Striatal microRNA controls cocaine intake through CREB signalling.

Cocaine addiction is characterized by a gradual loss of control over drug use, but the molecular mechanisms regulating vulnerability to this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with a history of extended access to cocaine. Striatal miR-212 decreases responsiveness to the motivational properties of cocaine by markedly amplifying the stimulatory effects of the drug on cAMP response element binding protein (CREB) signalling. This action occurs through miR-212-enhanced Raf1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (transducer of regulated CREB; also known as CRTC). Our findings indicate that striatal miR-212 signalling has a key role in determining vulnerability to cocaine addiction, reveal new molecular regulators that control the complex actions of cocaine in brain reward circuitries and provide an entirely new direction for the development of anti-addiction therapeutics based on the modulation of noncoding RNAs.

MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function.

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys.

BACKGROUND: Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) region of the Huntingtin (HTT) gene. The clinical features of HD are characterized by cognitive, psychological, and motor deficits. Molecular instability, a core component in neurological disease progression, can be comprehensively evaluated through longitudinal transcriptomic profiling. Development of animal models amenable to longitudinal examination enables distinct disease-associated mechanisms to be identified. RESULTS: Here we report the first longitudinal study of transgenic monkeys with genomic integration of various lengths of the human HTT gene and a range of polyQ repeats. With this unique group of transgenic HD nonhuman primates (HD monkeys), we profiled over 47,000 transcripts from peripheral blood collected over a 2 year timespan from HD monkeys and age-matched wild-type control monkeys. CONCLUSIONS: Messenger RNAs with expression patterns which diverged with disease progression in the HD monkeys considerably facilitated our search for transcripts with diagnostic or therapeutic potential in the blood of human HD patients, opening up a new avenue for clinical investigation.

Clonal Neoantigen: Emerging "Mechanism-based" Biomarker of Immunotherapy Response.

Clonal mutations represent the initiating molecular defects related to cellular transition of a normal phenotype to a malignant phenotype. Molecular genomic assessment utilizing next generation and whole exome sequencing is now being increasingly applied to biomarker determination to refine the use of targeted immune therapies. Case examples followed by retrospective study assessment have convincingly demonstrated clonal neoantigens provide a relevant predictor of response to checkpoint inhibition. A meta-analysis, by Litchfield et al., of over 1000 cancer patients from 12 landmark trials demonstrated no clinical benefit to checkpoint inhibitor (CPI) therapy in correlation to high subclonal tumor mutational burden (TMB), whereas high clonal TMB was found to be significantly correlated with better overall survival (p = 0.000000029). We discuss the mechanism of clonal vs. subclonal neoantigen targeting relationship to homologous recombination proficient (HRP) profile, evidence of preclinical and clinical benefit related to clonal neoantigens, and review a novel developing therapy called Vigil®, designed to expand the clonal neoantigen targeting effector cell populations. Vigil® is an autologous cellular immunotherapy which is designed to carry the full set of personal clonal neoantigens. Phase 2b results demonstrate a durable recurrence-free survival (RFS) and overall survival (OS) advantage for Vigil® in a subset ovarian cancer population with an HRP cancer profile.

MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction.

N-methyl-D-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice. Consistent with a role for miR-219 in NMDA receptor signaling, we identify calcium/calmodulin-dependent protein kinase II gamma subunit (CaMKIIgamma), a component of the NMDA receptor signaling cascade, as a target of miR-219. In vivo inhibition of miR-219 by specific antimiR in the murine brain significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression of behavioral aberrations associated with NMDA receptor hypofunction.

Network based analysis identifies TP53m-BRCA1/2wt-homologous recombination proficient (HRP) population with enhanced susceptibility to Vigil immunotherapy.

