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1.  Stereo- and Chemodivergent NHC-Promoted Functionalisation of Arylalkylketenes with Chloral** 
Stereo- and chemodivergent enantioselective reaction pathways are observed upon treatment of alkylarylketenes and trichloroacetaldehyde (chloral) with N-heterocyclic carbenes, giving selectively either β-lactones (up to 88:12 dr, up to 94 % ee) or α-chloroesters (up to 94 % ee). Either 2-arylsubstitution or an α-branched iPr alkyl substituent within the ketene favours the chlorination pathway, allowing chloral to be used as an electrophilic chlorinating reagent in asymmetric catalysis.
doi:10.1002/chem.201503308
PMCID: PMC4648049  PMID: 26406613
asymmetric catalysis; chlorination reactions; ketenes; lactones; stereodivergent reactions
2.  Modeling genome coverage in single-cell sequencing 
Bioinformatics  2014;30(22):3159-3165.
Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material.
Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries.
Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq.
Contact: andrewds@usc.edu
Supplementary information: Supplementary material is available at Bioinformatics online.
doi:10.1093/bioinformatics/btu540
PMCID: PMC4221128  PMID: 25107873
3.  A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis 
Nature  2015;520(7547):358-362.
Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites1. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions2–5. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extra-vascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.
doi:10.1038/nature14403
PMCID: PMC4634366  PMID: 25855289
4.  The Amordad database engine for metagenomics 
Bioinformatics  2014;30(20):2949-2955.
Motivation: Several technical challenges in metagenomic data analysis, including assembling metagenomic sequence data or identifying operational taxonomic units, are both significant and well known. These forms of analysis are increasingly cited as conceptually flawed, given the extreme variation within traditionally defined species and rampant horizontal gene transfer. Furthermore, computational requirements of such analysis have hindered content-based organization of metagenomic data at large scale.
Results: In this article, we introduce the Amordad database engine for alignment-free, content-based indexing of metagenomic datasets. Amordad places the metagenome comparison problem in a geometric context, and uses an indexing strategy that combines random hashing with a regular nearest neighbor graph. This framework allows refinement of the database over time by continual application of random hash functions, with the effect of each hash function encoded in the nearest neighbor graph. This eliminates the need to explicitly maintain the hash functions in order for query efficiency to benefit from the accumulated randomness. Results on real and simulated data show that Amordad can support logarithmic query time for identifying similar metagenomes even as the database size reaches into the millions.
Availability and implementation: Source code, licensed under the GNU general public license (version 3) is freely available for download from http://smithlabresearch.org/amordad
Contact: andrewds@usc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu405
PMCID: PMC4184256  PMID: 24974201
5.  Dietary patterns and changes in body composition in children between 9 and 11 years 
Food & Nutrition Research  2014;58:10.3402/fnr.v58.22769.
Objective
Childhood obesity is rising and dietary intake is a potentially modifiable factor that plays an important role in its development. We aim to investigate the association between dietary patterns, obtained through principal components analysis and gains in fat and lean mass in childhood.
Design
Diet diaries at 10 years of age collected from children taking part in the Avon Longitudinal Study of Parents and Children. Body composition was assessed using dual-energy X-ray absorptiometry at 9 and 11.
Setting
Longitudinal birth cohort.
Subjects
3911 children with complete data.
Results
There was an association between the Health Aware (positive loadings on high-fiber bread, and fruits and vegetables; negative loadings on chips, crisps, processed meat, and soft drinks) pattern score and decreased fat mass gain in girls. After adjusting for confounders, an increase of 1 standard deviation (sd) in this score led to an estimated 1.2% decrease in fat mass gain in valid-reporters and 2.1% in under-reporters. A similar decrease was found only in under-reporting boys. There was also an association between the Packed Lunch (high consumption of white bread, sandwich fillings, and snacks) pattern score and decreased fat mass gain (1.1% per sd) in valid-reporting but not under-reporting girls. The main association with lean mass gain was an increase with Packed Lunch pattern score in valid-reporting boys only.
