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1.  MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer 
Oncogene  2012;32(35):4139-4147.
The miR-106b-25 microRNA cluster is a candidate oncogene in human prostate cancer. Here, we report that microRNAs encoded by miR-106b-25 are up-regulated in both primary tumors and distant metastasis. Moreover, increased tumor miR-106b expression was associated with disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expression in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio = 4.1; 95% confidence interval: 1.6 to 12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that caspase-7 is a direct target of miR-106b, which was confirmed by Western blot analysis and a 3'UTR reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and caspase-7 expression were inhibited. Further analyses showed that caspase-7 is down-regulated in primary prostate tumors and metastatic lesions across multiple datasets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and disease outcome and may do so by altering apoptosis- and focal adhesion-related pathways.
doi:10.1038/onc.2012.424
PMCID: PMC3530025  PMID: 22986525
prostate cancer; microRNA; oncogene; apoptosis
2.  Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells 
Carcinogenesis  2013;34(8):1929-1939.
Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.
doi:10.1093/carcin/bgt138
PMCID: PMC3731808  PMID: 23619298
3.  An integrated understanding of the physiological response to elevated extracellular phosphate 
Journal of cellular physiology  2013;228(7):1536-1550.
Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.
doi:10.1002/jcp.24312
PMCID: PMC3702686  PMID: 23280476
Inorganic phosphate; FGF receptor signaling; immediate early genes; angiogenesis; AP-1
4.  Performance comparison of SNP detection tools with illumina exome sequencing data—an assessment using both family pedigree information and sample-matched SNP array data 
Nucleic Acids Research  2014;42(12):e101.
To apply exome-seq-derived variants in the clinical setting, there is an urgent need to identify the best variant caller(s) from a large collection of available options. We have used an Illumina exome-seq dataset as a benchmark, with two validation scenarios—family pedigree information and SNP array data for the same samples, permitting global high-throughput cross-validation, to evaluate the quality of SNP calls derived from several popular variant discovery tools from both the open-source and commercial communities using a set of designated quality metrics. To the best of our knowledge, this is the first large-scale performance comparison of exome-seq variant discovery tools using high-throughput validation with both Mendelian inheritance checking and SNP array data, which allows us to gain insights into the accuracy of SNP calling through such high-throughput validation in an unprecedented way, whereas the previously reported comparison studies have only assessed concordance of these tools without directly assessing the quality of the derived SNPs. More importantly, the main purpose of our study was to establish a reusable procedure that applies high-throughput validation to compare the quality of SNP discovery tools with a focus on exome-seq, which can be used to compare any forthcoming tool(s) of interest.
doi:10.1093/nar/gku392
PMCID: PMC4081058  PMID: 24831545
5.  Interleukin-27 treated human macrophages induce the expression of novel microRNAs which may mediate anti-viral properties 
Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an Interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.
doi:10.1016/j.bbrc.2013.03.046
PMCID: PMC3700531  PMID: 23535375
Interleukin-27; macrophages; MicroRNAs; HIV; HSV-1; HSV-2; HHV-8
6.  Pathway-Specific Engineered Mouse Allograft Models Functionally Recapitulate Human Serous Epithelial Ovarian Cancer 
PLoS ONE  2014;9(4):e95649.
The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1–deficient tumors and development of relevant biomarkers.
doi:10.1371/journal.pone.0095649
PMCID: PMC3991711  PMID: 24748377
7.  HIV Populations Are Large and Accumulate High Genetic Diversity in a Nonlinear Fashion 
Journal of Virology  2013;87(18):10313-10323.
HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.
doi:10.1128/JVI.01225-12
PMCID: PMC3754011  PMID: 23678164
8.  Relationship Between Child Care Centers’ Compliance With Physical Activity Regulations and Children’s Physical Activity, New York City, 2010 
Introduction
Physical activity may protect against overweight and obesity among preschoolers, and the policies and characteristics of group child care centers influence the physical activity levels of children who attend them. We examined whether children in New York City group child care centers that are compliant with the city’s regulations on child physical activity engage in more activity than children in centers who do not comply.
Methods
A sample of 1,352 children (mean age, 3.39 years) served by 110 group child care centers in low-income neighborhoods participated. Children’s anthropometric data were collected and accelerometers were used to measure duration and intensity of physical activity. Multilevel generalized linear regression modeling techniques were used to assess the effect of center- and child-level factors on child-level physical activity.
