in MPTP treated monkeys.
Sixteen macaques had MRI and baseline PETs using 6-[18F]fluorodopa (FD), [11C] dihydrotetrabenazine (DTBZ) and [11C] 2beta-carbomethoxy-3beta-4-fluorophenyltropane (CFT). MPTP (0 to 0.31 mg/kg) infused unilaterally via the internal carotid artery produced stable hemiparkinsonism by three weeks. After eight weeks, PETs were repeated and animals euthanized for striatal dopamine measurements and unbiased counts of tyrosine hydroxylase stained nigral cells.
Striatal uptake for each radiotracer (FD, DTBZ, CFT) correlated with stereologic nigral cell counts only for nigral loss < 50% (r2= 0.84; r2= 0.86; r2= 0.87, p<0.001 respectively; n=10). In contrast, striatal uptake correlated with striatal dopamine over the full range of dopamine depletion (r2= 0.95; r2= 0.94; r2= 0.94, p<0.001; n=16). Interestingly, indices of striatal uptake of FD, DTBZ and CFT correlated strongly with each other (r2=0.98, p<0.001).
Tracer uptake correlated with nigral neurons only when nigral loss < 50%. This along with previous work demonstrating that nigral cell counts correlate strongly with parkinsonism ratings may explain discordant results between neuroimaging and clinical endpoints. Furthermore, strong correlations among striatal uptake for these tracers support lack of differential regulation of decarboxylase activity (FD), vesicular monoamine transporter type 2 (DTBZ), and dopamine transporter (CFT) within 2 months after nigrostriatal injury.
PMCID: PMC3631302  PMID: 23423933
6.  Using Targeted Virotherapy to Treat a Resistant Ewing Sarcoma Model: From the Bedside to the Bench and Back 
The Scientific World Journal  2014;2014:171439.
Metastatic Ewing sarcoma (EWS) is often resistant to current multimodal chemotherapeutic regimens. Oncolytic virus therapy (OV) is a novel therapeutic platform whereby viruses can selectively infect as well as replicate in and kill tumor cells, while sparing normal tissues. The purpose of this study is to investigate the efficacy of the biotherapeutic oncolytic agent, vesicular stomatitis virus (VSVΔM51), to kill EWS cells that are resistant to conventional therapy. Our hypothesis is that systemic delivery of VSVΔM51 can demonstrate tumor-specific killing of resistant EWS cells, as well as a significant decrease of tumor burden in EWS bearing mice. Methods. A biopsy sample was obtained from a patient with metastatic EWS and was used to establish a novel EWS cell line. In vitro assays evaluated the oncolytic effect of vesicular stomatitis virus (VSVΔM51) on this cell line. EWS xenograft mice model bearing either lung or subcutaneous tumors was established to evaluate the antitumor specific oncolytic effect of VSVΔM51 after local and systemic delivery. Results. The established EWS cell line shared similar molecular and genetic traits to the patient's original tumor specimen. VSVΔM51 effectively infected and killed EWS cells in vitro. In vivo, VSVΔM51 selectively infected and killed EWS and led to significant delay in tumor growth. Conclusion. This study has been designed to implement a translational link between the bedside and the bench, where a specific challenging clinical scenario guided this basic science research. This research demonstrated that a sarcoma, which is resistant to current conventional standard therapies, is still susceptible to an alternative therapeutic platform, such as OV. Adding OV to the armamentarium of sarcoma treatment can enhance the future therapeutic approach towards these cancer patients.
PMCID: PMC3930196  PMID: 24672293
7.  Microtubule-associated protein tau is essential for long-term depression in the hippocampus 
The microtubule-associated protein tau is a principal component of neurofibrillary tangles, and has been identified as a key molecule in Alzheimer's disease and other tauopathies. However, it is unknown how a protein that is primarily located in axons is involved in a disease that is believed to have a synaptic origin. To investigate a possible synaptic function of tau, we studied synaptic plasticity in the hippocampus and found a selective deficit in long-term depression (LTD) in tau knockout mice in vivo and in vitro, an effect that was replicated by RNAi knockdown of tau in vitro. We found that the induction of LTD is associated with the glycogen synthase kinase-3-mediated phosphorylation of tau. These observations demonstrate that tau has a critical physiological function in LTD.
