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1.  Transcriptional and Epigenetic Dynamics During Specification of Human Embryonic Stem Cells 
Cell  2013;153(5):1149-1163.
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole genome bisulfite sequencing, chromatin immunoprecipitation-sequencing and RNA-sequencing reveals unique events associated with specification towards each lineage. Dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements bound by pluripotency factors or activated in specific lineages. In addition, we identified germ layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches leading to more faithful differentiation strategies as well as provide insights into the rewiring of human regulatory programs during cellular transitions.
PMCID: PMC3709577  PMID: 23664763
2.  Impact of sequencing depth in ChIP-seq experiments 
Nucleic Acids Research  2014;42(9):e74.
In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.
PMCID: PMC4027199  PMID: 24598259
3.  Genome-wide chromatin state transitions associated with developmental and environmental cues 
Cell  2013;152(3):642-654.
Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence and reprogramming.
PMCID: PMC3563935  PMID: 23333102
4.  Effects of fetal antiepileptic drug exposure 
Meador, K.J. | Baker, G.A. | Browning, N. | Cohen, M.J. | Bromley, R.L. | Clayton-Smith, J. | Kalayjian, L.A. | Kanner, A. | Liporace, J.D. | Pennell, P.B. | Privitera, M. | Loring, D.W. | Labiner, David | Moon, Jennifer | Sherman, Scott | Combs Cantrell, Deborah T. | Silver, Cheryl | Goyal, Monisha | Schoenberg, Mike R. | Pack, Alison | Palmese, Christina | Echo, Joyce | Meador, Kimford J. | Loring, David | Pennell, Page | Drane, Daniel | Moore, Eugene | Denham, Megan | Epstein, Charles | Gess, Jennifer | Helmers, Sandra | Henry, Thomas | Motamedi, Gholam | Flax, Erin | Bromfield, Edward | Boyer, Katrina | Dworetzky, Barbara | Cole, Andrew | Halperin, Lucila | Shavel-Jessop, Sara | Barkley, Gregory | Moir, Barbara | Harden, Cynthia | Tamny-Young, Tara | Lee, Gregory | Cohen, Morris | Penovich, Patricia | Minter, Donna | Moore, Layne | Murdock, Kathryn | Liporace, Joyce | Wilcox, Kathryn | Kanner, Andres | Nelson, Michael N. | Rosenfeld, William | Meyer, Michelle | Clayton-Smith, Jill | Mawer, George | Kini, Usha | Martin, Roy | Privitera, Michael | Bellman, Jennifer | Ficker, David | Baade, Lyle | Liow, Kore | Baker, Gus | Booth, Alison | Bromley, Rebecca | Casswell, Miranda | Barrie, Claire | Ramsay, Eugene | Arena, Patricia | Kalayjian, Laura | Heck, Christianne | Padilla, Sonia | Miller, John | Rosenbaum, Gail | Wilensky, Alan | Constantino, Tawnya | Smith, Julien | Adab, Naghme | Veling-Warnke, Gisela | Sam, Maria | O'Donovan, Cormac | Naylor, Cecile | Nobles, Shelli | Santos, Cesar | Holmes, Gregory L. | Druzin, Maurice | Morrell, Martha | Nelson, Lorene | Finnell, Richard | Yerby, Mark | Adeli, Khosrow | Wells, Peter | Browning, Nancy | Blalock, Temperance | Crawford, Todd | Hendrickson, Linda | Jolles, Bernadette | Kunchai, Meghan Kelly | Loblein, Hayley | Ogunsola, Yinka | Russell, Steve | Winestone, Jamie | Wolff, Mark | Zaia, Phyllis | Zajdowicz, Thad
Neurology  2012;78(16):1207-1214.
To examine outcomes at age 4.5 years and compare to earlier ages in children with fetal antiepileptic drug (AED) exposure.
The NEAD Study is an ongoing prospective observational multicenter study, which enrolled pregnant women with epilepsy on AED monotherapy (1999–2004) to determine if differential long-term neurodevelopmental effects exist across 4 commonly used AEDs (carbamazepine, lamotrigine, phenytoin, or valproate). The primary outcome is IQ at 6 years of age. Planned analyses were conducted using Bayley Scales of Infant Development (BSID at age 2) and Differential Ability Scale (IQ at ages 3 and 4.5).
