1 4313 157 MICRORNAS AS CANDIDATES FOR BIPOLAR DISORDER BIOMARKERS. BIPOLAR DISORDER (BD) IS A COMMON, RECURRING PSYCHIATRIC ILLNESS WITH UNKNOWN PATHOGENESIS. MUCH LIKE OTHER PSYCHIATRIC DISEASES, BD SUFFERS FROM THE CHRONIC LACK OF RELIABLE BIOMARKERS AND INNOVATIVE PHARMACOLOGICAL INTERVENTIONS. BETTER CHARACTERIZATION OF CLINICAL PROFILES, EXPERIMENTAL MEDICINE, GENOMIC DATA MINING, AND THE UTILIZATION OF EXPERIMENTAL MODELS, INCLUDING STEM CELL AND GENETICALLY MODIFIED MICE, ARE SUGGESTED WAYS FORWARD. ENVIRONMENT, INCLUDING EARLY CHILDHOOD EXPERIENCES, HAS BEEN DOCUMENTED TO MODULATE THE RISK FOR THE DEVELOPMENT OF PSYCHIATRIC DISORDERS VIA EPIGENETIC MECHANISMS. KEY EPIGENETIC REGULATORS, MICRORNAS (MIRNAS, MIRS), GOVERN NORMAL NEURONAL FUNCTIONING AND SHOW ALTERED EXPRESSION IN DIVERSE BRAIN PATHOLOGIES. WE OBSERVED SIGNIFICANT ALTERATIONS OF EXOSOMAL MIR-29C LEVELS IN PREFRONTAL CORTEX (BRODMANN AREA 9, BA9) OF BD PATIENTS. WE ALSO DEMONSTRATED THAT EXOSOMES EXTRACTED FROM THE ANTERIOR CINGULATE CORTEX (BA24), A CRUCIAL AREA FOR MODULATING EMOTIONAL EXPRESSION AND AFFECT, HAVE INCREASED LEVELS OF MIR-149 IN BD PATIENTS COMPARED TO CONTROLS. BECAUSE MIR-149 HAS BEEN SHOWN TO INHIBIT GLIAL PROLIFERATION, WE HYPOTHESIZED THAT INCREASED MIR-149 EXPRESSION IN BA24-DERIVED EXOSOMES MAY BE CONSISTENT WITH THE PREVIOUSLY REPORTED REDUCED GLIAL CELL NUMBERS IN BA24 OF PATIENTS DIAGNOSED WITH FAMILIAL BD. QPCR ANALYSIS OF LASER-MICRODISSECTED NEURONAL AND GLIAL CELLS FROM BA24 CORTICAL SAMPLES OF BD PATIENTS VERIFIED THAT THE GLIAL, BUT NOT NEURONAL, POPULATION EXHIBITS SIGNIFICANTLY INCREASED MIR-149 EXPRESSION. THESE FINDINGS SUPPORT NEURON-GLIA INTERACTION AS A POSSIBLE TARGET MECHANISM IN BD, IMPLICATED BY OTHERS IN NEUROIMAGING, POSTMORTEM, AND IN VIVO STUDIES OF THE PATHOLOGICAL CHANGES MEDIATED BY GLIAL CELLS. 2021 2 4328 41 MICRORNAS, STEM CELLS IN BIPOLAR DISORDER, AND LITHIUM THERAPEUTIC APPROACH. BIPOLAR DISORDER (BD) IS A SEVERE, CHRONIC, AND DISABLING NEUROPSYCHIATRIC DISORDER CHARACTERIZED BY RECURRENT MOOD DISTURBANCES (MANIA/HYPOMANIA AND DEPRESSION, WITH OR WITHOUT MIXED FEATURES) AND A CONSTELLATION OF COGNITIVE, PSYCHOMOTOR, AUTONOMIC, AND ENDOCRINE ABNORMALITIES. THE ETIOLOGY OF BD IS MULTIFACTORIAL, INCLUDING BOTH BIOLOGICAL AND EPIGENETIC FACTORS. RECENTLY, MICRORNAS (MIRNAS), A CLASS OF EPIGENETIC REGULATORS OF GENE EXPRESSION PLAYING A CENTRAL ROLE IN