PMCID: PMC3843876  PMID: 24298146
Alzheimer's disease; hippocampus; synaptic plasticity; long-term depression; tau
8.  Molecular Typing of Environmental and Clinical Strains of Vibrio vulnificus Isolated in the Northeastern USA 
PLoS ONE  2013;8(12):e83357.
Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as ‘LI’ and ‘LII’), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.
PMCID: PMC3875459  PMID: 24386187
9.  A conserved eEF2 coding variant in SCA26 leads to loss of translational fidelity and increased susceptibility to proteostatic insult 
Human Molecular Genetics  2012;21(26):5472-5483.
The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of −1 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases.
PMCID: PMC3516132  PMID: 23001565
10.  Inhibition of Nutlin-Resistant HDM2 Mutants by Stapled Peptides 
PLoS ONE  2013;8(11):e81068.
Pharmacological modulation of p53 activity is an attractive therapeutic strategy in cancers with wild-type p53. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2, a key negative regulator of p53 and blocks its activity. We have described resistance mutations in HDM2 that selectively reduce affinity for Nutlin but not p53. In the present communication, we show that stapled peptides targeting the same region of HDM2 as Nutlin are refractory to these mutations, and display reduced discrimination between the wild-type and mutant HDM2s with regards to functional abrogation of interaction with p53. The larger interaction footprint afforded by stapled peptides suggests that this class of ligands may prove comparatively more resilient to acquired resistance in a clinical setting.
PMCID: PMC3835680  PMID: 24278380
11.  Comparison of Superior Level Facet Joint Violations During Open and Percutaneous Pedicle Screw Placement 
Neurosurgery  2012;71(5):962-970.
Superior-level facet joint violation by pedicle screws may result in increased stress to the level above the instrumentation and may contribute to adjacent segment disease (ASD). Previous studies have evaluated facet joint violations in open or percutaneous screw cases, but there are no reports describing a direct institutional comparison.
To compare the incidence of superior-level facet violation for open versus percutaneous pedicle screws, and evaluate patient and surgical factors that impact this outcome.
We reviewed 279 consecutive patients who underwent an index instrumented lumbar fusion from 2007 to 2011 for degenerative spine disease with stenosis with or without spondylolisthesis. We used a CT grading system which represents progressively increasing grades of facet joint violation. Patient and surgical factors were evaluated to determine their impact on facet violation.
Our cohort consisted of 126 open and 153 percutaneous cases. Percutaneous procedures had a higher overall violation grade (p=0.018) and greater incidence of high-grade violations (p=0.0059) compared to open procedures. Bivariate analysis showed significantly greater violations in percutaneous cases for age<65, obesity, pedicle screws at L4, and 1- and 2-level surgeries. Multivariate analysis showed the percutaneous approach and depth of the spine to be independent risk factors for high-grade violations.
This study demonstrates greater facet violations for percutaneously placed pedicle screws compared to open.
PMCID: PMC3477296  PMID: 22843132
facet joint violation; open; percutaneous; adjacent segment disease; depth of spine
12.  Multiphoton tomographic imaging: a potential optical biopsy tool for detecting gastrointestinal inflammation and neoplasia 
Endoscopy is widely used to detect and remove premalignant lesions with the goal of preventing gastrointestinal (GI) cancers. Because current endoscopes do not provide cellular resolution, all suspicious lesions are biopsied and subjected to histological evaluation. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and cost. Here we explore the use of multiphoton microscopy (MPM), an optical biopsy tool that relies on intrinsic tissue emissions, to evaluate pathology in both experimental and human GI specimens, using hematoxylin and eosin (H&E)-stained sections from these tissues for comparison. After evaluating the entire normal mouse GI tract, MPM was used to investigate disease progression in mouse models of colitis and colorectal carcinogenesis. MPM provided sufficient histological detail to identify all relevant substructures in ex vivo normal GI tissue, visualize both acute and resolving stages of colitis, and show the progression of colorectal carcinogenesis. Next, ex vivo specimens from human subjects with celiac sprue, inflammatory bowel disease, and colorectal neoplasia were imaged by MPM. Finally, colonic mucosa in live anesthetized rats was imaged in vivo using a flexible endoscope prototype. In both animal models and human specimens, MPM images demonstrated a striking similarity to the results of H&E staining, as demonstrated by the 100% concordance achieved by the study pathologists’ diagnoses. In summary, MPM is a promising technique that accurately visualizes histology in fresh, unstained tissues. Our findings support the continued development of MPM as a technology to enhance the early detection of GI pathologies including premalignant lesions.
