Early rearing history influences oxytocin receptor epigenetic regulation in rhesus macaques.

Adaptations to stress can occur through epigenetic processes and may be a conduit for informing offspring of environmental challenge. We employed ChIP-sequencing for H3K4me3 to examine effects of early maternal deprivation (peer-rearing, PR) in archived rhesus macaque hippocampal samples (male, n = 13). Focusing on genes with roles in stress response and behavior, we assessed the effects of rearing on H3K4me3 binding by ANOVA. We found decreased H3K4me3 binding at genes critical to behavioral stress response, the most robust being the oxytocin receptor gene OXTR, for which we observed a corresponding decrease in RNA expression. Based on this finding, we performed behavioral analyses to determine whether a gain-of-function nonsynonymous OXTR SNP interacted with early stress to influence relevant behavioral stress reactivity phenotypes (n = 194), revealing that this SNP partially rescued the PR phenotype. PR infants exhibited higher levels of separation anxiety and arousal in response to social separation, but infants carrying the alternative OXTR allele did not exhibit as great a separation response. These data indicate that the oxytocin system is involved in social-separation response and suggest that epigenetic down-modulation of OXTR could contribute to behavioral differences observed in PR animals. Epigenetic changes at OXTR may represent predictive adaptive responses that could impart readiness to respond to environmental challenge or maintain proximity to a caregiver but also contribute to behavioral pathology. Our data also demonstrate that OXTR polymorphism can permit animals to partially overcome the detrimental effects of early maternal deprivation, which could have translational implications for human psychiatric disorders.

Genetic background and epigenetic modifications in the core of the nucleus accumbens predict addiction-like behavior in a rat model.

This study provides a demonstration in the rat of a clear genetic difference in the propensity for addiction-related behaviors following prolonged cocaine self-administration. It relies on the use of selectively bred high-responder (bHR) and low-responder (bLR) rat lines that differ in several characteristics associated with "temperament," including novelty-induced locomotion and impulsivity. We show that bHR rats exhibit behaviors reminiscent of human addiction, including persistent cocaine-seeking and increased reinstatement of cocaine seeking. To uncover potential underlying mechanisms of this differential vulnerability, we focused on the core of the nucleus accumbens and examined expression and epigenetic regulation of two transcripts previously implicated in bHR/bLR differences: fibroblast growth factor (FGF2) and the dopamine D2 receptor (D2). Relative to bHRs, bLRs had lower FGF2 mRNA levels and increased association of a repressive mark on histones (H3K9me3) at the FGF2 promoter. These differences were apparent under basal conditions and persisted even following prolonged cocaine self-administration. In contrast, bHRs had lower D2 mRNA under basal conditions, with greater association of H3K9me3 at the D2 promoter and these differences were no longer apparent following prolonged cocaine self-administration. Correlational analyses indicate that the association of H3K9me3 at D2 may be a critical substrate underlying the propensity to relapse. These findings suggest that low D2 mRNA levels in the nucleus accumbens core, likely mediated via epigenetic modifications, may render individuals more susceptible to cocaine addiction. In contrast, low FGF2 levels, which appear immutable even following prolonged cocaine exposure, may serve as a protective factor.

Learning Experimental Design through Targeted Student-Centric Journal Club with Screencasting.

Knowledge and application of experimental design principles are essential components of scientific methodology, and experience with these skills is fundamental for participating in scientific research. However, undergraduates often enter the research laboratory with little training in designing and interpreting their own experiments. In the context of a research university laboratory, we designed a journal club training exercise to address this need. Students were instructed on methods for interpreting scientific literature using a screencast, a digital recording of a slide presentation narrated by an instructor. Students subsequently examined a series of research publications with a focus on the experimental designs and data interpretation in a two-session group discussion journal club format. We have found this approach to be an efficient and productive method for engaging students in learning about principles of experimental design and further preparing them for success in laboratory research.

Basal microRNA expression patterns in reward circuitry of selectively bred high-responder and low-responder rats vary by brain region and genotype.