Results
Centers’ compliance with the regulation of obtaining at least 60 minutes of total physical activity per day was positively associated with children’s levels of moderate to vigorous physical activity (MVPA); compliance with the regulation of obtaining at least 30 minutes of structured activity was not associated with increased levels of MVPA. Children in centers with a dedicated outdoor play space available also spent more time in MVPA. Boys spent more time in MVPA than girls, and non-Hispanic black children spent more time in MVPA than Hispanic children.
Conclusion
To increase children’s level of MVPA in child care, both time and type of activity should be considered. Further examination of the role of play space availability and its effect on opportunities for engaging in physical activity is needed.
doi:10.5888/pcd11.130432
PMCID: PMC4208988  PMID: 25321630
9.  Evaluation Design of New York City’s Regulations on Nutrition, Physical Activity, and Screen Time in Early Child Care Centers 
This article describes the multi-method cross-sectional design used to evaluate New York City Department of Health and Mental Hygiene’s regulations of nutrition, physical activity, and screen time for children aged 3 years or older in licensed group child care centers. The Center Evaluation Component collected data from a stratified random sample of 176 licensed group child care centers in New York City. Compliance with the regulations was measured through a review of center records, a facility inventory, and interviews of center directors, lead teachers, and food service staff. The Classroom Evaluation Component included an observational and biometric study of a sample of approximately 1,400 children aged 3 or 4 years attending 110 child care centers and was designed to complement the center component at the classroom and child level. The study methodology detailed in this paper may aid researchers in designing policy evaluation studies that can inform other jurisdictions considering similar policies.
doi:10.5888/pcd11.130431
PMCID: PMC4208991  PMID: 25321635
10.  Measurement of Compliance With New York City’s Regulations on Beverages, Physical Activity, and Screen Time in Early Child Care Centers 
Introduction
Policy interventions designed to change the nutrition environment and increase physical activity in child care centers are becoming more common, but an understanding of the implementation of these interventions is yet to be developed. The objective of this study was to explore the extent and consistency of compliance with a policy intervention designed to promote nutrition and physical activity among licensed child care centers in New York City.
Methods
We used a multimethod cross-sectional approach and 2 independent components of data collection (Center Evaluation Component and Classroom Evaluation Component). The methods were designed to evaluate the impact of regulations on beverages served, physical activity, and screen time at child care centers. We calculated compliance scores for each evaluation component and each regulation and percentage agreement between compliance in the center and classroom components.
Results
Compliance with certain requirements of the beverage regulations was high and fairly consistent between components, whereas compliance with the physical activity regulation varied according to the data collection component. Compliance with the regulation on amount and content of screen time was high and consistent.
Conclusion
Compliance with the physical activity regulation may be a more fluid, day-to-day issue, whereas compliance with the regulations on beverages and television viewing may be easier to control at the center level. Multiple indicators over multiple time points may provide a more complete picture of compliance — especially in the assessment of compliance with physical activity policies.
doi:10.5888/pcd11.130433
PMCID: PMC4208992  PMID: 25321634
11.  MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis 
Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell–like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.
doi:10.1172/JCI71180
PMCID: PMC3871244  PMID: 24316975
12.  Knowledge and Theme Discovery across Very Large Biological Data Sets Using Distributed Queries: A Prototype Combining Unstructured and Structured Data 
PLoS ONE  2013;8(12):e80503.
As the discipline of biomedical science continues to apply new technologies capable of producing unprecedented volumes of noisy and complex biological data, it has become evident that available methods for deriving meaningful information from such data are simply not keeping pace. In order to achieve useful results, researchers require methods that consolidate, store and query combinations of structured and unstructured data sets efficiently and effectively. As we move towards personalized medicine, the need to combine unstructured data, such as medical literature, with large amounts of highly structured and high-throughput data such as human variation or expression data from very large cohorts, is especially urgent. For our study, we investigated a likely biomedical query using the Hadoop framework. We ran queries using native MapReduce tools we developed as well as other open source and proprietary tools. Our results suggest that the available technologies within the Big Data domain can reduce the time and effort needed to utilize and apply distributed queries over large datasets in practical clinical applications in the life sciences domain. The methodologies and technologies discussed in this paper set the stage for a more detailed evaluation that investigates how various data structures and data models are best mapped to the proper computational framework.
doi:10.1371/journal.pone.0080503
PMCID: PMC3846626  PMID: 24312478
13.  A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS 
PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.
doi:10.4172/2153-0602.1000136
PMCID: PMC3811116  PMID: 24179701
PacBio; CCS read; quality control (QC); pass number; quality value (QV); SVM regression; assembly
14.  Guanine Holes Are Prominent Targets for Mutation in Cancer and Inherited Disease 
PLoS Genetics  2013;9(9):e1003816.
Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G•C bp in the context of all 64 5′-NGNN-3′ motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.
Author Summary
A large number of DNA mutations identified in cells from patients with cancer or human inherited disease were analyzed to address a fundamental issue in human pathology, viz, the mutational mechanisms that cause irreversible changes to DNA. By using bioinformatics and computational methods, we found that mutations do not occur randomly, but instead affect specific bases, most often guanines flanked by other guanines or adenines. We attribute this effect to electron transfer, a chemical reaction known to underlie basic biological processes such as cellular respiration and photosynthesis. Certain types of carcinogens, oxidants or radiation can interact with DNA and abstract an electron. Our results imply that the ensuing sites of electron loss can migrate from their original position in the DNA to neighboring guanines where they become trapped, leading to further chemical modifications that may eventually result in mutations. Many of the mutations known to be important for tumor growth (driver mutations), as well as passenger mutations and mutations associated with inherited disease, appear to be caused by electron transfer. Beyond pathological mutations, electron transfer may represent a universal mechanism by which genetic changes occur in all life forms to drive population fitness over evolutionary time.
doi:10.1371/journal.pgen.1003816
PMCID: PMC3784513  PMID: 24086153
15.  Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles 
Nucleic Acids Research  2013;41(21):9732-9740.
Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.
doi:10.1093/nar/gkt762
PMCID: PMC3834830  PMID: 23975201
16.  PKCγ receptor mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress in the spinal cord of rats 
Molecular Pain  2013;9:35.
Background
Clinical studies indicate that patients with post-traumatic stress disorder (PTSD) frequently share comorbidity with numerous chronic pain conditions. However, the sustained effects of PTSD-like stress over time on visceral nociception and hyperalgesia have been rarely studied, and the underlying mechanisms of stress-induced modulation of visceral hyperalgesia remain elusive. The purpose of this study was to investigate the characterization of visceral nociception and hyperalgesia over time in rats exposed to PTSD-like stress, and to explore the potential role of protein kinase C gamma (PKCγ) in mediating visceral hyperalgesia following exposure to PTSD-like stress.
Results
On day 1, the rats exposed to single-prolonged stress (SPS, an established animal model for PTSD) exhibited an analgesic response and its visceromotor response (VMR) to graded colorectal distention (CRD) at 40 and 60 mmHg was reduced compared with the control group (all P < 0.05). On day 6, the VMR returned to the baseline value. However, as early as 7 days after SPS, VMR dramatically increased compared with its baseline value and that in the controls (all P < 0.001) and this increase persisted for 28 days, with the peak on day 9. Abdominal withdrawal reflex (AWR) scores were higher in SPS rats than in controls on days 7, 9, 14, 21 and 28 (all P < 0.001). Intrathecal administration of GF109203X (an inhibitor of PKC gamma), attenuated the SPS-induced increase in both VMR and AWR scores on days 7, 14, 21 and 28 (all P < 0.05). PKCγ protein expression determined by immunofluorescence was reduced in the spinal cord within 3 days after the exposure to SPS (P < 0.01), which returned to normal levels between days 4 and 6, and significantly increased from day 7, and this increase was maintained on days 14, 21, and 28 (all P < 0.001), with the peak on day 9. In addition, Western blotting showed a consistent trend in the changes of PKCγ protein expression.