Multivariate intent-to-treat (n = 310) and completer (n = 209) analyses of age 4.5 IQ revealed significant effects for AED group. IQ for children exposed to valproate was lower than each other AED. Adjusted means (95% confidence intervals) were carbamazepine 106 (102–109), lamotrigine 106 (102–109), phenytoin 105 (102–109), valproate 96 (91–100). IQ was negatively associated with valproate dose, but not other AEDs. Maternal IQ correlated with child IQ for children exposed to the other AEDs, but not valproate. Age 4.5 IQ correlated with age 2 BSID and age 3 IQ. Frequency of marked intellectual impairment diminished with age except for valproate (10% with IQ <70 at 4.5 years). Verbal abilities were impaired for all 4 AED groups compared to nonverbal skills.
Adverse cognitive effects of fetal valproate exposure persist to 4.5 years and are related to performances at earlier ages. Verbal abilities may be impaired by commonly used AEDs. Additional research is needed.
PMCID: PMC3324322  PMID: 22491865
5.  Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells 
Cell  2011;147(7):1628-1639.
Hundreds of Chromatin Regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs co-localize in characteristic patterns at distinct chromatin environments, genes of coherent functions and distal regulatory elements. When comparing between cell types, CRs redistribute to different loci, but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.
PMCID: PMC3312319  PMID: 22196736
6.  Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types 
PLoS Genetics  2011;7(12):e1002389.
DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.
Author Summary
Epigenetic modifications including DNA methylation at the position 5 of the cytosine base provide regulatory information to the genome sequence. The primary target of cytosine methylation in mammals is the CpG dinucleotide. However, previous studies in the mouse and more recent work in humans have highlighted the presence of non-CpG methylation in pluripotent cells. Currently, little is known about the role of this type of DNA methylation. We sought to further characterize non-CpG methylation by employing a comprehensive data set of genome-scale methylation maps across various human cell types. Our analysis reveals that non-CpG methylation varies dramatically between pluripotent cells and is closely linked to CpG methylation. Moreover, we show that depletion of the de novo DNA methyltransferases results in a global reduction of non-CpG methylation levels. Taken together, these findings further advance our understanding of cytosine methylation and describe its distribution among a large number of human cell types.
PMCID: PMC3234221  PMID: 22174693
7.  Systematic analysis of chromatin state dynamics in nine human cell types 
Nature  2011;473(7345):43-49.
Chromatin profiling has emerged as a powerful means for genome annotation and detection of regulatory activity. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell type-specificities, and their functional interactions. Focusing on cell type-specific patterns of promoters and enhancers, we define multi-cell activity profiles for chromatin state, gene expression, regulatory motif enrichment, and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for interpreting genome-wide association studies. Top-scoring disease SNPs are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus proposing a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.
PMCID: PMC3088773  PMID: 21441907
8.  Accelerated repetitive transcranial magnetic stimulation (aTMS) for treatment-resistant depression 
Depression and anxiety  2010;27(10):960-963.
Repetitive transcranial magnetic stimulation (rTMS) has shown safety and efficacy for treatment-resistant depression (TRD) but requires daily treatment over 4–6 weeks. Accelerated TMS, with all treatments delivered over a few days, would have significant advantages in terms of access and patient acceptance.
Open-label accelerated TMS (aTMS), consisting of 15 rTMS sessions administered over 2 days, was tested in 14 depressed patients not responding to at least one antidepressant medication. Effects on depression, anxiety and cognition were assessed the day following treatment, then after 3 and 6 weeks.
No seizure activity was observed, and only one patient had a serious adverse event (increased suicidal ideation). Two patients failed to complete a full course of aTMS treatments, and 36% did not complete all study visits. Depression and anxiety significantly decreased following aTMS treatments, and improvements persisted 3 and 6 weeks later. Response rates immediately following treatment, and at 3 and 6 weeks, were 43%, 36% and 36%, respectively. Remission rates at the same timepoints were 29%, 36% and 29%.