Conclusions
There is a small association between dietary patterns and change in fat mass in mid-childhood. Differences between under- and valid-reporters emphasize the need to consider valid-reporters separately in such studies.
doi:10.3402/fnr.v58.22769
PMCID: PMC4090365  PMID: 25018688
dietary patterns; principal components analysis; ALSPAC; body composition; valid-reporters
6.  Asymmetric Isothiourea-Catalysed Formal [3+2] Cycloadditions of Ammonium Enolates with Oxaziridines 
A highly enantioselective Lewis base-catalysed formal [3+2] cycloaddition of ammonium enolates and oxaziridines to give stereodefined oxazolidin-4-ones in high yield is described. Employing an enantioenriched oxaziridine in this process leads to a matched/mis-matched effect with the isothiourea catalyst and allowed the synthesis of either syn- or anti-stereodefined oxazolidin-4-ones in high d.r., yield and ee. Additionally, the oxazolidin-4-one products have been derivatised to afford functionalised enantioenriched building blocks.
doi:10.1002/chem.201501271
PMCID: PMC4531819  PMID: 26073559
asymmetric synthesis; heterocycles; Lewis base; organocatalysis; oxaziridines
7.  Stereodivergent Organocatalytic Intramolecular Michael Addition/Lactonization for the Asymmetric Synthesis of Substituted Dihydrobenzofurans and Tetrahydrofurans 
A stereodivergent asymmetric Lewis base catalyzed Michael addition/lactonization of enone acids into substituted dihydrobenzofuran and tetrahydrofuran derivatives is reported. Commercially available (S)-(−)-tetramisole hydrochloride gives products with high syn diastereoselectivity in excellent enantioselectivity (up to 99:1 d.r.syn/anti, 99 % eesyn), whereas using a cinchona alkaloid derived catalyst gives the corresponding anti-diastereoisomers as the major product (up to 10:90 d.r.syn/anti, 99 % eeanti).
doi:10.1002/chem.201402684
PMCID: PMC4517160  PMID: 24989672
asymmetric catalysis; cinchona alkaloid; isothiourea; Michael addition; organocatalysis; oxygen heterocycles; stereodivergent
8.  Rate and Equilibrium Constants for the Addition of N-Heterocyclic Carbenes into Benzaldehydes: A Remarkable 2-Substituent Effect** 
Rate and equilibrium constants for the reaction between N-aryl triazolium N-heterocyclic carbene (NHC) precatalysts and substituted benzaldehyde derivatives to form 3-(hydroxybenzyl)azolium adducts under both catalytic and stoichiometric conditions have been measured. Kinetic analysis and reaction profile fitting of both the forward and reverse reactions, plus onwards reaction to the Breslow intermediate, demonstrate the remarkable effect of the benzaldehyde 2-substituent in these reactions and provide insight into the chemoselectivity of cross-benzoin reactions.
doi:10.1002/anie.201501840
PMCID: PMC4510784  PMID: 25908493
2-substituent effect; kinetics; mechanistic studies; N-heterocyclic carbenes; organocatalysis
9.  An epigenetic memory of pregnancy in the mouse mammary gland 
Cell reports  2015;11(7):1102-1109.
SUMMARY
Pregnancy is the major modulator of mammary gland activity. It induces a tremendous expansion of the mammary epithelium and the generation of alveolar structures for milk production. Anecdotal evidence from multiparous humans, indicates that the mammary gland may react less strongly to the first pregnancy than it does to subsequent pregnancies. Here we verify that the mouse mammary gland responds more robustly to a second pregnancy, indicating that the gland retains a long-term memory of pregnancy. A comparison of genome-wide profiles of DNA methylation in isolated mammary cell types reveals substantial and long lasting alterations. Since these alterations are maintained in the absence of the signal that induced them, we term them epigenetic. The majority of alterations in DNA methylation affect sites occupied by the Stat5a transcription factor and mark specific genes that are upregulated during pregnancy. We postulate that the epigenetic memory of a first pregnancy primes the activation of gene expression networks that promote mammary gland function in subsequent reproductive cycles. More broadly, our data indicate that physiological experience can broadly alter epigenetic states, functionally modifying the capacity of the affected cells to respond to later stimulatory events.