PMCID: PMC3491145  PMID: 22961775
13.  Experimental Placebo Analgesia Changes Resting-State Alpha Oscillations  
PLoS ONE  2013;8(10):e78278.
The lack of clear understanding of the pathophysiology of chronic pain could explain why we currently have only a few effective treatments. Understanding how pain relief is realised during placebo analgesia could help develop improved treatments for chronic pain. Here, we tested whether experimental placebo analgesia was associated with altered resting-state cortical activity in the alpha frequency band of the electroencephalogram (EEG). Alpha oscillations have been shown to be influenced by top-down processes, which are thought to underpin the placebo response.
Seventy-three healthy volunteers, split into placebo or control groups, took part in a well-established experimental placebo procedure involving treatment with a sham analgesic cream. We recorded ongoing (resting) EEG activity before, during, and after the sham treatment.
We show that resting alpha activity is modified by placebo analgesia. Post-treatment, alpha activity increased significantly in the placebo group only (p < 0.001). Source analysis suggested that this alpha activity might have been generated in medial components of the pain network, including dorsal anterior cingulate cortex, medial prefrontal cortex, and left insula.
These changes are consistent with a cognitive state of pain expectancy, a key driver of the placebo analgesic response. The manipulation of alpha activity may therefore present an exciting avenue for the development of treatments that directly alter endogenous processes to better control pain.
PMCID: PMC3795660  PMID: 24147129
14.  Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae 
Nucleic Acids Research  2013;42(1):e2.
Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.
PMCID: PMC3874202  PMID: 24106087
15.  Low Nigrostriatal Reserve for Motor Parkinsonism in Nonhuman Primates 
Experimental neurology  2012;237(2):355-362.
Nigrostriatal reserve refers to the threshold of neuronal injury to dopaminergic cell bodies and their terminal fields required to produce parkinsonian motor deficits. Inferential studies have estimated striatal dopamine reserve to be at least 70%. Knowledge of this threshold is critical for planning interventions to prevent symptom onset or reverse nigrostriatal injury sufficient to restore function in people with Parkinson disease. In this study, we determine the nigrostriatal reserve in a non-human primate model that mimics the motor manifestations of Parkinson disease.
Fifteen macaque monkeys received unilateral randomized doses of the selective dopaminergic neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We compared blinded validated ratings of parkinsonism to in vitro measures of striatal dopamine and unbiased stereologic counts of nigral neurons after tyrosine hydroxylase immunostaining.
The percent of residual cell counts in lesioned nigra correlated linearly with the parkinsonism score at 2 months (r = −0.87, p <0.0001). The parkinsonism score at 2 months correlated linearly with the percent residual striatal dopamine (r = −0.77, p = 0.016) followed by a flooring effect once nigral cell loss exceeded 50%. A reduction of about 14 to 23% of nigral neuron counts or 14 to 37% of striatal dopamine was sufficient to induce mild parkinsonism.
The nigral cell body and terminal field injury needed to produce parkinsonian motor manifestations may be much less than previously thought.
PMCID: PMC3443325  PMID: 22836146
Parkinsonism; MPTP; substantia nigra; dopamine
16.  Comparison of the Genome Sequences of “Candidatus Portiera aleyrodidarum” Primary Endosymbionts of the Whitefly Bemisia tabaci B and Q Biotypes 
“Candidatus Portiera aleyrodidarum” is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whitefly Bemisia tabaci. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.
PMCID: PMC3591977  PMID: 23315735
17.  Combined Effects of Ephedrine-Containing Dietary Supplements, Caffeine, and Nicotine on Morphology and Ultrastructure of Rat Hearts 
Journal of Caffeine Research  2012;2(3):123-132.