Accelerated TMS demonstrated an excellent safety profile with efficacy comparable to that achieved daily rTMS in other trials. Limitations primarily include open-label treatment and a small sample size.
PMCID: PMC3020591  PMID: 20734360
Depression; Transcranial Magnetic Stimulation; Repetitive; Clinical Trial
9.  Bupropion Decreases Resting Motor Threshold: A Case Report 
Brain stimulation  2009;3(3):177-180.
Bupropion is associated with a dose-related increased seizure risk. This effect could correlate with a change in motor cortex excitability. Transcranial magnetic stimulation (TMS) can assess changes in motor cortical excitability by measuring resting motor threshold (RMT).
RMT was determined before and during two weeks concomitant administration of bupropion at two different doses (150 mg per day and 300 mg per day) in a 41 year old female enrolled in a study of repetitive TMS (rTMS) for the treatment of depression.
RMT was significantly lower when the patient took 300 mg per day of bupropion compared to no bupropion and 150 mg per day of bupropion. When bupropion was reduced to 150 mg, RMT returned to the pre-medication level.
Bupropion 300 mg per day increased cortical excitability as demonstrated by decreased RMT. This finding emphasizes the importance of assessing RMT regularly during rTMS treatment – especially in the context of new or changed doses of medications.
PMCID: PMC2905603  PMID: 20633447
Bupropion; Resting Motor Threshold; repetitive Transcranial Magnetic Stimulation; Cortical Excitability
10.  Enhanced expression of mitochondrial superoxide dismutase leads to prolonged in vivo cell cycle progression and up-regulation of mitochondrial thioredoxin 
Free radical biology & medicine  2010;48(11):1501-1512.
Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6 fold increase in MnSOD activities leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation, and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase of the mitochondrial form of thioredoxin (thioredoxin 2), but not of several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.
PMCID: PMC2945707  PMID: 20188820
MnSOD; partial hepatectomy; mitochondria; thioredoxin 2; liver regeneration; cell cycle progression
11.  Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications 
Nature biotechnology  2010;28(10):1097-1105.
Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.
PMCID: PMC2955169  PMID: 20852635
DNA methylation; Sequencing; Bisulfite
12.  Cerebral Cortex Plasticity After 90 Days of Bed Rest: Data from TMS and fMRI 
Microgravity animal models have demonstrated corticospinal plasticity; however, little is understood of its functional significance. In this pilot study, we explored corticospinal plasticity in a bed rest model. We hypothesized that the lack of weight bearing would induce cortical reorganization correlating with performance.
Four subjects underwent functional MRI (fMRI), transcranial magnetic stimulation (TMS), and functional mobility testing (FMT) before and after 90 d of bed rest. Recruitment curves (RC) were created by measuring motor evoked potentials over a range of TMS intensities with changes in the slope of the RC reflecting changes in corticospinal excitability.
Significant leg RC slope decreases were observed on post-bed rest day 1 (P1) (t(2805) = −4.14, P < 0.0001), P2 (t(2805) = −6.59, P < 0.0001), P3 (t(2805) = −6.15, P < 0.0001), P5 (t(2805) = −7.93, P < 0.0001), P8 (t(2805) = −3.30, P = 0.001), and P12 (t(2805)= −3.33, P = 0.0009), suggesting a group decrease in corticospinal excitability in the immediate post-bed rest period with recovery approaching baseline over the following 2 wk. Significant effects were observed for hand RC slopes only for P2 (t(2916) = 1.97, P = 0.049), P3 (t(2916) = −2.12, P = 0.034), and P12 (t(2916) = −2.19, P = 0.029); no significant effects were observed for days P0 (t(2916) = −1.32, ns), P1 (t(2916) = 1.00, ns), P5 (t(2916) = −0.21, ns), or P8 (t(2916) = −0.27, ns). fMRI showed no change in activation for the hand but an increase in activation post-bed rest for the leg. On an individual basis, a more heterogeneous response was found which showed a potential association with performance on FMT.
Results of this research include a better understanding of the cortical plasticity associated with leg disuse and may lead to applications in patient and astronaut rehabilitation.
