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1.  Excitatory/inhibitory synaptic imbalance leads to hippocampal hyperexcitability in mouse models of Tuberous Sclerosis 
Neuron  2013;78(3):510-522.
Neural circuits are regulated by activity-dependent feedback systems that tightly control network excitability and which are thought to be crucial for proper brain development. Defects in the ability to establish and maintain network homeostasis may be central to the pathogenesis of neurodevelopmental disorders. Here we examine the function of the Tuberous Sclerosis Complex (TSC)-mTOR signaling pathway, a common target of mutations associated with epilepsy and autism spectrum disorder, in regulating activity-dependent processes in the mouse hippocampus. We find that TSC/mTOR is a central component of a positive feedback loop that promotes network activity by repressing inhibitory synapses onto excitatory neurons. In Tsc1 KO neurons, weakened inhibition caused by deregulated mTOR alters the balance of excitatory and inhibitory synaptic transmission leading to hippocampal hyperexcitability. These findings identify the TSC/mTOR pathway as a novel regulator of neural network activity and have implications for the neurological dysfunction in disorders exhibiting deregulated mTOR signaling.
PMCID: PMC3690324  PMID: 23664616
2.  The Triform algorithm: improved sensitivity and specificity in ChIP-Seq peak finding 
BMC Bioinformatics  2012;13:176.
Chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-Seq) is the most frequently used method to identify the binding sites of transcription factors. Active binding sites can be seen as peaks in enrichment profiles when the sequencing reads are mapped to a reference genome. However, the profiles are normally noisy, making it challenging to identify all significantly enriched regions in a reliable way and with an acceptable false discovery rate.
We present the Triform algorithm, an improved approach to automatic peak finding in ChIP-Seq enrichment profiles for transcription factors. The method uses model-free statistics to identify peak-like distributions of sequencing reads, taking advantage of improved peak definition in combination with known characteristics of ChIP-Seq data.
Triform outperforms several existing methods in the identification of representative peak profiles in curated benchmark data sets. We also show that Triform in many cases is able to identify peaks that are more consistent with biological function, compared with other methods. Finally, we show that Triform can be used to generate novel information on transcription factor binding in repeat regions, which represents a particular challenge in many ChIP-Seq experiments. The Triform algorithm has been implemented in R, and is available via
PMCID: PMC3480842  PMID: 22827163
ChIP-Seq; Peak finding; Benchmark; Repeats
3.  Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of Retinitis Pigmentosa 
Nature Neuroscience  2008;12(1):44-52.
Retinitis Pigmentosa (RP) is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. While most mutations that cause RP are in rod photoreceptor (PR) -specific genes, cone PRs die as well. To understand the mechanism of non-autonomous cone death, four mouse models harboring mutations in rod-specific genes were analyzed. Changes in the insulin/mTOR pathway that coincided with the activation of autophagy during the period of cone death were found. We thus either increased or decreased the insulin level and measured the survival of cones in one of the models. Mice treated systemically with insulin had prolonged cone survival, while depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in RP could, at least in part, be due to the starvation of cones.
PMCID: PMC3339764  PMID: 19060896
4.  RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug 
BMC Genomics  2012;13:6.
Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.
We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.
We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.
PMCID: PMC3273426  PMID: 22226239
5.  Age-Associated Disruption of Molecular Clock Expression in Skeletal Muscle of the Spontaneously Hypertensive Rat 
PLoS ONE  2011;6(11):e27168.
It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.
PMCID: PMC3208587  PMID: 22076133
6.  JTK_CYCLE: an efficient non-parametric algorithm for detecting rhythmic components in genome-scale datasets 
Journal of biological rhythms  2010;25(5):372-380.
Circadian rhythms are oscillations of physiology, behavior, and metabolism that have period lengths of 24 hours. In several model organisms and man, circadian clock genes have been characterized and found to be transcription factors. Because of this, researchers have used microarrays to characterize global regulation of gene expression and algorithmic approaches to detect cycling. Here we present a new algorithm, JTK_CYCLE, designed to efficiently identify and characterize cycling variables in large datasets. Compared to COSOPT and the Fisher’s G test, two commonly used methods for detecting cycling transcripts, JTK_CYCLE distinguishes between rhythmic and non-rhythmic transcripts more reliably and efficiently. We also show that JTK_CYCLE’s increased resistance to outliers results in considerably greater sensitivity and specificity. Moreover, JTK_CYCLE accurately measures the period, phase, and amplitude of cycling transcripts, facilitating downstream analyses. Finally, it is several orders of magnitude faster than COSOPT, making it ideal for large scale data sets. We used JTK_CYCLE to analyze legacy data sets including NIH3T3 cells, which have comparatively low amplitude. JTK_CYCLE’s improved power led to the identification of a novel cluster of RNA-interacting genes whose abundance is under clear circadian regulation. These data suggest that JTK_CYCLE is an ideal tool for identifying and characterizing oscillations in genome-scale datasets.
PMCID: PMC3119870  PMID: 20876817
Circadian Rhythms; Biological Oscillations; Statistical Methods; Systems Biology; Genomics; Microarrays
7.  Accurate Expression Profiling of Very Small Cell Populations 
PLoS ONE  2010;5(12):e14418.
Expression profiling, the measurement of all transcripts of a cell or tissue type, is currently the most comprehensive method to describe their physiological states. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from nanograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts.
Methods and Findings
We provide evidence that currently available methods for expression profiling of very small cell populations are prone to technical noise and therefore cannot be used efficiently as discovery tools. Furthermore, we present Pico Profiling, a new expression profiling method from as few as ten cells, and we show that this approach is as informative as standard techniques from thousands to millions of cells. The central component of Pico Profiling is Whole Transcriptome Amplification (WTA), which generates expression profiles that are highly comparable to those produced by others, at different times, by standard protocols or by Real-time PCR. We provide a complete workflow from RNA isolation to analysis of expression profiles.
Pico Profiling, as presented here, allows generating an accurate expression profile from cell populations as small as ten cells.
PMCID: PMC3010985  PMID: 21203435
8.  Synergistic Function of E2F7 and E2F8 is Essential for Cell Survival and Embryonic Development 
Developmental cell  2008;14(1):62-75.
The novel E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and hetero-dimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.
PMCID: PMC2253677  PMID: 18194653
E2Fs; embryo development; apoptosis; cell cycle; transcriptional regulation
9.  Big Results from Small Samples: Evaluation of Amplification Protocols for Gene Expression Profiling 
Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.
PMCID: PMC2062549  PMID: 17595311
microarray; gene expression; RNA amplification; small sample; GeneChip; quantitative PCR
10.  Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays 
BMC Genomics  2007;8:111.
Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.
We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.
A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.
PMCID: PMC1868757  PMID: 17470268
11.  Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform 
Nucleic Acids Research  2006;34(3):e17.
DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.
PMCID: PMC1361623  PMID: 16464820

Results 1-11 (11)