Mental health disorders involving altered reward, emotionality, and anxiety are thought to result from the interaction of individual predisposition (genetic factors) and personal experience (environmental factors), although the mechanisms that contribute to an individual's vulnerability to these disorders remain poorly understood. We used an animal model of individual variation [inbred high-responder/low-responder (bHR-bLR) rodents] known to vary in reward, anxiety, and emotional processing to examine neuroanatomical expression patterns of microRNAs (miRNAs). Laser capture microdissection was used to dissect the prelimbic cortex and the nucleus accumbens core and shell prior to analysis of basal miRNA expression in bHR and bLR male rats. These studies identified 187 miRNAs differentially expressed by genotype in at least one brain region, 10 of which were validated by qPCR. Four of these 10 qPCR-validated miRNAs demonstrated differential expression across multiple brain regions, and all miRNAs with validated differential expression between genotypes had lower expression in bHR animals compared with bLR animals. microRNA (miR)-484 and miR-128a expression differences between the prelimbic cortex of bHR and bLR animals were validated by semiquantitative in situ hybridization. miRNA expression analysis independent of genotype identified 101 miRNAs differentially expressed by brain region, seven of which validated by qPCR. Dnmt3a mRNA, a validated target of miR-29b, varied in a direction opposite that of miR-29b's differential expression between bHR and bLR animals. These data provide evidence that basal central nervous system miRNA expression varies in the bHR-bLR model, implicating microRNAs as potential epigenetic regulators of key neural circuits and individual differences associated with mental health disorders.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

OBJECTIVE: Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. METHODS: Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. RESULTS: Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. INTERPRETATION: These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.

Fibroblast growth factor-2 (FGF2) augmentation early in life alters hippocampal development and rescues the anxiety phenotype in vulnerable animals.

Individuals with mood disorders exhibit alterations in the fibroblast growth factor system, including reduced hippocampal fibroblast growth factor-2 (FGF2). It is difficult, however, to pinpoint whether these alterations are a cause or consequence of the disorder. The present study asks whether FGF2 administered the day after birth has long-lasting effects on hippocampal development and emotionality. We show that early-life FGF2 shifts the pace of neurogenesis, with an early acceleration around weaning followed by a deceleration in adulthood. This, in turn, results in a denser dentate gyrus with more neurons. To assess the impact of early-life FGF2 on emotionality, we use rats selectively bred for differences in locomotor response to novelty. Selectively bred low-responder (bLR) rats show low levels of novelty-induced locomotion and exhibit high levels of anxiety- and depression-like behavior compared with their selectively bred high-responder counterparts. Early-life FGF2 decreased anxiety-like behavior in highly anxious bLRs without altering other behaviors and without affecting high-responder rats. Laser capture microscopy of the dentate gyrus followed by microarray analysis revealed genes that were differentially expressed in bLRs exposed to early-life FGF2 vs. vehicle-treated bLRs. Some of the differentially expressed genes that have been positively associated with anxiety were down-regulated, whereas genes that promote cell survival were up-regulated. Overall, these results show a key role for FGF2 in the developmental trajectory of the hippocampus as well as the modulation of anxiety-like behavior in adulthood, and they point to potential downstream targets for the treatment of anxiety disorders.

Negative regulation of Yap during neuronal differentiation.

Regulated proliferation and cell cycle exit are essential aspects of neurogenesis. The Yap transcriptional coactivator controls proliferation in a variety of tissues during development, and this activity is negatively regulated by kinases in the Hippo signaling pathway. We find that Yap is expressed in mitotic mouse retinal progenitors and it is downregulated during neuronal differentiation. Forced expression of Yap prolongs proliferation in the postnatal mouse retina, whereas inhibition of Yap by RNA interference (RNAi) decreases proliferation and increases differentiation. We show Yap is subject to post-translational inhibition in the retina, and also downregulated at the level of mRNA expression. Using a cell culture model, we find that expression of the proneural basic helix-loop-helix (bHLH) transcription factors Neurog2 or Ascl1 downregulates Yap mRNA levels, and simultaneously inhibits Yap protein via activation of the Lats1 and/or Lats2 kinases. Conversely, overexpression of Yap prevents proneural bHLH proteins from initiating cell cycle exit. We propose that mutual inhibition between proneural bHLH proteins and Yap is an important regulator of proliferation and cell cycle exit during mammalian neurogenesis.

Neurotransmission-related gene expression in the frontal pole is altered in subjects with bipolar disorder and schizophrenia.