PMCID: PMC2861654  PMID: 20058735
functional MRI (fMRI); transcranial magnetic stimulation (TMS); motor; performance
13.  Mice Deficient in Both Mn Superoxide Dismutase and Glutathione Peroxidase-1 Have Increased Oxidative Damage and a Greater Incidence of Pathology but No Reduction in Longevity 
To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1−/−, Sod2+/−Gpx1+/−, Sod2+/−Gpx1−/−, and wild-type control mice. Oxidative damage was elevated in Sod2+/−Gpx1−/− mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2+/−Gpx1−/− mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.
PMCID: PMC2781787  PMID: 19776219
Oxidative stress; Longevity
14.  Overexpression of Mn Superoxide Dismutase Does Not Increase Life Span in Mice 
Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4–6 months) and old (26–28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.
PMCID: PMC2759571  PMID: 19633237
Oxidative damage; Mn superoxide dismutase; Pathology; Aging
15.  Accuracy and Precision of MR Blood Oximetry Based On the Long Paramagnetic Cylinder Approximation of Large Vessels 
An accurate non-invasive method to measure hemoglobin oxygen saturation (%HbO2) of deep-lying vessels without catheterization would have many clinical applications. Quantitative MRI may be the only imaging modality that can address this difficult and important problem. MR susceptometry-based oximetry for measuring blood oxygen saturation in large vessels models the vessel as a long paramagnetic cylinder immersed in an external field. The intravascular magnetic susceptibility relative to surrounding muscle tissue is a function of HbO2 and can be quantified with a field mapping pulse sequence. In this work, the method’s accuracy and precision was investigated theoretically on the basis of an analytical expression for the arbitrarily oriented cylinder, as well as experimentally in phantoms and in vivo in the femoral artery and vein at 3T field strength. Errors resulting from vessel tilt, non-circularity of vessel cross-section, and induced magnetic field gradients were evaluated and methods for correction designed and implemented. Hemoglobin saturation was measured at successive vessel segments, differing in geometry such as eccentricity and vessel tilt but constant blood oxygen saturation levels, as a means to evaluate measurement consistency. The average standard error and coefficient of variation of measurements in phantoms were less than 2% with tilt correction alone, in agreement with theory, suggesting that high accuracy and reproducibility can be achieved while ignoring non-circularity for tilt angles up to about 30°. In vivo, repeated measurements of %HbO2 in the femoral vessels yielded a coefficient of variation of less than 5%. In conclusion, the data suggest that %HbO2 can be measured reproducibly in vivo in large vessels of the peripheral circulation on the basis of the paramagnetic cylinder approximation of the incremental field.
PMCID: PMC2753239  PMID: 19526517
16.  PTK6 Regulates IGF-1-Induced Anchorage-Independent Survival 
PLoS ONE  2010;5(7):e11729.
Proteins that are required for anchorage-independent survival of tumor cells represent attractive targets for therapeutic intervention since this property is believed to be critical for survival of tumor cells displaced from their natural niches. Anchorage-independent survival is induced by growth factor receptor hyperactivation in many cell types. We aimed to identify molecules that critically regulate IGF-1-induced anchorage-independent survival.
Methods and Results
We conducted a high-throughput siRNA screen and identified PTK6 as a critical component of IGF-1 receptor (IGF-1R)-induced anchorage-independent survival of mammary epithelial cells. PTK6 downregulation induces apoptosis of breast and ovarian cancer cells deprived of matrix attachment, whereas its overexpression enhances survival. Reverse-phase protein arrays and subsequent analyses revealed that PTK6 forms a complex with IGF-1R and the adaptor protein IRS-1, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation. PTK6 is highly expressed not only in the previously reported Her2+ breast cancer subtype, but also in high grade ER+, Luminal B tumors and high expression is associated with adverse outcomes.
These findings highlight PTK6 as a critical regulator of anchorage-independent survival of breast and ovarian tumor cells via modulation of IGF-1 receptor signaling, thus supporting PTK6 as a potential therapeutic target for multiple tumor types. The combined genomic and proteomic approaches in this report provide an effective strategy for identifying oncogenes and their mechanism of action.