Conclusions
The modified SPS alters visceral sensitivity to CRD, and contributes to the maintenance of visceral hyperalgesia, which is associated with enhanced PKCγ expression in the spinal cord. Functional blockade of the PKCγ receptors attenuates SPS-induced visceral hyperalgesia. Thus, the present study identifies a specific molecular mechanism for visceral hyperalgesia which may pave the way for novel therapeutic strategies for PTSD-like conditions.
doi:10.1186/1744-8069-9-35
PMCID: PMC3751645  PMID: 23837410
Visceral hyperalgesia; Protein kinase C gamma; Post-traumatic stress disorder; Single-prolonged stress; Colorectal distention; Visceromotor response; Spinal cord
17.  System-Level Change in Cultural and Linguistic Competence (CLC): How Changes in CLC are Related to Service Experience Outcomes in Systems of Care 
As US demographic trends shift toward more diversity, it becomes increasingly necessary to address differential needs of diverse groups of youth in mental health service systems. Cultural and linguistic competence (CLC) is essential to providing the most appropriate mental health services to youth and their families. The successful implementation of CLC often begins at the system level. Though various factors may affect change and system-level factors set the tone for broad acceptance of CLC within systems, there is limited empirical evidence linking culturally competent practices to outcomes. The purpose of the present study was to examine system-level CLC changes over time within systems of care and their associations with service experiences among youth and their families. Participants were 4,512 youth and their families enrolled in the national evaluation of the Children’s Mental Health Initiative (CMHI). Results suggest that implementation of CLC at the system level improves over time in funded systems of care. Further, variation exists in specific system-level components of CLC. In addition, the changes in CLC at the system level are related to family/caregiver participation in treatment. Implications for supporting positive changes in CLC among systems of care communities, and specific strategies for community psychologists, are discussed.
doi:10.1007/s10464-011-9442-1
PMCID: PMC3182295  PMID: 21553095
Cultural and linguistic competence; Children’s mental health; Mental health services; System of care
18.  Intraclass Correlation Associated with Therapists: Estimates and Applications in Planning Psychotherapy Research 
Cognitive behaviour therapy  2011;40(1):10.1080/16506073.2010.520731.
It is essential that outcome research permit clear conclusions to be drawn about the efficacy of interventions. The common practice of nesting therapists within conditions can pose important methodological challenges that affect interpretation, particularly if the study is not powered to account for the nested design. An obstacle to the optimal design of these studies is lack of data about the intraclass correlation coefficient (ICC), which measures the statistical dependencies introduced by nesting. To begin the development of a public database of ICC estimates, the authors report ICCs for a variety outcomes reported in 20 psychotherapy outcome studies. The magnitude of the N = 495 ICC estimates varied widely across measures and studies. The authors provide recommendations regarding how to select and aggregate ICC estimates for power calculations and show how researchers can use ICC estimates to choose the number of patients and therapists that will optimize power. Attention to these recommendations will strengthen the validity of inferences drawn from psychotherapy studies that nest therapists within conditions.
doi:10.1080/16506073.2010.520731
PMCID: PMC3650614  PMID: 21337212
Statistical dependence; therapists effects; intraclass correlation; power; psychotherapy research
19.  SysBioCube: A Data Warehouse and Integrative Data Analysis Platform Facilitating Systems Biology Studies of Disorders of Military Relevance 
SysBioCube is an integrated data warehouse and analysis platform for experimental data relating to diseases of military relevance developed for the US Army Medical Research and Materiel Command Systems Biology Enterprise (SBE). It brings together, under a single database environment, pathophysio-, psychological, molecular and biochemical data from mouse models of post-traumatic stress disorder and (pre-) clinical data from human PTSD patients.. SysBioCube will organize, centralize and normalize this data and provide an access portal for subsequent analysis to the SBE. It provides new or expanded browsing, querying and visualization to provide better understanding of the systems biology of PTSD, all brought about through the integrated environment. We employ Oracle database technology to store the data using an integrated hierarchical database schema design. The web interface provides researchers with systematic information and option to interrogate the profiles of pan-omics component across different data types, experimental designs and other covariates.
PMCID: PMC3814498  PMID: 24303294
20.  IL-1R–MyD88 signaling in keratinocyte transformation and carcinogenesis 
The Journal of Experimental Medicine  2012;209(9):1689-1702.
Keratinocyte MyD88 is a component of an IL-1α–IL-1R autocrine loop that drives Ras-mediated transformation in vitro and contributes to skin tumor formation in vivo.
Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. In this study, we report that mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective wild-type (WT) controls. MyD88−/− or IL-1R−/− keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Although RAS-expressing MyD88−/− keratinocytes form only a few small tumors in orthotopic grafts, IL-1R–deficient RAS-expressing keratinocytes retain the ability to form tumors in orthotopic grafts. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1α, IL-1R, and MyD88 leading to phosphorylation of IκBα and NF-κB activation. Blocking IL-1α–mediated NF-κB activation in RAS-expressing WT keratinocytes reverses the differentiation defect and inhibits proinflammatory gene expression. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes.
doi:10.1084/jem.20101355
PMCID: PMC3428947  PMID: 22908325
21.  Transcription signatures encoded by ultraconserved genomic regions in human prostate cancer 
Molecular Cancer  2013;12:13.