The frontal pole (Brodmann area 10, BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning prior to the age of onset for bipolar disorder (BP) and schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ postmortem samples (n = 72). We chose this method for its high sensitivity to detect low-level expression. We then strengthened our findings by performing a meta-analysis of publicly released BA10 microarray data (n = 101) and identified sources of convergence with our qPCR results. To improve interpretation, we leveraged the unusually large database of clinical metadata accompanying our samples to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly available datasets. Using these convergent sources of evidence, we identified 20 neurotransmission-related genes that were differentially expressed in BP and SCHIZ in BA10. These results included a large diagnosis-related decrease in two important therapeutic targets with low levels of expression, HTR2B and DRD4, as well as other findings related to dopaminergic, GABAergic and astrocytic function. We also observed that therapeutics may produce a differential expression that opposes diagnosis effects. In contrast, substances of abuse showed similar effects on BA10 gene expression as BP and SCHIZ, potentially amplifying diagnosis-related dysregulation.

Glucocorticoid treatment of astrocytes results in temporally dynamic transcriptome regulation and astrocyte-enriched mRNA changes in vitro.

While general effects of glucocorticoids are well established, the specific cellular mechanisms by which these hormones exert tissue-dependent effects continue to be elaborated. Diseases that demonstrate altered glucocorticoid signaling have been associated with alterations in astrocytes, yet relatively little is known about the effects of glucocorticoids upon this cell type. We have analyzed mRNA expression patterns following glucocorticoid treatment of mouse primary astrocyte cultures. Microarray analysis of cortical astrocyte cultures treated with dexamethasone over an eight-point, 24 h time course identified 854 unique genes with ≥twofold change in mRNA expression at one or more time points. Clustering analysis associated subsets of these mRNA expression changes with gene ontology categories known to be impacted by glucocorticoids. Numerous mRNAs regulated by dexamethasone were also regulated by the natural ligand corticosterone; all of the mRNAs regulated ≥twofold by corticosterone were substantially attenuated by cotreatment with the glucocorticoid receptor antagonist RU486. Of the mRNAs demonstrating ≥twofold expression change in response to both glucocorticoids, 33 mRNAs were previously associated with glucocorticoid regulation, and 36 mRNAs were novel glucocorticoid targets. All genes tested by qPCR for glucocorticoid regulation in cortical astrocyte cultures were also regulated by glucocorticoids in hippocampal astrocyte cultures (18/18). Interestingly, a portion of glucocorticoid-regulated genes were astrocyte enriched; the percentage of astrocyte-enriched genes per total number of regulated genes was highest for the early time points and steadily decreased over the time course. These findings suggest that astrocytes in vitro may initially deploy cell type-specific patterns of mRNA regulatory responses to glucocorticoids and subsequently activate additional cell type-independent responses.

Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase.

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal ß-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)ß. PKIß-targeted shRNA constructs both reduced the levels of PKIß expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIß protein, and this rescue required the PKA-specific inhibitory sequence of the PKIß protein. PKIß played a very specific role in the Ascl1-mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIß. Our results define a novel requirement for PKIß and its inhibition of PKA during neuronal differentiation of P19 cells.

The rhesus macaque is three times as diverse but more closely equivalent in damaging coding variation as compared to the human.

BACKGROUND: As a model organism in biomedicine, the rhesus macaque (Macaca mulatta) is the most widely used nonhuman primate. Although a draft genome sequence was completed in 2007, there has been no systematic genome-wide comparison of genetic variation of this species to humans. Comparative analysis of functional and nonfunctional diversity in this highly abundant and adaptable non-human primate could inform its use as a model for human biology, and could reveal how variation in population history and size alters patterns and levels of sequence variation in primates. RESULTS: We sequenced the mRNA transcriptome and H3K4me3-marked DNA regions in hippocampus from 14 humans and 14 rhesus macaques. Using equivalent methodology and sampling spaces, we identified 462,802 macaque SNPs, most of which were novel and disproportionately located in the functionally important genomic regions we had targeted in the sequencing. At least one SNP was identified in each of 16,797 annotated macaque genes. Accuracy of macaque SNP identification was conservatively estimated to be >90%. Comparative analyses using SNPs equivalently identified in the two species revealed that rhesus macaque has approximately three times higher SNP density and average nucleotide diversity as compared to the human. Based on this level of diversity, the effective population size of the rhesus macaque is approximately 80,000 which contrasts with an effective population size of less than 10,000 for humans. Across five categories of genomic regions, intergenic regions had the highest SNP density and average nucleotide diversity and CDS (coding sequences) the lowest, in both humans and macaques. Although there are more coding SNPs (cSNPs) per individual in macaques than in humans, the ratio of dN/dS is significantly lower in the macaque. Furthermore, the number of damaging nonsynonymous cSNPs (have damaging effects on protein functions from PolyPhen-2 prediction) in the macaque is more closely equivalent to that of the human. CONCLUSIONS: This large panel of newly identified macaque SNPs enriched for functionally significant regions considerably expands our knowledge of genetic variation in the rhesus macaque. Comparative analysis reveals that this widespread, highly adaptable species is approximately three times as diverse as the human but more closely equivalent in damaging variation.