Thus far immunotherapy has had limited impact on ovarian cancer. Vigil (a novel DNA-based multifunctional immune-therapeutic) has shown clinical benefit to prolong relapse-free survival (RFS) and overall survival (OS) in the BRCA wild type and HRP populations. We further analyzed molecular signals related to sensitivity of Vigil treatment. Tissue from patients enrolled in the randomized double-blind trial of Vigil vs. placebo as maintenance in frontline management of advanced resectable ovarian cancer underwent DNA polymorphism analysis. Data was generated from a 981 gene panel to determine the tumor mutation burden and classify variants using Ingenuity Variant Analysis software (Qiagen) or NIH ClinVar. Only variants classified as pathogenic or likely pathogenic were included. STRING application (version 1.5.1) was used to create a protein-protein interaction network. Topological distance and probability of co-mutation were used to calculated the C-score and cumulative C-score (cumC-score). Kaplan-Meier analysis was used to determine the relationship between gene pairs with a high cumC-score and clinical parameters. Improved relapse free survival in Vigil treated patients was found for the TP53m-BRCAwt-HRP group compared to placebo (21.1 months versus 5.6 months p = 0.0013). Analysis of tumor mutation burden did not reveal statistical benefit in patients receiving Vigil versus placebo. Results suggest a subset of ovarian cancer patients with enhanced susceptibility to Vigil immunotherapy. The hypothesis-generating data presented invites a validation study of Vigil in target identified populations, and supports clinical consideration of STRING-generated network application to biomarker characterization with other cancer patients targeted with Vigil.

Placental microRNA expression in pregnancies complicated by preeclampsia.

OBJECTIVE: The role of posttranscription regulation in preeclampsia is largely unknown. We investigated preeclampsia-related placental microRNA (miRNA) expression using microarray and confirmatory quantitative real-time polymerase chain reaction experiments. STUDY DESIGN: Placental expressions of characterized and novel miRNAs (1295 probes) were measured in samples collected from 20 preeclampsia cases and 20 controls. Differential expression was evaluated using Student t test and fold change analyses. In pathway analysis, we examined functions/functional relationships of targets of differentially expressed miRNAs. RESULTS: Eight miRNAs were differentially expressed (1 up-regulated and 7 down-regulated) among preeclampsia cases compared with controls. These included previously identified candidates (miR-210, miR-1, and a miRNA in the 14q32.31 cluster region) and others that are novel (miR-584 and miR-34c-5p). These miRNAs target genes that participate in organ/system development (cardiovascular and reproductive system), immunologic dysfunction, cell adhesion, cell cycle, and signaling. CONCLUSION: Expression of miRNAs that target genes in diverse pathophysiological processes is altered in the setting of preeclampsia.

Descriptive study of California egg layer premises and analysis of risk factors for Salmonella enterica serotype enteritidis as characterized by manure drag swabs.

This cross-sectional, double-blind study reports the prevalence of Salmonella enterica serotype enteritidis (SE) on California egg layer premises using single vs. pooled manure drag swabs and presents a description of egg production and management systems in the state and an initial analysis of risk factors for SE. The study included 91% of all known eligible egg premises in California, representing the majority of eggs produced in the state. The overall prevalence of SE on California egg layer premises was 10.5%, while 1.1% of all rows sampled were positive for SE. The percentage of positive rows for SE on any premises never exceeded 25% of the 16 swabs collected per premises. A description of egg production and management on California egg layer premises is presented. Statistically significant associations for SE were not evident and were limited because of sample size and the low prevalence of SE on California egg layer premises. Several biological and management factors, such as flock health, stage of production, manure management, ventilation, and watering systems, show trend associations with premises positive for SE and require further investigation. Manure drag swabs serve as a useful tool to validate the core components of an egg quality assurance program for SE based on process control principles.

The occurrence and distribution of Salmonella enteritidis and other serovars on California egg laying premises: a comparison of two sampling methods and two culturing techniques.

Between the summer of 1998 and the winter of 2000, Salmonella analysis was performed on 2128 single and 532 pooled manure drag swabs obtained from 133 California commercial egg laying farms. The isolation of Salmonella from all rows and from all flocks using single or pooled swabs was 80% and 92%, respectively. Hence, there was no statistical difference between single vs. pooled swabs in terms of identifying Salmonella on a row or flock basis. A total of 14 serogroups comprising 44 serotypes were isolated from 123 of 133 farms. When the top 10 serotypes were considered, there was no significant difference in the range of serotypes isolated by the two culturing methods. The overall S. enteritidis prevalence for California flocks was 10.5% (14/133). The overall row prevalence for S. enteritidis for all the farms was 1.1% (24/2128), and the overall pool prevalence was 2.4% (13/532). Sixty percent (12/20) of the S. enteritidis isolates from the positive farms were phage type 4, and 40% (8/20) represented five other phage types (1, 6B, 7, 8, and 28).

