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1.  A Nonparametric Statistical Method That Improves Physician Cost of Care Analysis 
Health Services Research  2012;47(6):2398-2417.
Objective
To develop a compositing method that demonstrates improved performance compared with commonly used tests for statistical analysis of physician cost of care data.
Data Source
Commercial preferred provider organization (PPO) claims data for internists from a large metropolitan area.
Study Design
We created a nonparametric composite performance metric that maintains risk adjustment using the Wilcoxon rank-sum (WRS) test. We compared the resulting algorithm to the parametric observed-to-expected ratio, with and without a statistical test, for stability of physician cost ratings among different outlier trimming methods and across two partially overlapping time periods.
Principal Findings
The WRS algorithm showed significantly greater within-physician stability among several typical outlier trimming and capping methods. The algorithm also showed significantly greater within-physician stability when the same physicians were analyzed across time periods.
Conclusions
The nonparametric algorithm described is a more robust and more stable methodology for evaluating physician cost of care than commonly used observed-to-expected ratio techniques. Use of such an algorithm can improve physician cost assessment for important current applications such as public reporting, pay for performance, and tiered benefit design.
doi:10.1111/j.1475-6773.2012.01415.x
PMCID: PMC3523381  PMID: 22524195
Statistical methods; physician profiling; nonparametric statistics; cost-efficiency; efficiency index
2.  Developmental Profiles of the Murine Palatal Methylome 
BACKGROUND
Environmental factors contribute to the etiology of cleft palate (CP). Identification of genes that are methylated during development of the secondary palate will contribute to a better understanding of the gene-environment link contributing to CP.
METHODS
Genomic DNA fragments from secondary palate tissue from gestational days (GDs) 12 to 14 were subjected to Selective Enrichment of Methylated DNA (SEMD) and used to probe NimbleGen 2.1M mouse promoter arrays. Input (control) and SEMD samples were labeled with Cy3 and Cy5, respectively, and used for array hybridization (three arrays per GD). Data were analyzed using the Bioconductor package Ringo. Gene methylation was verified by pyrosequencing analysis and expression by quantitative real-time PCR.
RESULTS
A total of 5577 methylated genes were identified during palate development: (1) 74% of genes were methylated on all three GDs; (2) CpG islands accounted for only 30% of methylated regions of interest (MRIs); (3) location of MRIs was more often observed in gene bodies (73%) than in promoters; (4) evaluation of MRIs on GDs 12–14 revealed no significant differentially methylated regions; (5) DAVID analysis of MRIs revealed that the cadherin and Wnt signaling pathways, as well as pathways involved in proteoglycan synthesis, were significantly enriched for methylated genes.
CONCLUSIONS
Our prior studies identified differentially expressed mRNAs and micro-RNAs in the developing palate. The current study complements these studies by identifying genes whose expression may be altered as a result of DNA methylation.
doi:10.1002/bdra.23126
PMCID: PMC3776452  PMID: 23554260
cleft palate; CpG methylation; craniofacial; epigenetics; extra-cellular matrix; promoter microarray; pyrosequencing; secondary palate; Wnt signaling
3.  Epigenetic regulation of Sox4 during palate development 
Epigenomics  2013;5(2):131-146.
Aim
Identification of genes that contribute to secondary palate development provide a better understanding of the etiology of palatal clefts. Gene-expression profiling of the murine palate from gestational days 12–14 (GD12–14), a critical period in palate development, identified Sox4 as a differentially expressed gene. In this study, we have examined if the differential expression of Sox4 in the palate is due to changes in DNA methylation.
Materials & methods
In situ hybridization analysis was used to localize the expression of Sox4 in the developing murine secondary palate. CpG methylation profiling of a 1.8-kb upstream region of Sox4 in the secondary palate from GD12–14 and transfection analysis in murine embryonic maxillary mesenchymal cells using Sox4 deletion, mutant and in vitro methylated plasmid constructs were used to identify critical CpG residues regulating Sox4 expression in the palate.