The serotonin transporter gene is a substrate for age and stress dependent epigenetic regulation in rhesus macaque brain: potential roles in genetic selection and gene × environment interactions.

In humans, it has been demonstrated that the serotonin transporter linked polymorphic region (5-HTTLPR) genotype moderates risk in the face of adversity. One mechanism by which stress could interact with genotype is via epigenetic modifications. We wanted to examine whether stress interacted with genotype to predict binding of a histone 3 protein trimethylated at lysine 3 (H3K4me3) that marks active promoters. The brains (N = 61) of male rhesus macaques that had been reared in the presence or absence of stress were archived and the hippocampusi dissected. Chromatin immunoprecipitation was performed with an antibody against H3K4me3 followed by sequencing on a SolexaG2A. The effects of age, genotype (5-HTTLPR long/long vs. short), and stress exposure (peer-reared vs. mother-reared) on levels of H3K4me3 binding were determined. We found effects of age and stress exposure. There was a decline in H3K4me3 from preadolescence to postadolescence and lower levels in peer-reared monkeys and no effects of genotype. When we controlled for age, however, we found that there were effects of 5-HTTLPR genotype and rearing condition on H3K4me3 binding. In a larger sample, we observed that cerebrospinal fluid 5-hydroxyindoleacetic acid levels were subject to interactive effects among age, rearing history, and genotype. Genes containing both genetic selection and epigenetic regulation may be particularly important in stress adaptation and development. We find evidence for selection at the solute carrier family C6 member 4 gene and observe epigenetic reorganization according to genotype, stress, and age. These data suggest that developmental stage may moderate effects of stress and serotonin transporter genotype in the emergence of alternative adaptation strategies and in the vulnerability to developmental or psychiatric disorders.

Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription.

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

Genome-wide association and meta-analysis of bipolar disorder in individuals of European ancestry.

Bipolar disorder (BP) is a disabling and often life-threatening disorder that affects approximately 1% of the population worldwide. To identify genetic variants that increase the risk of BP, we genotyped on the Illumina HumanHap550 Beadchip 2,076 bipolar cases and 1,676 controls of European ancestry from the National Institute of Mental Health Human Genetics Initiative Repository, and the Prechter Repository and samples collected in London, Toronto, and Dundee. We imputed SNP genotypes and tested for SNP-BP association in each sample and then performed meta-analysis across samples. The strongest association P value for this 2-study meta-analysis was 2.4 x 10(-6). We next imputed SNP genotypes and tested for SNP-BP association based on the publicly available Affymetrix 500K genotype data from the Wellcome Trust Case Control Consortium for 1,868 BP cases and a reference set of 12,831 individuals. A 3-study meta-analysis of 3,683 nonoverlapping cases and 14,507 extended controls on >2.3 M genotyped and imputed SNPs resulted in 3 chromosomal regions with association P approximately 10(-7): 1p31.1 (no known genes), 3p21 (>25 known genes), and 5q15 (MCTP1). The most strongly associated nonsynonymous SNP rs1042779 (OR = 1.19, P = 1.8 x 10(-7)) is in the ITIH1 gene on chromosome 3, with other strongly associated nonsynonymous SNPs in GNL3, NEK4, and ITIH3. Thus, these chromosomal regions harbor genes implicated in cell cycle, neurogenesis, neuroplasticity, and neurosignaling. In addition, we replicated the reported ANK3 association results for SNP rs10994336 in the nonoverlapping GSK sample (OR = 1.37, P = 0.042). Although these results are promising, analysis of additional samples will be required to confirm that variant(s) in these regions influence BP risk.