Cigarette smokers have an increased risk for coronary artery disease. Nicotine present in cigarettes can adversely affect the cardiovascular system via stimulation of both sympathetic and parasympathetic neurons. Caffeine, another cardiovascular and central nervous system (CNS) stimulant, is commonly found in Ephedra and Ephedra-free dietary supplements. These caffeine-containing supplements also have been linked to cardiovascular toxicities. Although no longer on the U.S market, Ephedra-containing supplements are another source of cardiovascular and CNS stimulants, namely the ephedrine alkaloids. Together caffeine, nicotine, and ephedrine can individually stress the cardiovascular system, and an overlap of these agents is predicted in smokers and dieters. To understand the collective effects of these stimulants on the heart morphology and ultrastructure, rats were exposed to synthetic combinations of nicotine (0.2 mg/kg/day), ephedrine (0–30 mg/kg/day), and/or caffeine (0–24 mg/kg/day) as well as an extract from a caffeine-containing Ephedra supplement (Metabolife 356). After exposure for 3 days, the hearts were removed and examined for hypersensitivity myocarditis and myocardial necrosis. None of the drugs tested alone affected heart tissue morphology, nor were atypical cardiac cells observed. However, in combination, significant interactions were found between caffeine and ephedrine; the interventricular septum was most susceptible, with a significant increase in atypical cardiac cells observed. Nicotine pretreatment caused greater susceptibility to cardiotoxicity associated with combinations of caffeine + ephedrine or Metabolife, particularly in the left ventricle wall. These results indicate that sympathomimetic combinations present in Ephedra supplements may have produced cardiotoxicity reported in consumers of these products. Moreover, the presence of nicotine exacerbates these toxic effects.
PMCID: PMC3621369
18.  Linking Stochastic Fluctuations in Chromatin Structure and Gene Expression 
PLoS Biology  2013;11(8):e1001621.
Electron microscopy of single gene molecules and mathematical modeling shows that a promoter stochastically transitions between transcriptionally favorable and unfavorable nucleosome configurations, providing a mechanism for transcriptional bursting.
The number of mRNA and protein molecules expressed from a single gene molecule fluctuates over time. These fluctuations have been attributed, in part, to the random transitioning of promoters between transcriptionally active and inactive states, causing transcription to occur in bursts. However, the molecular basis of transcriptional bursting remains poorly understood. By electron microscopy of single PHO5 gene molecules from yeast, we show that the “activated” promoter assumes alternative nucleosome configurations at steady state, including the maximally repressive, fully nucleosomal, and the maximally non-repressive, nucleosome-free, configuration. We demonstrate that the observed probabilities of promoter nucleosome configurations are obtained from a simple, intrinsically stochastic process of nucleosome assembly, disassembly, and position-specific sliding; and we show that gene expression and promoter nucleosome configuration can be mechanistically coupled, relating promoter nucleosome dynamics and gene expression fluctuations. Together, our findings suggest a structural basis for transcriptional bursting, and offer new insights into the mechanism of transcriptional regulation and the kinetics of promoter nucleosome transitions.
Author Summary
In eukaryotes, such as plants, fungi, and animals, the DNA is wrapped around basic protein cores called nucleosomes at more or less regular intervals. This wrapping discourages transcription, the first step in gene expression. By isolating PHO5 gene molecules from yeast cells and analyzing their structure by electron microscopy, we provide evidence that the “nucleosomes” completely unwrap and then re-wrap in an intrinsically stochastic manner. Only nucleosomes that wrap the regulatory sequences of the gene (promoter) were observed to unspool; no such unspooling was found across the body of the gene. Random unwrapping and re-wrapping generates an ensemble of alternative promoter nucleosome configurations, some conducive to transcription, others not. Mounting evidence suggests that transcription occurs in bursts, where transcripts are released in close succession, interrupted by intervals of transcriptional inactivity; this may lead to significant stochastic fluctuations in gene expression. Although the mechanism of this behavior is not understood, our findings now provide a structural basis for it, suggesting that spooling and unspooling of promoter DNA from the nucleosomes determines the fundamental frequency of transcriptional bursting.
PMCID: PMC3735467  PMID: 23940458
19.  Integrative Modeling of eQTLs and Cis-Regulatory Elements Suggests Mechanisms Underlying Cell Type Specificity of eQTLs 
PLoS Genetics  2013;9(8):e1003649.