Results
Spatiotemporal analysis revealed that Sox4 is expressed in the medial edge epithelium and presumptive rugae-forming regions of the palate from GD12 to GD13. Following palatal shelf fusion on GD14, Sox4 was expressed exclusively in the epithelia of the palatal rugae, structures that serve as signaling centers for the anteroposterior extension of the palate, and that are thought to serve as neural stem cell niches. Methylation of a 1.8-kb region upstream of Sox4, containing the putative promoter, completely eliminated promoter activity. CpG methylation profiling of the 1.8-kb region identified a CpG-poor region (DMR4) that exhibited significant differential methylation during palate development, consistent with changes in Sox4 mRNA expression. Changes in the methylation of DMR4 were attributed primarily to CpGs 83 and 85.
Conclusion
Our studies indicate that Sox4 is an epigenetically regulated gene that likely integrates multiple signaling systems for mediating palatal fusion, palatal extension and/or the maintenance of the neural stem cell niche in the rugae.
doi:10.2217/epi.13.1
PMCID: PMC3776497  PMID: 23566091
cleft palate; CpG methylation; epigenetics; medial edge epithelium; orofacial; rugae; secondary palate; Sox4
4.  Gene expression changes in the secondary palate and mandible of Prdm16−/− mice 
Cell and tissue research  2012;351(3):445-452.
Loss of Prdm16 expression in the mouse leads to a complete cleft of the secondary palate. In the current report, changes in gene expression in the secondary palates of Prdm16−/− fetuses were determined in an attempt to reveal the mechanism(s) leading to failure of palate closure in these mice. Defined, pathway-based, PCR arrays were conducted to analyze the expression of genes associated with the extracellular matrix and the TGF-β and BMP signaling networks, perturbations of which can lead to palatal clefting. Loss of Prdm16 expression in the secondary palate leads to alterations in numerous genes within these groups, many of which have been linked to chondrogenesis and osteogenesis. The expression of several genes linked to bone development was significantly changed in the developing secondary palate. Analysis of gene expression in the mandibles of Prdm16−/− fetuses revealed similar alterations in the same gene set. These data suggest that one function of Prdm16 is the regulation of genes that play a role in differentiation of mesenchymal cells into chondro/osteocytes.
doi:10.1007/s00441-012-1525-2
PMCID: PMC3584240  PMID: 23149718
craniofacia; mandible; palate; chondrogenesis; osteogenesis
5.  Postreactivation Glucocorticoids Impair Recall of Established Fear Memory 
Pavlovian fear conditioning provides one of the best rodent models of acquired anxiety disorders, including posttraumatic stress disorder. Injection of a variety of drugs after training in fear-conditioning paradigms can impair consolidation of fear memories. Indeed, early clinical trials suggest that immediate administration of such drugs after a traumatic event may decrease the risk of developing posttraumatic stress disorder in humans (Pitman et al., 2002; Vaiva et al., 2003). The use of such a treatment is limited by the difficulty of treating every patient at risk and by the difficulty in predicting which patients will experience chronic adverse consequences. Recent clinical trials suggest that administration of glucocorticoids may have a beneficial effect on established posttraumatic stress disorder (Aerni et al., 2004) and specific phobia (Soravia et al., 2006). Conversely, glucocorticoid administration after training is known to enhance memory consolidation (McGaugh and Roozendaal, 2002; Roozendaal, 2002). From a clinical perspective, enhancement of a fear memory or a reactivated fear memory would not be desirable. We report here that when glucocorticoids are administered immediately after reactivation of a contextual fear memory, subsequent recall is significantly diminished. Additional experiments support the interpretation that glucocorticoids not only decrease fear memory retrieval but, in addition, augment consolidation of fear memory extinction rather than decreasing reconsolidation. These findings provide a rodent model for a potential treatment of established acquired anxiety disorders in humans, as suggested by others (Aerni et al., 2004; Schelling et al., 2004), based on a mechanism of enhanced extinction.