PMCID: PMC2909213  PMID: 20668531
17.  Genes contributing to prion pathogenesis 
The Journal of general virology  2008;89(Pt 7):1777-1788.
Prion diseases are caused by conversion of a normally folded, nonpathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPC molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analyzed prion incubation times in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 gene candidates, because their products either colocalize with PrP, are associated with Alzheimer’s disease, are elevated during prion disease, or function in PrP-mediated signaling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernable effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of amyloid beta (A4) precursor protein (App) or interleukin 1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13%, 16%, and 19%, respectively.
PMCID: PMC2828448  PMID: 18559949
18.  A streamlined sub-cloning procedure to transfer shRNA from a pSM2 vector to a pGIPZ lentiviral vector 
RNA interference (RNAi) is a widely used molecular biology technique to investigate the importance of specific genes in molecular pathways. Since mammalian cells are equipped with endogenous RNAi processing machinery, it has become common practice to transfect constructs that encode for short hairpin RNAs that are then cleaved to form the active RNAi sequences that bind to target mRNAs. Given the profit potential of this research approach, companies have developed retroviral libraries of shRNA constructs targeting the majority of the human genes. Recent technologic advances have allowed the rapid improvement of the vectors carrying the shRNA constructs while the silencing sequences remain the same. Therefore, sub-cloning of shRNA sequences from more obsolete vectors to newer vectors is a straightforward way to take advantage of newer delivery technologies. We describe here a streamlined procedure to transfer shRNA sequences from the pSM2 retroviral vector to a newer pGIPZ vector that is more stable, contains a GFP cassette and allows the preparation of high titer viral particles for transduction of cells and in vivo use. We demonstrate that our protocol provides a cost-effective and fast method to successfully sub-clone shRNA from a pSM2 retroviral vector to a pGIPZ lentiviral vector making it a useful tool for the investigators that have purchased pSM2 vectors in the past and wish now to upgrade their constructs by inserting them in more versatile vectors.
PMCID: PMC2995545  PMID: 21139985
Sub-cloning; shRNA; RNA interference; lentivirus; retrovirus; pSM2; pGIPZ; pTRIPZ
19.  The in vivo Gene Expression Signature of Oxidative Stress 
Physiological genomics  2008;34(1):112-126.
How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes, CuZn-Superoxide Dismutase (Sod1) and Glutathione Peroxidase-1 (Gpx1).
Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3 to 6 hours in wild type mice without any lethality. In contrast, treating Sod1−/− or Gpx1−/− mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classical antioxidant genes, though long-term oxidative stress in the Sod1−/− mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor, Nrf2. The main finding of our study is that the common response to elevated oxidative stress, with diquat treatment in wild type, Gpx1−/−, Sod1−/− mice and in untreated Sod1−/− mice, is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53-target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.
PMCID: PMC2532791  PMID: 18445702
Oxidative Stress; Gene Expression; p53-target genes; Sod1; Gpx1
20.  The overexpression of major antioxidant enzymes does not extend the lifespan of mice 
Aging Cell  2008;8(1):73-75.
We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.
PMCID: PMC2667893  PMID: 19077044
aging; antioxidant enzymes; transgenic and knockout mice
21.  An Open Study of Repetitive Transcranial Magnetic Stimulation in Treatment-Resistant Depression with Parkinson’s Disease 
Major depression is a common concomitant of chronic central nervous system disorders, notably Parkinson’s disease (PD). Repetitive transcranial magnetic stimulation (rTMS) has been investigated as a potential treatment for depression in PD and for the movement disorder of PD, but comprehensive testing in multiple areas of performance has seldom been carried out within a single study. We studied the effect of left dorsolateral prefrontal rTMS on several different functional domains.
Fourteen PD patients with treatment-resistant depression entered an open, 10-day inpatient study of 10-Hertz rTMS, undergoing extensive psychiatric, neuropsychological, and motor testing from baseline to 6 weeks after treatment. Motor testing included a defined “off” state.
rTMS was well-tolerated. Highly significant improvement in depression scores was seen three days and 3-6 weeks after treatment. Improvement was also found in anxiety, movement scores (especially in the off state), and some neuropsychological measures. We found no evidence of increased risk from rTMS in this population.