BRAIN DEVELOPMENT AND PLASTICITY, HAVE BEEN RELATED TO SEVERAL NEUROPSYCHIATRIC DISORDERS, INCLUDING BD. MOREOVER, AN ALTERATION IN THE NUMBER/DISTRIBUTION AND DIFFERENTIATION POTENTIAL OF NEURAL STEM CELLS HAS ALSO BEEN DESCRIBED, SIGNIFICANTLY AFFECTING BRAIN HOMEOSTASIS AND NEUROPLASTICITY. THIS REVIEW AIMED TO EVALUATE THE MOST RELIABLE SCIENTIFIC EVIDENCE ON MIRNAS AS BIOMARKERS FOR THE DIAGNOSIS OF BD AND ASSESS THEIR IMPLICATIONS IN RESPONSE TO MOOD STABILIZERS, SUCH AS LITHIUM. NEURAL STEM CELL DISTRIBUTION, REGULATION, AND DYSFUNCTION IN THE ETIOLOGY OF BD ARE ALSO DISSECTED. 2022 3 4370 43 MIRNAS AND THEIR ROLE IN THE CORRELATION BETWEEN SCHIZOPHRENIA AND CANCER (REVIEW). SCHIZOPHRENIA (SZ) AND CANCER (CA) HAVE A BROAD SPECTRUM OF CLINICAL PHENOTYPES AND A COMPLEX BIOLOGICAL BACKGROUND, IMPLICATING A LARGE NUMBER OF GENETIC AND EPIGENETIC FACTORS. SZ IS A CHRONIC NEURODEVELOPMENTAL DISORDER SIGNIFIED BY AN INCREASE IN THE EXPRESSION OF APOPTOTIC MOLECULAR SIGNALS, WHEREAS CA IS CONVERSELY CHARACTERIZED BY AN INCREASE IN APPROPRIATE MOLECULAR SIGNALING THAT STIMULATES UNCONTROLLED CELL PROLIFERATION. THE RATHER LOW RISK OF DEVELOPING CA IN PATIENTS SUFFERING FROM SZ IS A HYPOTHESIS THAT IS STILL UNDER DEBATE. RECENT EVIDENCE HAS INDICATED THAT MICRORNAS (MIRNAS OR MIRS), A LARGE GROUP OF SMALL NON?CODING OLIGONOUCLEOTIDES, MAY PLAY A SIGNIFICANT ROLE IN THE DEVELOPMENT OF CA AND MAJOR PSYCHIATRIC DISORDERS, SUCH AS SZ, BIPOLAR DISORDER, AUTISM SPECTRUM DISORDERS, SUICIDALITY AND DEPRESSION, THROUGH THEIR INTERFERENCE WITH THE EXPRESSION OF MULTIPLE GENES. FOR INSTANCE, THE POSSIBLE ROLE OF LET?7, MIR?98 AND MIR?183 AS BIOMARKERS FOR CA AND SZ WAS INVESTIGATED IN OUR PREVIOUS RESEARCH STUDIES. THEREFORE, FURTHER INVESTIGATIONS ON THE EXPRESSION PROFILES OF THESE REGULATORY, SMALL RNA MOLECULES AND THE MOLECULAR PATHWAYS THROUGH WHICH THEY EXERT THEIR CONTROL MAY PROVIDE A PLAUSIBLE EXPLANATION AS TO WHETHER THERE IS A CORRELATION BETWEEN PSYCHIATRIC DISORDERS AND LOW RISK OF DEVELOPING CA. 2016 4 2534 41 EPIGENETICS IN BIPOLAR DISORDER: A CRITICAL REVIEW OF THE LITERATURE. INTRODUCTION: BIPOLAR DISORDER (BD) IS A CHRONIC, DISABLING DISEASE CHARACTERISED BY ALTERNATE MOOD EPISODES, SWITCHING THROUGH DEPRESSIVE AND MANIC/HYPOMANIC PHASES. MOOD STABILIZERS, IN PARTICULAR LITHIUM SALTS, CONSTITUTE THE CORNERSTONE OF THE TREATMENT IN THE ACUTE PHASE AS WELL AS FOR THE