doi:10.1523/JNEUROSCI.2397-06.2006
PMCID: PMC3917138  PMID: 16971540
learning; memory; reconsolidation; consolidation; fear conditioning; recall; extinction; glucocorticoid
7.  Neurobehavioral phenotype of C57BL/6J mice prenatally and neonatally exposed to cigarette smoke 
Although maternal cigarette smoking during pregnancy is a well-documented risk factor for a variety of adverse pregnancy outcomes, how prenatal cigarette smoke exposure affects postnatal neurobehavioral/cognitive development remains poorly defined. In order to investigate the cause of an altered behavioral phenotype, mice developmentally exposed to a paradigm of ‘active’ maternal cigarette smoke is needed. Accordingly, cigarette smoke exposed (CSE) and air-exposed C57BL/6J mice were treated for 6 h per day in paired inhalation chambers throughout gestation and lactation and were tested for neurobehavioral effects while controlling for litter effects. CSE mice exhibited less than normal anxiety in the elevated zero maze, transient hypoactivity during a 1 h locomotor activity test, had longer latencies on the last day of cued Morris water maze testing, impaired hidden platform learning in the Morris water maze during acquisition, reversal, and shift trials, and impaired retention for platform location on probe trials after reversal but not after acquisition or shift. CSE mice also showed a sexually dimorphic response in central zone locomotion to a methamphetamine challenge (males under-responded and females over-responded), and showed reduced anxiety in the light-dark test by spending more time on the light side. No differences on tests of marble burying, acoustic startle response with prepulse inhibition, Cincinnati water maze, matching-to-sample Morris water maze, conditioned fear, forced swim, or MK-801-induced locomotor activation were found. Collectively, the data indicate that developmental cigarette smoke exposure induces subnormal anxiety in a novel environment, impairs spatial learning and reference memory while sparing other behaviors (route-based learning, fear conditioning, and forced swim immobility). The findings add support to mounting evidence that developmental cigarette smoke exposure has long-term adverse effects on brain function.
doi:10.1016/j.ntt.2013.01.001
PMCID: PMC3593942  PMID: 23314114
behavior; cigarette smoke; tobacco; inhalation exposure; pregnancy; prenatal
8.  Developmental Epigenetics of the Murine Secondary Palate 
ILAR Journal  2012;53(3-4):240-252.
Orofacial clefts occur with a frequency of 1 to 2 per 1000 live births. Cleft palate, which accounts for 30% of orofacial clefts, is caused by the failure of the secondary palatal processes—medially directed, oral projections of the paired embryonic maxillary processes—to fuse. Both gene mutations and environmental effects contribute to the complex etiology of this disorder. Although much progress has been made in identifying genes whose mutations are associated with cleft palate, little is known about the mechanisms by which the environment adversely influences gene expression during secondary palate development. An increasing body of evidence, however, implicates epigenetic processes as playing a role in adversely influencing orofacial development. Epigenetics refers to inherited changes in phenotype or gene expression caused by processes other than changes in the underlying DNA sequence. Such processes include, but are not limited to, DNA methylation, microRNA effects, and histone modifications that alter chromatin conformation. In this review, we describe our current understanding of the possible role epigenetics may play during development of the secondary palate. Specifically, we present the salient features of the embryonic palatal methylome and profile the expression of numerous microRNAs that regulate protein-encoding genes crucial to normal orofacial ontogeny.
doi:10.1093/ilar.53.3-4.240
PMCID: PMC4021823  PMID: 23744964
cleft palate; CpG islands; DMR; methylation promoter arrays; methylome; microRNAs; secondary palate
9.  Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis 
Human Molecular Genetics  2012;21(13):2899-2911.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation–prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation–prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation–prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.
doi:10.1093/hmg/dds116
PMCID: PMC3373238  PMID: 22454397
10.  Behavioral characterization of P311 knockout mice 
Genes, brain, and behavior  2008;7(7):786-795.
P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses.
doi:10.1111/j.1601-183X.2008.00420.x
PMCID: PMC3632358  PMID: 18616608
Behavior; emotionality; learning; memory; mutant mice; P311
11.  Adenosine Inhibition of Mesopontine Cholinergic Neurons: Implications for EEG Arousal 
Science (New York, N.Y.)  1994;263(5147):689-692.