Graphical Abstract
doi:10.1016/j.celrep.2015.04.015
PMCID: PMC4439279  PMID: 25959817
10.  External Validation and Calibration of IVFpredict: A National Prospective Cohort Study of 130,960 In Vitro Fertilisation Cycles 
PLoS ONE  2015;10(4):e0121357.
Background
Accurately predicting the probability of a live birth after in vitro fertilisation (IVF) is important for patients, healthcare providers and policy makers. Two prediction models (Templeton and IVFpredict) have been previously developed from UK data and are widely used internationally. The more recent of these, IVFpredict, was shown to have greater predictive power in the development dataset. The aim of this study was external validation of the two models and comparison of their predictive ability.
Methods and Findings
130,960 IVF cycles undertaken in the UK in 2008–2010 were used to validate and compare the Templeton and IVFpredict models. Discriminatory power was calculated using the area under the receiver-operator curve and calibration assessed using a calibration plot and Hosmer-Lemeshow statistic. The scaled modified Brier score, with measures of reliability and resolution, were calculated to assess overall accuracy. Both models were compared after updating for current live birth rates to ensure that the average observed and predicted live birth rates were equal. The discriminative power of both methods was comparable: the area under the receiver-operator curve was 0.628 (95% confidence interval (CI): 0.625–0.631) for IVFpredict and 0.616 (95% CI: 0.613–0.620) for the Templeton model. IVFpredict had markedly better calibration and higher diagnostic accuracy, with calibration plot intercept of 0.040 (95% CI: 0.017–0.063) and slope of 0.932 (95% CI: 0.839–1.025) compared with 0.080 (95% CI: 0.044–0.117) and 1.419 (95% CI: 1.149–1.690) for the Templeton model. Both models underestimated the live birth rate, but this was particularly marked in the Templeton model. Updating the models to reflect improvements in live birth rates since the models were developed enhanced their performance, but IVFpredict remained superior.
Conclusion
External validation in a large population cohort confirms IVFpredict has superior discrimination and calibration for informing patients, clinicians and healthcare policy makers of the probability of live birth following IVF.
doi:10.1371/journal.pone.0121357
PMCID: PMC4390202  PMID: 25853703
11.  Coping with continuous human disturbance in the wild: insights from penguin heart rate response to various stressors 
BMC Ecology  2012;12:10.
Background
A central question for ecologists is the extent to which anthropogenic disturbances (e.g. tourism) might impact wildlife and affect the systems under study. From a research perspective, identifying the effects of human disturbance caused by research-related activities is crucial in order to understand and account for potential biases and derive appropriate conclusions from the data.
Results
Here, we document a case of biological adjustment to chronic human disturbance in a colonial seabird, the king penguin (Aptenodytes patagonicus), breeding on remote and protected islands of the Southern ocean. Using heart rate (HR) as a measure of the stress response, we show that, in a colony with areas exposed to the continuous presence of humans (including scientists) for over 50 years, penguins have adjusted to human disturbance and habituated to certain, but not all, types of stressors. When compared to birds breeding in relatively undisturbed areas, birds in areas of high chronic human disturbance were found to exhibit attenuated HR responses to acute anthropogenic stressors of low-intensity (i.e. sounds or human approaches) to which they had been subjected intensely over the years. However, such attenuation was not apparent for high-intensity stressors (i.e. captures for scientific research) which only a few individuals experience each year.