Genetic variants in cis-regulatory elements or trans-acting regulators frequently influence the quantity and spatiotemporal distribution of gene transcription. Recent interest in expression quantitative trait locus (eQTL) mapping has paralleled the adoption of genome-wide association studies (GWAS) for the analysis of complex traits and disease in humans. Under the hypothesis that many GWAS associations tag non-coding SNPs with small effects, and that these SNPs exert phenotypic control by modifying gene expression, it has become common to interpret GWAS associations using eQTL data. To fully exploit the mechanistic interpretability of eQTL-GWAS comparisons, an improved understanding of the genetic architecture and causal mechanisms of cell type specificity of eQTLs is required. We address this need by performing an eQTL analysis in three parts: first we identified eQTLs from eleven studies on seven cell types; then we integrated eQTL data with cis-regulatory element (CRE) data from the ENCODE project; finally we built a set of classifiers to predict the cell type specificity of eQTLs. The cell type specificity of eQTLs is associated with eQTL SNP overlap with hundreds of cell type specific CRE classes, including enhancer, promoter, and repressive chromatin marks, regions of open chromatin, and many classes of DNA binding proteins. These associations provide insight into the molecular mechanisms generating the cell type specificity of eQTLs and the mode of regulation of corresponding eQTLs. Using a random forest classifier with cell specific CRE-SNP overlap as features, we demonstrate the feasibility of predicting the cell type specificity of eQTLs. We then demonstrate that CREs from a trait-associated cell type can be used to annotate GWAS associations in the absence of eQTL data for that cell type. We anticipate that such integrative, predictive modeling of cell specificity will improve our ability to understand the mechanistic basis of human complex phenotypic variation.
Author Summary
When interpreting genome-wide association studies showing that specific genetic variants are associated with disease risk, scientists look for a link between the genetic variant and a biological mechanism behind that disease. One functional mechanism is that the genetic variant may influence gene transcription via a co-localized genomic regulatory element, such as a transcription factor binding site within an open chromatin region. Often this type of regulation occurs in some cell types but not others. In this study, we look across eleven gene expression studies with seven cell types and consider how genetic transcription regulators, or eQTLs, replicate within and between cell types. We identify pervasive allelic heterogeneity, or transcriptional control of a single gene by multiple, independent eQTLs. We integrate extensive data on cell type specific regulatory elements from ENCODE to identify general methods of transcription regulation through enrichment of eQTLs within regulatory elements. We also build a classifier to predict eQTL replication across cell types. The results in this paper present a path to an integrative, predictive approach to improve our ability to understand the mechanistic basis of human phenotypic variation.
PMCID: PMC3731231  PMID: 23935528
20.  Psychophysical Properties of Low-Frequency Hearing: Implications for Perceiving Speech and Music via Electric and Acoustic Stimulation 
We have investigated the psychophysical properties of low-frequency hearing, both before and after implantation, to see if we can account for the benefit to speech understanding and melody recognition of adding acoustic stimulation to electric stimulation. In this paper, we review our work and the work of others and describe preliminary results not previously published. We show (a) that it is possible to preserve normal or near-normal nonlinear cochlear processing in the implanted ear following electric and acoustic stimulation surgery – though this is not the typical outcome; (b) that although low-frequency frequency selectivity is generally disrupted following implantation, some degree of frequency selectivity can be preserved, and (c) that neither nonlinear cochlear processing nor frequency selectivity in the acoustic hearing ear is correlated with the gain in speech understanding afforded by combined electric and acoustic stimulation. In another set of experiments, we show that the value of preserving hearing in the implanted ear is best seen in complex listening environments in which binaural cues can play a role in perception.
PMCID: PMC3697738  PMID: 19955721
22.  Managing for Interactions between Local and Global Stressors of Ecosystems 
PLoS ONE  2013;8(6):e65765.
Global stressors, including climate change, are a major threat to ecosystems, but they cannot be halted by local actions. Ecosystem management is thus attempting to compensate for the impacts of global stressors by reducing local stressors, such as overfishing. This approach assumes that stressors interact additively or synergistically, whereby the combined effect of two stressors is at least the sum of their isolated effects. It is not clear, however, how management should proceed for antagonistic interactions among stressors, where multiple stressors do not have an additive or greater impact. Research to date has focussed on identifying synergisms among stressors, but antagonisms may be just as common. We examined the effectiveness of management when faced with different types of interactions in two systems – seagrass and fish communities – where the global stressor was climate change but the local stressors were different. When there were synergisms, mitigating local stressors delivered greater gains, whereas when there were antagonisms, management of local stressors was ineffective or even degraded ecosystems. These results suggest that reducing a local stressor can compensate for climate change impacts if there is a synergistic interaction. Conversely, if there is an antagonistic interaction, management of local stressors will have the greatest benefits in areas of refuge from climate change. A balanced research agenda, investigating both antagonistic and synergistic interaction types, is needed to inform management priorities.