Increased discharge activity of mesopontine cholinergic neurons participates in the production of electroencephalographic (EEG) arousal; such arousal diminishes as a function of the duration of prior wakefulness or of brain hyperthermia. Whole-cell and extracellular recordings in a brainstem slice show that mesopontine cholinergic neurons are under the tonic inhibitory control of endogenous adenosine, a neuromodulator released during brain metabolism. This inhibitory tone is mediated postsynaptically by an inwardly rectifying potassium conductance and by an inhibition of the hyperpolarization-activated current. These data provide a coupling mechanism linking neuronal control of EEG-arousal with the effects of prior wakefulness, brain hyperthermia, and the use of the adenosine receptor blockers caffeine and theophylline.
PMCID: PMC3612520  PMID: 8303279
12.  The Geometry of Locomotive Behavioral States in C. elegans 
PLoS ONE  2013;8(3):e59865.
We develop a new hidden Markov model-based method to analyze C elegans locomotive behavior and use this method to quantitatively characterize behavioral states. In agreement with previous work, we find states corresponding to roaming, dwelling, and quiescence. However, we also find evidence for a continuum of intermediate states. We suggest that roaming, dwelling, and quiescence may best be thought of as extremes which, mixed in any proportion, define the locomotive repertoire of C elegans foraging and feeding behavior.
doi:10.1371/journal.pone.0059865
PMCID: PMC3610905  PMID: 23555813
13.  Adenosine: A Mediator of the Sleep-Inducing Effects of Prolonged Wakefulness 
Science (New York, N.Y.)  1997;276(5316):1265-1268.
Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region.
PMCID: PMC3599777  PMID: 9157887
14.  STRAIN-SPECIFIC MODIFIER GENES GOVERNING CRANIOFACIAL PHENOTYPES 
BACKGROUND
The presence of strain-specific modifier genes is known to modulate the phenotype and pathophysiology of mice harboring genetically engineered mutations. Thus, identification of genetic modifier genes is requisite to understanding control of phenotypic expression. c-Ski is a transcriptional regulator. Ski−/− mice on a C57BL6J (B6) background exhibit facial clefting, while Ski−/− mice on a 129P3 (129) background present with exencephaly.
METHODS
In the present study, oligonucleotide-based gene expression profiling was utilized to identify potential strain-specific modifier gene candidates present in wild-type mice of B6 and 129 genetic backgrounds. Changes in gene expression were verified by TaqMan quantitative real-time PCR.
RESULTS
Steady-state levels of 89 genes demonstrated a significantly higher level of expression, and those of 68 genes demonstrated a significantly lower level of expression in the developing neural tubes from E8.5, B6 embryos when compared to expression levels in neural tubes derived from E8.5, 129 embryos.
CONCLUSIONS
Based on the results from the current comparative microarray study, and taking into consideration a number of relevant published reports, several potential strain-specific gene candidates, likely to modify the craniofacial phenotypes in various knockout mouse models have been identified.
doi:10.1002/bdra.22890
PMCID: PMC3302963  PMID: 22371338
Neural tube; embryo; mouse; modifier gene; microarray
15.  MmPalateMiRNA, an R package compendium illustrating analysis of miRNA microarray data 
Background
MicroRNAs (miRNAs) constitute the largest family of noncoding RNAs involved in gene silencing and represent critical regulators of cell and tissue differentiation. Microarray expression profiling of miRNAs is an effective means of acquiring genome-level information of miRNA activation and inhibition, as well as the potential regulatory role that these genes play within a biological system. As with mRNA expression profiling arrays, miRNA microarrays come in a variety of platforms from numerous manufacturers, and there are a multitude of techniques available for reducing and analyzing these data.
Results
In this paper, we present an analysis of a typical two-color miRNA microarray experiment using publicly available packages from R and Bioconductor, the open-source software project for the analysis of genomic data. Covered topics include visualization, normalization, quality checking, differential expression, cluster analysis, miRNA target identification, and gene set enrichment analysis. Many of these tools carry-over from the analysis of mRNA microarrays, but with some notable differences that require special attention. The paper is presented as a “compendium” which, along with the accompanying R package MmPalateMiRNA, contains all of the experimental data and source code to reproduce the analyses contained in the paper.
Conclusions
The compendium presented in this paper will provide investigators with an access point for applying the methods available in R and Bioconductor for analysis of their own miRNA array data.
doi:10.1186/1751-0473-8-1
PMCID: PMC3654997  PMID: 23298515
16.  Chemokine-Mediated Migration of Mesencephalic Neural Crest Cells 
Cytokine  2011;56(3):760-768.