Conclusions
Habituation to anthropogenic sounds/approaches could be an adaptation to deal with chronic innocuous stressors, and beneficial from a research perspective. Alternately, whether penguins have actually habituated to anthropogenic disturbances over time or whether human presence has driven the directional selection of human-tolerant phenotypes, remains an open question with profound ecological and conservation implications, and emphasizes the need for more knowledge on the effects of human disturbance on long-term studied populations.
doi:10.1186/1472-6785-12-10
PMCID: PMC3543187  PMID: 22784366
Stress; Heart rate; Habituation; Selection; Seabird; Human disturbance; Long-term monitoring
12.  Prenatal exposure to maternal smoking and offspring DNA methylation across the lifecourse: findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) 
Human Molecular Genetics  2014;24(8):2201-2217.
Maternal smoking during pregnancy has been found to influence newborn DNA methylation in genes involved in fundamental developmental processes. It is pertinent to understand the degree to which the offspring methylome is sensitive to the intensity and duration of prenatal smoking. An investigation of the persistence of offspring methylation associated with maternal smoking and the relative roles of the intrauterine and postnatal environment is also warranted. In the Avon Longitudinal Study of Parents and Children, we investigated associations between prenatal exposure to maternal smoking and offspring DNA methylation at multiple time points in approximately 800 mother–offspring pairs. In cord blood, methylation at 15 CpG sites in seven gene regions (AHRR, MYO1G, GFI1, CYP1A1, CNTNAP2, KLF13 and ATP9A) was associated with maternal smoking, and a dose-dependent response was observed in relation to smoking duration and intensity. Longitudinal analysis of blood DNA methylation in serial samples at birth, age 7 and 17 years demonstrated that some CpG sites showed reversibility of methylation (GFI1, KLF13 and ATP9A), whereas others showed persistently perturbed patterns (AHRR, MYO1G, CYP1A1 and CNTNAP2). Of those showing persistence, we explored the effect of postnatal smoke exposure and found that the major contribution to altered methylation was attributed to a critical window of in utero exposure. A comparison of paternal and maternal smoking and offspring methylation showed consistently stronger maternal associations, providing further evidence for causal intrauterine mechanisms. These findings emphasize the sensitivity of the methylome to maternal smoking during early development and the long-term impact of such exposure.
doi:10.1093/hmg/ddu739
PMCID: PMC4380069  PMID: 25552657
13.  Leveraging cross-link modification events in CLIP-seq for motif discovery 
Nucleic Acids Research  2014;43(1):95-103.
High-throughput protein–RNA interaction data generated by CLIP-seq has provided an unprecedented depth of access to the activities of RNA-binding proteins (RBPs), the key players in co- and post-transcriptional regulation of gene expression. Motif discovery forms part of the necessary follow-up data analysis for CLIP-seq, both to refine the exact locations of RBP binding sites, and to characterize them. The specific properties of RBP binding sites, and the CLIP-seq methods, provide additional information not usually present in the classic motif discovery problem: the binding site structure, and cross-linking induced events in reads. We show that CLIP-seq data contains clear secondary structure signals, as well as technology- and RBP-specific cross-link signals. We introduce Zagros, a motif discovery algorithm specifically designed to leverage this information and explore its impact on the quality of recovered motifs. Our results indicate that using both secondary structure and cross-link modifications can greatly improve motif discovery on CLIP-seq data. Further, the motifs we recover provide insight into the balance between sequence- and structure-specificity struck by RBP binding.
doi:10.1093/nar/gku1288
PMCID: PMC4288180  PMID: 25505146
14.  Preferred Methods of Sexually Transmitted Infection Service Delivery Among an Urban Sample of Underserved Midwestern Men 
Sexually transmitted diseases  2014;41(2):129-132.