RNA-seq analysis to identify novel roles of scleraxis during embryonic mouse heart valve remodeling.

Heart valve disease affects up to 30% of the population and has been shown to have origins during embryonic development. Valvulogenesis begins with formation of endocardial cushions in the atrioventricular canal and outflow tract regions. Subsequently, endocardial cushions remodel, elongate and progressively form mature valve structures composed of a highly organized connective tissue that provides the necessary biomechanical function throughout life. While endocardial cushion formation has been well studied, the processes required for valve remodeling are less well understood. The transcription factor Scleraxis (Scx) is detected in mouse valves from E15.5 during initial stages of remodeling, and expression remains high until birth when formation of the highly organized mature structure is complete. Heart valves from Scx-/- mice are abnormally thick and develop fibrotic phenotypes similar to human disease by juvenile stages. These phenotypes begin around E15.5 and are associated with defects in connective tissue organization and valve interstitial cell differentiation. In order to understand the etiology of this phenotype, we analyzed the transcriptome of remodeling valves isolated from E15.5 Scx-/- embryos using RNA-seq. From this, we have identified a profile of protein and non-protein mRNAs that are dependent on Scx function and using bioinformatics we can predict the molecular functions and biological processes affected by these genes. These include processes and functions associated with gene regulation (methyltransferase activity, DNA binding, Notch signaling), vitamin A metabolism (retinoic acid biosynthesis) and cellular development (cell morphology, cell assembly and organization). In addition, several mRNAs are affected by alternative splicing events in the absence of Scx, suggesting additional roles in post-transcriptional modification. In summary, our findings have identified transcriptome profiles from abnormal heart valves isolated from E15.5 Scx-/- embryos that could be used in the future to understand mechanisms of heart valve disease in the human population.

Myocardial alternative RNA splicing and gene expression profiling in early stage hypoplastic left heart syndrome.

Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome.

Matrix metalloproteinase 9 expression: new regulatory elements.

Retinal ganglion cells apoptosis is linked to matrix metalloproteinase 9 (MMP-9) controlled changes of extracellular matrix. Abnormal expression of MMP-9 is associated with glaucomatous alterations. Thus, the knowledge of MMP-9 regulation is important for the understanding the pathogenesis of glaucoma. Here, we investigated the role of 3'-untranslated regions (3'-UTR) and microRNAs in MMP-9 regulation. We used in vitro mutagenesis and Luc reporter system to identify regulatory elements in the 3'-UTR of MMP-9. microRNAs were analyzed by qRT-PCR, and their role was investigated with inhibitors and mimics. We identified targets for miRNAs in 3'-UTR of MMP-9 involved in the regulation of MMP-9 expression. We then isolated miRNAs from the optic nerve A7 astrocytes and 293 T cells and confirmed the role of mi340 in the regulation using specific inhibitors and mimics. The results obtained show a new miRNA-mediated mechanism of MMP-9 expression regulation.

Wasp gene expression supports an evolutionary link between maternal behavior and eusociality.

The presence of workers that forgo reproduction and care for their siblings is a defining feature of eusociality and a major challenge for evolutionary theory. It has been proposed that worker behavior evolved from maternal care behavior. We explored this idea by studying gene expression in the primitively eusocial wasp Polistes metricus. Because little genomic information existed for this species, we used 454 sequencing to generate 391,157 brain complementary DNA reads, resulting in robust hits to 3017 genes from the honey bee genome, from which we identified and assayed orthologs of 32 honey bee behaviorally related genes. Wasp brain gene expression in workers was more similar to that in foundresses, which show maternal care, than to that in queens and gynes, which do not. Insulin-related genes were among the differentially regulated genes, suggesting that the evolution of eusociality involved major nutritional and reproductive pathways.