Clefts of the lip and/or palate are among the most prevalent birth defects affecting approximately 7000 newborns in the United States annually. Disruption of the developmentally programmed migration of neural crest cells (NCCs) into the orofacial region is thought to be one of the major causes of orofacial clefting. Signaling of the chemokine SDF-1 (Stromal Derived Factor-1) through its specific receptor, CXCR4, is required for the migration of many stem cell and progenitor cell populations from their respective sites of emergence to the regions where they differentiate into complex cell types, tissues and organs. In the present study, “transwell” assays of chick embryo mesencephalic (cranial) NCC migration and ex ovo whole embryo “bead implantation” assays were utilized to determine whether SDF-1/CXCR4 signaling mediates mesencephalic NCC migration. Results from this study demonstrate that attenuation of SDF-1 signaling, through the use of specific CXCR4 antagonists (AMD3100 and TN14003), disrupts the migration of mesencephalic NCCs into the orofacial region, suggesting a novel role for SDF-1/CXCR4 signaling in the directed migration of mesencephalic NCCs in the early stage embryo.
doi:10.1016/j.cyto.2011.09.014
PMCID: PMC3221884  PMID: 22015108
chemokine; chick embryo; cranial neural crest; CXCR4; orofacial development; SDF-1
17.  CNS Dopamine Transmission Mediated by Noradrenergic Innervation 
The Journal of Neuroscience  2012;32(18):6072-6080.
The pre-synaptic source of dopamine in the CA1 field of dorsal hippocampus is uncertain due to an anatomical mismatch between dopaminergic terminals and receptors. We show, in an in vitro slice preparation from C57BL6 male mice, that a dopamine (DA) D1 receptor (D1R) mediated enhancement in glutamate synaptic transmission occurs following release of endogenous DA with amphetamine exposure. It is assumed DA is released from terminals innervating from the ventral tegmental area (VTA) even though DA transporter (DAT) positive fibers are absent in hippocampus, a region with abundant D1Rs. It has been suggested this results from a lack of DAT expression on VTA terminals rather than a lack of these terminals per se. Neither a knockdown of tyrosine hydroxylase (TH) expression in the VTA by THsiRNA, delivered locally, by adeno-associated viral vector, nor localized pharmacological blockade of DAT to prevent amphetamine uptake into DA terminals, has any effect on the D1R synaptic, enhancement response to amphetamine. However, either a decrease in TH expression in the locus coeruleus (LC) or a blockade of the norepinephrine (NE) transporter prevents the DA mediated response, indicating LC terminals can release both NE and DA. These findings suggest noradrenergic fibers may be the primary source of DA release in hippocampus and corresponding DA mediated increase in synaptic transmission. Accordingly, these data imply the LC may have a role in DA transmission in the CNS in response to drugs of abuse, and potentially, under physiological conditions.
doi:10.1523/JNEUROSCI.6486-11.2012
PMCID: PMC3371362  PMID: 22553014
hippocampus; dopamine; locus coeruleus; ventral tegmental area; noradrenergic and dopaminergic
18.  MicroRNA GENE EXPRESSION SIGNATURES IN THE DEVELOPING NEURAL TUBE 
BACKGROUND
Neurulation requires precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube (NT) defects. MicroRNAs are key modulators of cell and tissue differentiation. In order to define potential roles of miRNAs in development of the murine NT, miRNA microarray analysis was conducted to establish expression profiles, and identify miRNA target genes and functional gene networks.
METHODS
miRNA expression profiles in murine embryonic NTs derived from gestational days 8.5, 9.0 and 9.5 were defined and compared utilizing miRXplore™ microarrays from Miltenyi Biotech GmbH. Gene expression changes were verified by TaqMan™ quantitative Real-Time PCR. clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined via Ingenuity Pathway Analysis.