Background
Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors.
Methods
Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2′deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.
Results
We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2′deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs.
Conclusions
This first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.
doi:10.1186/1476-4598-12-13
PMCID: PMC3626580  PMID: 23409773
Ultraconserved region; Gene expression; Prostate cancer
22.  Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA 
Retrovirology  2013;10:18.
Background
454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.
Results
We constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.
Conclusion
Standard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.
doi:10.1186/1742-4690-10-18
PMCID: PMC3599717  PMID: 23402264
454 pyrosequencing; HIV-1; Error rate; PCR induced recombination
23.  SRAdb: query and use public next-generation sequencing data from within R 
BMC Bioinformatics  2013;14:19.
Background
The Sequence Read Archive (SRA) is the largest public repository of sequencing data from the next generation of sequencing platforms including Illumina (Genome Analyzer, HiSeq, MiSeq, .etc), Roche 454 GS System, Applied Biosystems SOLiD System, Helicos Heliscope, PacBio RS, and others.
Results
SRAdb is an attempt to make queries of the metadata associated with SRA submission, study, sample, experiment and run more robust and precise, and make access to sequencing data in the SRA easier. We have parsed all the SRA metadata into a SQLite database that is routinely updated and can be easily distributed. The SRAdb R/Bioconductor package then utilizes this SQLite database for querying and accessing metadata. Full text search functionality makes querying metadata very flexible and powerful. Fastq files associated with query results can be downloaded easily for local analysis. The package also includes an interface from R to a popular genome browser, the Integrated Genomics Viewer.
Conclusions
SRAdb Bioconductor package provides a convenient and integrated framework to query and access SRA metadata quickly and powerfully from within R.
doi:10.1186/1471-2105-14-19
PMCID: PMC3560148  PMID: 23323543
24.  Consensus-based Recommendations for Research Priorities Related to Interventions to Safeguard Patient Safety in the Crowded Emergency Department 
This article describes the results of the Interventions to Safeguard Safety breakout session of the 2011 Academic Emergency Medicine (AEM) consensus conference entitled “Interventions to Assure Quality in the Crowded Emergency Department.” Using a multistep nominal group technique, experts in emergency department (ED) crowding, patient safety, and systems engineering defined knowledge gaps and priority research questions related to the maintenance of safety in the crowded ED. Consensus was reached for seven research priorities related to interventions to maintain safety in the setting of a crowded ED. Included among these are: 1) How do routine corrective processes and compensating mechanism change during crowding? 2) What metrics should be used to determine ED safety? 3) How can checklists ensure safer care and what factors contribute to their success or failure? 4) What constitutes safe staffing levels / ratios? 5) How can we align emergency medicine (EM)-specific patient safety issues with national patient safety issues? 6) How can we develop metrics and skills to recognize when an ED is getting close to catastrophic overload conditions? and 7) What can EM learn from experts and modeling from fields outside of medicine to develop innovative solutions? These priorities have the potential to inform future clinical and human factors research and extramural funding decisions related to this important topic.
doi:10.1111/j.1553-2712.2011.01234.x
PMCID: PMC3370301  PMID: 22168192
25.  Social Norms and Self-efficacy Among Heavy Using Adolescent Marijuana Smokers 
Psychology of Addictive Behaviors  2011;25(4):727-732.
Adolescence is a time in which individuals are particularly likely to engage in health-risk behaviors, with marijuana being the most prevalent illicit drug used. Perceptions of others’ use (i.e., norms) have previously been found to be related to increased marijuana use. Additionally, low refusal self-efficacy has been associated with increased marijuana consumption. This cross-sectional study examined the effects of normative perceptions and self-efficacy on negative marijuana outcomes for a heavy using adolescent population. A structural equation model was tested and supported such that significant indirect paths were present from descriptive norms to marijuana outcomes through self-efficacy. Implications for prevention and intervention with heavy using adolescent marijuana users are discussed.
doi:10.1037/a0024958
PMCID: PMC3342009  PMID: 21842969
marijuana; adolescent substance abuse; social norms; social learning theory

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