Developmental Programming: Prenatal Testosterone Excess on Liver and Muscle Coding and Noncoding RNA in Female Sheep.

Prenatal testosterone (T)-treated female sheep manifest peripheral insulin resistance, ectopic lipid accumulation, and insulin signaling disruption in liver and muscle. This study investigated transcriptional changes and transcriptome signature of prenatal T excess-induced hepatic and muscle-specific metabolic disruptions. Genome-wide coding and noncoding (nc) RNA expression in liver and muscle from 21-month-old prenatal T-treated (T propionate 100 mg intramuscular twice weekly from days 30-90 of gestation; term: 147 days) and control females were compared. Prenatal T (1) induced differential expression of messenger RNAs (mRNAs) in liver (15 down, 17 up) and muscle (66 down, 176 up) (false discovery rate < 0.05, absolute log2 fold change > 0.5); (2) downregulated mitochondrial pathway genes in liver and muscle; (3) downregulated hepatic lipid catabolism and peroxisome proliferator-activated receptor (PPAR) signaling gene pathways; (4) modulated noncoding RNA (ncRNA) metabolic processes gene pathway in muscle; and (5) downregulated 5 uncharacterized long noncoding RNA (lncRNA) in the muscle but no ncRNA changes in the liver. Correlation analysis showed downregulation of lncRNAs LOC114112974 and LOC105607806 was associated with decreased TPK1, and LOC114113790 with increased ZNF470 expression. Orthogonal projections to latent structures discriminant analysis identified mRNAs HADHA and SLC25A45, and microRNAs MIR154A, MIR25, and MIR487B in the liver and ARIH1 and ITCH and miRNAs MIR369, MIR10A, and MIR10B in muscle as potential biomarkers of prenatal T excess. These findings suggest downregulation of mitochondria, lipid catabolism, and PPAR signaling genes in the liver and dysregulation of mitochondrial and ncRNA gene pathways in muscle are contributors of lipotoxic and insulin-resistant hepatic and muscle phenotype. Gestational T excess programming of metabolic dysfunctions involve tissue-specific ncRNA-modulated transcriptional changes.

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription.

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.

Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time- and concentration-dependent manner. Interestingly, the gene encoding the cell cycle regulator Gadd45gamma was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45gamma as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45gamma promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45gamma promoter. Furthermore, we found that overexpression of Gadd45gamma itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.

Genetic Liability for Internalizing Versus Externalizing Behavior Manifests in the Developing and Adult Hippocampus: Insight From a Meta-analysis of Transcriptional Profiling Studies in a Selectively Bred Rat Model.

BACKGROUND: For more than 16 years, we have selectively bred rats for either high or low levels of exploratory activity within a novel environment. These bred high-responder (bHR) and bred low-responder (bLR) rats model temperamental extremes, exhibiting large differences in internalizing and externalizing behaviors relevant to mood and substance use disorders. METHODS: We characterized persistent differences in gene expression related to bHR/bLR phenotype across development and adulthood in the hippocampus, a region critical for emotional regulation, by meta-analyzing 8 transcriptional profiling datasets (microarray and RNA sequencing) spanning 43 generations of selective breeding (postnatal day 7: n = 22; postnatal day 14: n = 49; postnatal day 21: n = 21; adult: n = 46; all male). We cross-referenced expression differences with exome sequencing within our colony to pinpoint candidates likely to mediate the effect of selective breeding on behavioral phenotype. The results were compared with hippocampal profiling from other bred rat models. RESULTS: Genetic and transcriptional profiling results converged to implicate multiple candidate genes, including two previously associated with metabolism and mood: Trhr and Ucp2. Results also highlighted bHR/bLR functional differences in the hippocampus, including a network essential for neurodevelopmental programming, proliferation, and differentiation, centering on Bmp4 and Mki67. Finally, we observed differential expression related to microglial activation, which is important for synaptic pruning, including 2 genes within implicated chromosomal regions: C1qa and Mfge8. CONCLUSIONS: These candidate genes and functional pathways may direct bHR/bLR rats along divergent developmental trajectories and promote a widely different reactivity to the environment.

NGSQC: cross-platform quality analysis pipeline for deep sequencing data.