Semistructured interviews were completed with a sample of 25 men residing in an urban area of the Midwestern United States to elicit preferred methods of sexually transmitted infection service delivery. Results highlight the influence of stigma, social support, and perceived risk on sexually transmitted infection screening uptake and preferred methods of screening.
doi:10.1097/OLQ.0000000000000082
PMCID: PMC4148079  PMID: 24413494
15.  Using beta-binomial regression for high-precision differential methylation analysis in multifactor whole-genome bisulfite sequencing experiments 
BMC Bioinformatics  2014;15:215.
Background
Whole-genome bisulfite sequencing currently provides the highest-precision view of the epigenome, with quantitative information about populations of cells down to single nucleotide resolution. Several studies have demonstrated the value of this precision: meaningful features that correlate strongly with biological functions can be found associated with only a few CpG sites. Understanding the role of DNA methylation, and more broadly the role of DNA accessibility, requires that methylation differences between populations of cells are identified with extreme precision and in complex experimental designs.
Results
In this work we investigated the use of beta-binomial regression as a general approach for modeling whole-genome bisulfite data to identify differentially methylated sites and genomic intervals.
Conclusions
The regression-based analysis can handle medium- and large-scale experiments where it becomes critical to accurately model variation in methylation levels between replicates and account for influence of various experimental factors like cell types or batch effects.
doi:10.1186/1471-2105-15-215
PMCID: PMC4230021  PMID: 24962134
Epigenomics; Differential methylation; Beta-binomial regression
16.  Intergenerational genomic DNA methylation patterns in mouse hybrid strains 
Genome Biology  2014;15(5):R68.
Background
DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.
Results
We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes.
Conclusions
The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.
doi:10.1186/gb-2014-15-5-r68
PMCID: PMC4076608  PMID: 24887417
17.  Metastatic Melanoma: Lactate Dehydrogenase Levels and CT Imaging Findings of Tumor Devascularization Allow Accurate Prediction of Survival in Patients Treated with Bevacizumab1 
Radiology  2013;270(2):425-434.
Purpose
To predict survival in patients with metastatic melanoma by evaluating a combination of serum lactate dehydrogenase (LDH) level and initial computed tomographic (CT) findings of tumor devascularization after antiangiogenic therapy.
Materials and Methods
Consent was waived for this institutional review board–approved, retrospective, secondary analysis. Forty-four patients with metastatic melanoma received bevacizumab therapy in a randomized prospective phase II trial. Target lesions on the initial posttherapy CT images were evaluated by using Response Evaluation Criteria in Solid Tumors, the Choi criteria, and Morphology, Attenuation, Size, and Structure (MASS) criteria. Cox proportional hazards models were used to assess the association of baseline clinical variables including serum LDH and imaging findings with progression-free and overall survival. The receiver operating characteristic curve with area under the curve (AUC) was used to evaluate accuracy.
Results
In multivariate analysis, a high baseline serum LDH level was associated with decreased progression-free survival (hazard ratio = 1.29 for each increase of 100 IU/L; P = .002) and overall survival (hazard ratio = 1.44 for each increase of 100 IU/L; P = .001). Evaluation with MASS criteria of the first CT examination after therapy strongly predicted progression-free (P < .001) and overall (P < .001) survival. Baseline serum LDH level was moderately accurate for predicting progression-free survival at 9 months (AUC = 0.793) and overall survival at 18 months (AUC = 0.689). The combination of baseline serum LDH levels and evaluation with MASS criteria at the first CT examination after therapy had significantly higher accuracy for predicting progression-free survival at 9 months (AUC = 0.969) and overall survival at 18 months (AUC = 0.813) than did baseline serum LDH levels alone for prediction of progression-free survival (P = .020).
Conclusion
A combination of baseline serum LDH levels and evaluation with MASS criteria at the first CT examination after bevacizumab therapy had the highest accuracy for predicting survival in patients with metastatic melanoma.
doi:10.1148/radiol.13130776
PMCID: PMC3985552  PMID: 24072776
18.  The Effects of Mary Rose Conservation Treatment on Iron Oxidation Processes and Microbial Communities Contributing to Acid Production in Marine Archaeological Timbers 
PLoS ONE  2014;9(2):e84169.