RESULTS
miRXplore™ chips enabled examination of 609 murine miRNAs. Expression of approximately 12% of these was detected in murine embryonic NTs. Clustering analysis revealed several developmentally regulated expression clusters among these expressed genes. Target analysis of differentially expressed miRNAs enabled identification of numerous target genes associated with cellular processes essential for normal NT development. Utilization of Ingenuity Pathway Analysis revealed interactive biological networks which connected differentially expressed miRNAs with their target genes, and highlighted functional relationships.
CONCLUSIONS
The present study defined unique gene expression signatures of a range of miRNAs in the developing NT during the critical period of NT morphogenesis. Analysis of miRNA target genes and gene interaction pathways revealed that specific miRNAs may direct expression of numerous genes encoding proteins which have been shown to be indispensable for normal neurulation. This study is the first to identify miRNA expression profiles and their potential regulatory networks in the developing mammalian NT.
doi:10.1002/bdra.20819
PMCID: PMC3165006  PMID: 21770019
neural tube; embryo; mouse; microRNA; microarray; neurulation
19.  Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory 
Neuropsychopharmacology  2011;36(8):1545-1556.
To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories.
doi:10.1038/npp.2011.61
PMCID: PMC3138668  PMID: 21508930
CBP; histone acetylation; long-term memory; long-term potentiation; learning & memory; molecular & cellular neurobiology; plasticity; neurogenetics; CBP; chromatin, histone acetylation; hippocampus
20.  Genetic and Clinical Features of Progranulin-Associated Frontotemporal Lobar Degeneration 
Archives of neurology  2011;68(4):488-497.
Objective
To assess the relative frequency of unique mutations and their associated characteristics in 97 individuals with mutations in progranulin (GRN), an important cause of frontotemporal lobar degeneration (FTLD).
Participants and Design
A 46-site International Frontotemporal Lobar Degeneration Collaboration was formed to collect cases of FTLD with TAR DNA-binding protein of 43-kDa (TDP-43)–positive inclusions (FTLD-TDP). We identified 97 individuals with FTLD-TDP with pathogenic GRN mutations (GRN+ FTLD-TDP), assessed their genetic and clinical characteristics, and compared them with 453 patients with FTLD-TDP in which GRN mutations were excluded (GRN− FTLD-TDP). No patients were known to be related. Neuropathologic characteristics were confirmed as FTLD-TDP in 79 of the 97 GRN+ FTLDTDP cases and all of the GRN− FTLD-TDP cases.
Results
Age at onset of FTLD was younger in patients with GRN+ FTLD-TDP vs GRN− FTLD-TDP (median, 58.0 vs 61.0 years; P<.001), as was age at death (median, 65.5 vs 69.0 years; P<.001). Concomitant motor neuron disease was much less common in GRN+ FTLDTDP vs GRN− FTLD-TDP (5.4% vs 26.3%; P<.001). Fifty different GRN mutations were observed, including 2 novel mutations: c.139delG (p.D47TfsX7) and c.378C>A (p.C126X). The 2 most common GRN mutations were c.1477C>T (p.R493X, found in 18 patients, representing 18.6% of GRN cases) and c.26C>A (p.A9D, found in 6 patients, representing 6.2% of cases). Patients with the c.1477C>T mutation shared a haplotype on chromosome 17; clinically, they resembled patients with other GRN mutations. Patients with the c.26C>A mutation appeared to have a younger age at onset of FTLD and at death and more parkinsonian features than those with other GRN mutations.
Conclusion
GRN+ FTLD-TDP differs in key features from GRN− FTLD-TDP.
doi:10.1001/archneurol.2011.53
PMCID: PMC3160280  PMID: 21482928
21.  Stereoselective Effects of 4-Hydroxynonenal in Cultured Mouse Hepatocytes 
Chemical research in toxicology  2010;23(10):1601-1607.