Further controlled trials of rTMS in PD appear worthwhile, and should include a defined “off” state.
TMS may be beneficial for depressed PD patients in multiple functional domains.
PMCID: PMC2121115  PMID: 17714987
Transcranial magnetic stimulation; depression; Parkinson disease
22.  SIT4 Regulation of Mig1p-Mediated Catabolite Repression in Saccharomyces cerevisiae* 
FEBS letters  2007;581(29):5658-5663.
E153 is a respiratory deficient mutant of Saccharomyces cerevisiae with a mutation in the active site of the Sit4p protein phosphatase. Measurements of mitochondrial respiration and cytochromes indicate that the mutation suppresses glucose repression. The escape from catabolite repression is accompanied by a marked reduction of the transcriptional repressor Mig1p. The presence of normal levels of MIG1 mRNA in the mutant and its association with the polysome fraction suggests that depletion of Mig1p is the result of protein degradation. This study shows that in addition to phosphorylation by Snf1p, the transcriptional repressor activity of Mig1p is also regulated by a post-transcriptional Sit4p-dependent pathway. Our evidence suggests that this pathway involves turnover of Mig1p.
PMCID: PMC2247712  PMID: 18022394
Saccharomyces cerevisiae; SIT4; MIG1; catabolite repression; mitochondria; respiration
23.  A Novel Technique for Imaging with Inhomogeneous Fields 
We introduce a simple, efficient, low-SAR method for magnetic resonance imaging in the presence of a static field with a permanent, and possibly large gradient. The technique, which is called slant-slice imaging is essentially a spin-echo imaging sequence except that the imaging slice is oriented such that the static field gradient can be used in conjunction with applied gradients during readout. Data is collected for two dimensional slices. Unlike single point imaging techniques, entire lines of k-space are acquired with each readout. The slant-slice pulse sequence is used to obtain high quality images, using a clinical scanner to simulate a static field with a large permanent gradient. The effects of the inhomogeneity are quantified by two parameters ν and q, which are useful for assessing the utility of a magnet design for 3D-MR imaging.
PMCID: PMC1847800  PMID: 16962800
nuclear magnetic resonance; imaging; inhomogeneous fields; slant-slice imaging; SNR; SAR
24.  Combination rapid transcranial magnetic stimulation in treatment refractory depression 
High frequency (>1 Hz) repetitive transcranial magnetic stimulation (rTMS) applied to the left prefrontal cortex and low frequency (≤1 Hz) rTMS applied to the right prefrontal cortex have shown antidepressant effects. However, the clinical significance of these effects has often been modest. It was hypothesized that a combination of these two techniques might act synergistically and result in more clinically relevant antidepressant effects. Sixty-two subjects with treatment-resistant major depression (an average of 8 failed medication trials) were randomized to receive combination right low frequency (1 Hz)/left high frequency (10 Hz) rTMS over the dorsolateral prefrontal cortex at 110% of the motor threshold vs sham rTMS. Subjects were treated for 2 weeks (10 weekday sessions) and received 1600 stimulations during each treatment session. Subjects receiving combination treatment were further randomized to receive different orders of treatment: right low frequency first (Slow Right) vs left high frequency first (Fast Left). There were no statistical differences in the active vs sham treatment arms in the primary outcome variable, the Hamilton Depression Rating Scale (HDRS). However compared with subjects in the Sham and Slow Right arms, there was a trend for subjects in the Fast Left arm to show improvement in the HDRS, the Beck Depression Inventory, and the Brief Psychotic Rating Scale with increased number of treatments. The Fast Left arm also showed significant improvement in both blinded clinician and self-ratings of global improvement. These differences were hypothesized to be due to the decreased number of failed medication trials for subjects in Fast Left arm. Neuropsychological performance was not significantly different between the sham and active rTMS arms. Future studies should increase the number of treatment sessions and focus on subjects with moderate treatment resistance.
PMCID: PMC2671728  PMID: 19412449
transcranial magnetic stimulation; treatment resistant depression
25.  Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study 
BMC Genomics  2004;5:26.
Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip® technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.
The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.
This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.
PMCID: PMC415544  PMID: 15113399

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