The Tudor warship the Mary Rose has reached an important transition point in her conservation. The 19 year long process of spraying with polyethylene glycol (PEG) has been completed (April 29th 2013) and the hull is air drying under tightly controlled conditions. Acidophilic bacteria capable of oxidising iron and sulfur have been previously identified and enriched from unpreserved timbers of the Mary Rose, demonstrating that biological pathways of iron and sulfur oxidization existed potentially in this wood, before preservation with PEG. This study was designed to establish if the recycled PEG spray system was a reservoir of microorganisms capable of iron and sulfur oxidization during preservation of the Mary Rose. Microbial enrichments derived from PEG impregnated biofilm collected from underneath the Mary Rose hull, were examined to better understand the processes of cycling of iron. X-ray absorption spectroscopy was utilised to demonstrate the biological contribution to production of sulfuric acid in the wood. Using molecular microbiological techniques to examine these enrichment cultures, PEG was found to mediate a shift in the microbial community from a co-culture of Stenotrophomonas and Brevunidimonas sp, to a co-culture of Stenotrophomonas and the iron oxidising Alicyclobacillus sp. Evidence is presented that PEG is not an inert substance in relation to the redox cycling of iron. This is the first demonstration that solutions of PEG used in the conservation of the Mary Rose are promoting the oxidation of ferrous iron in acidic solutions, in which spontaneous abiotic oxidation does not occur in water. Critically, these results suggest PEG mediated redox cycling of iron between valence states in solutions of 75% PEG 200 and 50% PEG 2000 (v/v) at pH 3.0, with serious implications for the future use of PEG as a conservation material of iron rich wooden archaeological artefacts.
doi:10.1371/journal.pone.0084169
PMCID: PMC3929279  PMID: 24586230
19.  On the Value of Intra-Motif Dependencies of Human Insulator Protein CTCF 
PLoS ONE  2014;9(1):e85629.
The binding affinity of DNA-binding proteins such as transcription factors is mainly determined by the base composition of the corresponding binding site on the DNA strand. Most proteins do not bind only a single sequence, but rather a set of sequences, which may be modeled by a sequence motif. Algorithms for de novo motif discovery differ in their promoter models, learning approaches, and other aspects, but typically use the statistically simple position weight matrix model for the motif, which assumes statistical independence among all nucleotides. However, there is no clear justification for that assumption, leading to an ongoing debate about the importance of modeling dependencies between nucleotides within binding sites. In the past, modeling statistical dependencies within binding sites has been hampered by the problem of limited data. With the rise of high-throughput technologies such as ChIP-seq, this situation has now changed, making it possible to make use of statistical dependencies effectively. In this work, we investigate the presence of statistical dependencies in binding sites of the human enhancer-blocking insulator protein CTCF by using the recently developed model class of inhomogeneous parsimonious Markov models, which is capable of modeling complex dependencies while avoiding overfitting. These findings lead to a more detailed characterization of the CTCF binding motif, which is only poorly represented by independent nucleotide frequencies at several positions, predominantly at the 3′ end.
doi:10.1371/journal.pone.0085629
PMCID: PMC3899044  PMID: 24465627
20.  Genomic Analyses Reveal Broad Impact of miR-137 on Genes Associated with Malignant Transformation and Neuronal Differentiation in Glioblastoma Cells 
PLoS ONE  2014;9(1):e85591.
miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137 – among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.