4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 μM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. caspase-3-dependent cleavage of glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 hrs. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 hours after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.
doi:10.1021/tx100190k
PMCID: PMC3017229  PMID: 20873854
22.  Essential Role for Vav GEFs in Brain-derived Neurotrophic Factor (BDNF)-induced Dendritic Spine Growth and Synapse Plasticity 
Brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, regulate a wide range of cellular processes, including dendritic spine formation and functional synapse plasticity. However, the signaling mechanisms that link BDNF-activated TrkB to F-actin remodeling enzymes and dendritic spine morphological plasticity remain poorly understood. We report here that BDNF/TrkB signaling in neurons activates the Vav family of Rac/RhoA guanine nucleotide exchange factors (GEFs) through a novel TrkB kinase-dependent mechanism. We find that Vav is required for BDNF-stimulated Rac-GTP production in cortical and hippocampal neurons. Vav is partially enriched at excitatory synapses in the postnatal hippocampus, but does not appear to be required for normal dendritic spine density. Rather, we observe significant reductions in both BDNF-induced, rapid dendritic spine head growth and in CA3-CA1 theta burst stimulated (TBS) long-term potentiation (LTP) in Vav-deficient mouse hippocampal slices, suggesting that Vav-dependent regulation of dendritic spine morphological plasticity facilitates normal functional synapse plasticity.
doi:10.1523/JNEUROSCI.0685-11.2011
PMCID: PMC3183742  PMID: 21880903
23.  PRDM16 Expression in the Developing Mouse Embryo 
Acta histochemica  2009;113(2):150-155.
SUMMARY
PRDM16 is a member of the PR domain-containing protein family and is associated with various disease states including myelodysplastic syndrome and adult T cell leukemia, as well as developmental abnormalities such as cleft palate. It is also known to act as a regulator of cell differentiation. Expression analysis of PRDM16 is limited, especially within the developing embryo. The current study evaluated the temporal and spatial localization of PRDM16 during early mouse development (embryonic days 8.5–14.5). PRDM16 was first detected on E9.5 in a limited number of tissues and by E14.5, was expressed in a broad range of developing tissues including those of the brain, lung, kidney, and gastrointestinal tract. The expression pattern is consistent with a role for PRDM16 in the development of multiple tissues. Collectively, these studies are the first to characterize the expression of the PRDM16 gene during early murine development.
doi:10.1016/j.acthis.2009.09.006
PMCID: PMC2891916  PMID: 19853285
embryo; development; MEL1; PRDM16; transcription factor
24.  TGFβ-1 and Wnt-3a interact to induce unique gene expression profiles in murine embryonic palate mesenchymal cells 
Development of the secondary palate in mammals is a complex process under the control of numerous growth and differentiation factors that regulate key processes such as cell proliferation, synthesis of extracellular matrix molecules, and epithelial-mesenchymal transdifferentiation. Alterations in any one of these processes either through genetic mutation or environmental insult have the potential to lead to clefts of the secondary palate. Members of the TGFβ family of cytokines are crucial mediators of these processes and emerging evidence supports a pivotal role for members of the Wnt family of secreted growth and differentiation factors. Previous work in this laboratory demonstrated cross-talk between the Wnt and TGFβ signaling pathways in cultured mouse embryonic palate mesenchymal cells. In the current study we tested the hypothesis that unique gene expression profiles are induced in murine embryonic palate mesenchymal cells as a result of this cross-talk between the TGFβ and Wnt signal transduction pathways.
doi:10.1016/j.reprotox.2010.10.006
PMCID: PMC3138487  PMID: 20955781
embryo; palate; TGFβ; Wnt; microarray; gene expression
25.  Adenosine: front and center in linking nutrition and metabolism to neuronal activity 
The Journal of Clinical Investigation  2011;121(7):2548-2550.
Many individuals with epilepsy benefit from consuming a ketogenic diet, which is similar to the more commonly known Atkins diet. The underlying molecular reason for this has not been determined. However, in this issue of the JCI, Masino et al. have elucidated the mechanism responsible for the antiepileptic effects of the ketogenic diet in mice. The diet is shown to decrease expression of the enzyme adenosine kinase (Adk), which is responsible for clearing the endogenous antiepileptic agent adenosine (Ado) from the extracellular CNS space. Decreased expression of Adk results in increased extracellular Ado, activation of inhibitory Ado A1 receptors, and decreased seizure generation, the desired therapeutic effect. The authors’ work serves to emphasize the importance of controlling Adk expression, not only as the mechanism of action of the ketogenic diet, but also as a potential target of future therapies.
doi:10.1172/JCI58391
PMCID: PMC3223847  PMID: 21701073

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