PMCID: PMC3680442  PMID: 23776542
23.  BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion 
Cellular Signalling  2012;24(5):1012-1022.
Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor β Receptor (TGFβR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFβR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFβR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFβR3 in BMP2 signaling in epicardial cells. Whereas TGFβ induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFβ-stimulated differentiation. Tgfbr3−/− cells respond to BMP2 indicating that TGFβR3 is not required. However, Tgfbr3−/− cells show decreased invasion in response to BMP2 and overexpression of TGFβR3 in Tgfbr3−/− cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFβR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3+/+ or Tgfbr3−/− cells rescued with TGFβR3 decreased BMP2-stimulated invasion confirming a requirement for TGFβR3/GIPC interaction. Our results reveal the relative roles of TGFβR3-dependent and TGFβR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFβR3 in mediating BMP2-stimulated invasion.
PMCID: PMC3288519  PMID: 22237159
epithelial to mesenchymal transformation; transforming growth factor beta; bone morphogenic protein; epicardium; invasion; differentiation; coronary vessels
24.  Fluorosugar Chain Termination Agents as Probes of the Sequence Specificity of a Carbohydrate Polymerase 
Naturally occurring carbohydrate polymers are ubiquitous. They are assembled by polymerizing glycosyltransferases, which can generate polysaccharide products with repeating sequence patterns. The fidelity of enzymes of this class is unknown. We report a method for testing the fidelity of carbohydrate polymerase pattern deposition: we synthesized fluorosugar donors and used them as chain termination agents. The requisite nucleotide fluorosugars could be produced from a single intermediate using the Jacobsen catalyst in a kinetically controlled separation of diastereomers. The resulting fluorosugar donors were used by galactofuranosyltransferase GlfT2 from Myco-bacterium tuberculosis (M. tb), and the data indicate that this enzyme mediates the cell wall galactan production through a sequence-specific polymerization.
PMCID: PMC3338147  PMID: 22458542
25.  The effect of radiation therapy techniques (IMRT vs. 3DCRT) on outcome in patients with intermediate risk rhabdomyosarcoma enrolled in COG D9803—A report from Children’s Oncology Group 
To compare dosimetric parameters of intensity modulated radiation therapy (IMRT) and three-dimensional conformal radiation therapy (3DCRT) in patients with intermediate risk rhabdomyosarcoma (RMS), and to analyze their effect on local-regional control (LRC) and failure-free survival (FFS).
Methods and Materials
The study population consisted of 375 patients enrolled on Children’s Oncology Group protocol D9803, receiving IMRT or 3DCRT. Dosimetric data was collected from 179 patients with an available composite plan. Chi-square or Fisher’s exact tests were used to compare patient characteristics and radiotherapy (RT) parameters between the two groups. Time-to- event outcomes were estimated using the Kaplan-Meier method and compared using log-rank tests. Cox analysis was used to examine the effect of RT technique on FFS after adjusting for primary site and risk group.
The median follow-up time was 5.7 and 4.2 years for patients receiving 3DCRT and IMRT respectively. No differences in 5 year failure of LRC (18% vs. 15%) and FFS (72% vs. 76%) were noted between the two groups. Multivariate analysis revealed no association between RT technique and FFS. Patients with primary tumors in parameningeal (PM) sites were more likely to receive IMRT than 3DCRT. IMRT became more common during the later years of the study. Patients receiving IMRT were more likely to receive >50Gy, photon energy of ≤6MV, and >5 RT fields than those who received 3DCRT. There was improved coverage of the IMRT planning target volume (PTV) by the prescription dose compared to the 3DCRT with similar target dose heterogeneity.
IMRT improved target dose coverage when compared to 3DCRT, though an improvement in LRC or FFS could not be demonstrated in this population. Future studies comparing integral dose to non-target tissue and late RT toxicity between the two groups is warranted.
PMCID: PMC3154985  PMID: 21470795
Rhabdomyosarcoma; Intermediate risk; IMRT/3DCRT

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