doi:10.1371/journal.pone.0085591
PMCID: PMC3899048  PMID: 24465609
21.  Increased plasma levels of soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of VEGF in overweight/obese women 
The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity, and overweight/obesity. We previously reported that exercise induced a circulating angiostatic phenotype characterized by increased sFlt-1 and endostatin and decreased unbound-VEGF in men. However, there is no data on women. The present study determines the following: 1) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound-VEGF in the circulation of adult female volunteers; 2) whether overweight/obese women have a higher plasma level of unbound-VEGF than lean women. 72 African American and Caucasian adult women volunteers aged from 18–44 were enrolled into the exercise study. All the participants walked on a treadmill for 30 minutes at a moderate intensity (55–59% heart rate reserve), and oxygen consumption (VO2) was quantified by utilizing a metabolic cart. We had the blood samples before and immediately after exercise from 63 participants. ELISA assays (R&D Systems) showed that plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 minutes), significantly higher than basal levels, 54.5±3.3 pg/ml, before exercise (P < 0.01; n=63). There was no significant difference in the % increase of sFlt-1 levels after exercise between African American and Caucasian (P=0.533) or between lean and overweight/obese women (P=0.892). There was no significant difference in plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. Basal plasma levels of unbound-VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than basal levels of unbound-VEGF in lean women, 27.34±4.99 pg/ml (P < 0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing breast cancer progression and that VEGF is an important biomarker in obesity and obesity-related cancer progression.
doi:10.1097/CEJ.0b013e328353ed81
PMCID: PMC3449013  PMID: 22609636
Exercise; Young adult women; Overweight/obese; sFlt-1; Endostatin; VEGF
22.  Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA 
Molecular and Cellular Biology  2013;33(14):2683-2690.
Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.
doi:10.1128/MCB.00220-13
PMCID: PMC3700126  PMID: 23671186
23.  A profile of transcriptomic changes in the rd10 mouse model of retinitis pigmentosa 
Molecular Vision  2014;20:1612-1628.
Purpose
Retinitis pigmentosa (RP) is a photoreceptor disease that affects approximately 100,000 people in the United States. Treatment options are limited, and the prognosis for most patients is progressive vision loss. Unfortunately, understanding of the molecular underpinnings of RP initiation and progression is still limited. However, the development of animal models of RP, coupled with high-throughput sequencing, has provided an opportunity to study the underlying cellular and molecular changes in this disease.
Methods
Using RNA-Seq, we present the first retinal transcriptome analysis of the rd10 murine model of retinal degeneration.
Results
Our data confirm the loss of rod-specific transcripts and the increased relative expression of Müller-specific transcripts, emphasizing the important role of reactive gliosis and innate immune activation in RP. Moreover, we report substantial changes in relative isoform usage among neuronal differentiation and morphogenesis genes, including a marked shift to shorter transcripts.
Conclusions
Our analyses implicate remodeling of the inner retina and possible Müller cell dedifferentiation.
PMCID: PMC4235044  PMID: 25489233
24.  A Reference Methylome Database and Analysis Pipeline to Facilitate Integrative and Comparative Epigenomics 
PLoS ONE  2013;8(12):e81148.
DNA methylation is implicated in a surprising diversity of regulatory, evolutionary processes and diseases in eukaryotes. The introduction of whole-genome bisulfite sequencing has enabled the study of DNA methylation at a single-base resolution, revealing many new aspects of DNA methylation and highlighting the usefulness of methylome data in understanding a variety of genomic phenomena. As the number of publicly available whole-genome bisulfite sequencing studies reaches into the hundreds, reliable and convenient tools for comparing and analyzing methylomes become increasingly important. We present MethPipe, a pipeline for both low and high-level methylome analysis, and MethBase, an accompanying database of annotated methylomes from the public domain. Together these resources enable researchers to extract interesting features from methylomes and compare them with those identified in public methylomes in our database.
doi:10.1371/journal.pone.0081148
PMCID: PMC3855694  PMID: 24324667
25.  scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints 
Gene therapy  2012;20(6):670-677.
With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.
doi:10.1038/gt.2012.81
PMCID: PMC3577988  PMID: 23151520
Osteoarthritis; Self-complementary Adeno-Associated Virus; Interleukin-1 Receptor Antagonist; Synovium; Cartilage; Equine

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