BACKGROUND: While the accuracy and precision of deep sequencing data is significantly better than those obtained by the earlier generation of hybridization-based high throughput technologies, the digital nature of deep sequencing output often leads to unwarranted confidence in their reliability. RESULTS: The NGSQC (Next Generation Sequencing Quality Control) pipeline provides a set of novel quality control measures for quickly detecting a wide variety of quality issues in deep sequencing data derived from two dimensional surfaces, regardless of the assay technology used. It also enables researchers to determine whether sequencing data related to their most interesting biological discoveries are caused by sequencing quality issues. CONCLUSIONS: Next generation sequencing platforms have their own share of quality issues and there can be significant lab-to-lab, batch-to-batch and even within chip/slide variations. NGSQC can help to ensure that biological conclusions, in particular those based on relatively rare sequence alterations, are not caused by low quality sequencing.

Preparation and characterization of (CNSSS)2(A)2 (A = AsF6(-), SbF6(-), Sb2F11(-)) containing the O2-like 5,5'-bis(1,2,3,4-trithiazolium) dication: the second example of a simple nonsterically hindered main-group diradical that retains its paramagnetism in the solid state.

The reaction of NC-CN with a 1:1 mixture of S(4)(MF(6))(2) and S(8)(MF(6))(2) (M = As, Sb) (stoichiometrically equivalent to four "S(3)MF(6)" units) results in the quantitative formation of S(3)NCCNS(3)(MF(6))(2) [7(MF(6))(2)], which is the thermodynamic sink in this reaction. The Sb(2)F(11)(-) salt 7(Sb(2)F(11))(2) is prepared by the addition of an excess of SbF(5) to 7(AsF(6))(2). Crystal structure determinations for all three salts show that 7(2+) can be viewed as two R-CNS(3)(+) radical cations joined together by a C-C single bond. The two rings are coplanar and in a trans orientation due to electrostatic N(delta-)...S(delta+) interactions. The classically bonded alternative (quinoidal structure), in which the octet rule is obeyed, is not observed and is much higher in energy based on calculated estimates and a simple comparison of pi bond energies. Calculated molecular orbitals (MOs) support this, showing that the MO corresponding to the quinoidal structure lies higher in energy than the nearly degenerate singly occupied MOs of 7(2+). The vibrational spectra of 7(2+) in all salts were assigned based on a normal-coordinate analysis and theoretical vibrational frequencies calculated at the PBE0/6-31G* level. In the solid state, 7(2+) is a planar disjoint diradical with essentially degenerate open-shell singlet and triplet states. The disjoint nature of the diradical cation 7(2+) is established by magnetic susceptibility studies of the Sb(2)F(11)(-) salt doped in an isomorphous diamagnetic host material (CNSNS)(2)(Sb(2)F(11))(2) [10(Sb(2)F(11))(2)]. Intramolecular spin coupling is extremely weak corresponding to a singlet-triplet gap (DeltaE(ST) = 2J) of <+/-2 cm(-1). CASPT2[12,12]/6-311G* calculations support a triplet ground state with a small singlet-triplet gap. The single-crystal electron paramagnetic resonance (EPR) of 7(Sb(2)F(11))(2) doped in 10(Sb(2)F(11))(2) is in agreement with the triplet state arising from the weak coupling between the unpaired electrons residing in p(pi) orbitals in each of the rings. Variable-temperature susceptibility data for bulk samples of 7(A)(2) (A = SbF(6)(-), AsF(6)(-), Sb(2)F(11)(-)) are analyzed by employing both 1D chain and 2D sheet magnetic models. These studies reveal significant intermolecular exchange approximating that of a 1D chain for the SbF(6)(-) salt with |J| = 32 cm(-1). The exchange coupling is on the same order of magnitude as that for the AsF(6)(-) salt, although in this case it is likely that there are complex exchange pathways where no particular one is dominant. Intermolecular exchange in the Sb(2)F(11)(-) salt is an order of magnitude weaker. In solution, the EPR spectrum of 7(2+) shows a broad triplet resonance as well as a sharp resonance that is tentatively attributed to a rotomer of the 7(2+)/anion pair, which is likely the origin of the green species given on dissolution of the red 7(2+) salts in SO(2)/AsF(3)/MF(5). We account for the many similarities between O(2) and 7(2+), which are the only simple nonsterically hindered nonmetal diradicals to retain their paramagnetism in the solid state. 7(2+) is also the first isolable, essentially sulfur-based diradical as evidenced by calculated spin densities.