PREVENTION OF RECURRENCES. THE PATHOPHYSIOLOGY OF BD AND THE MECHANISMS OF ACTION OF MOOD STABILIZERS REMAIN LARGELY UNKNOWN BUT SEVERAL PIECES OF EVIDENCE POINT TO GENE X ENVIRONMENT INTERACTIONS. EPIGENETICS, DEFINED AS THE REGULATION OF GENE EXPRESSION WITHOUT GENETIC CHANGES, COULD BE THE MOLECULAR SUBSTRATE OF THESE INTERACTIONS. IN THIS LITERATURE REVIEW, WE SUMMARIZE THE MAIN EPIGENETIC FINDINGS ASSOCIATED WITH BD AND RESPONSE TO MOOD STABILIZERS. METHODS: WE SEARCHED PUBMED, AND EMBASE DATABASES AND CLASSIFIED THE ARTICLES DEPENDING ON THE EPIGENETIC MECHANISMS (DNA METHYLATION, HISTONE MODIFICATIONS AND NON-CODING RNAS). RESULTS: WE PRESENT THE DIFFERENT EPIGENETIC MODIFICATIONS ASSOCIATED WITH BD OR WITH MOOD-STABILIZERS. THE MAJOR REPORTED MECHANISMS WERE DNA METHYLATION, HISTONE METHYLATION AND ACETYLATION, AND NON-CODING RNAS. OVERALL, THE ASSESSMENTS ARE POORLY HARMONIZED AND THE RESULTS ARE MORE LIMITED THAN IN OTHER PSYCHIATRIC DISORDERS (E.G. SCHIZOPHRENIA). HOWEVER, THE NATURE OF BD AND ITS TREATMENT OFFER EXCELLENT OPPORTUNITIES FOR EPIGENETIC RESEARCH: CLEAR IMPACT OF ENVIRONMENTAL FACTORS, CLINICAL VARIATION BETWEEN MANIC OR DEPRESSIVE EPISODES RESULTING IN POSSIBLE IDENTIFICATION OF STATE AND TRAITS BIOMARKERS, DOCUMENTED IMPACT OF MOOD-STABILIZERS ON THE EPIGENOME. CONCLUSION: EPIGENETIC IS A GROWING AND PROMISING FIELD IN BD THAT MAY SHED LIGHT ON ITS PATHOPHYSIOLOGY OR BE USEFUL AS BIOMARKERS OF RESPONSE TO MOOD-STABILIZER. 2021 5 2909 39 GENE EXPRESSION PROFILING IN FIBROMYALGIA INDICATES AN AUTOIMMUNE ORIGIN OF THE DISEASE AND OPENS NEW AVENUES FOR TARGETED THERAPY. FIBROMYALGIA IS A CHRONIC DISORDER CHARACTERIZED BY WIDESPREAD PAIN AND BY SEVERAL NON-PAIN SYMPTOMS. AUTOIMMUNITY, SMALL FIBER NEUROPATHY AND NEUROINFLAMMATION HAVE BEEN SUGGESTED TO BE INVOLVED IN THE PATHOGENESIS OF THE DISEASE. WE HAVE INVESTIGATED THE GENE EXPRESSION PROFILE IN PERIPHERAL BLOOD MONONUCLEAR CELLS OBTAINED FROM TEN PATIENTS AND TEN HEALTHY SUBJECTS. OF THE 545,500 TRANSCRIPTS ANALYZED, 1673 RESULTED MODULATED IN FIBROMYALGIC PATIENTS. THE MAJORITY OF THESE GENES ARE INVOLVED IN BIOLOGICAL PROCESSES AND PATHWAYS LINKED TO THE CLINICAL MANIFESTATIONS OF THE DISEASE. MOREOVER, GENES INVOLVED IN IMMUNOLOGICAL PATHWAYS CONNECTED TO INTERLEUKIN-17 AND TO TYPE I INTERFERON SIGNATURES WERE ALSO MODULATED, SUGGESTING THAT AUTOIMMUNITY PLAYS A ROLE IN THE DISEASE. WE THEN AIMED AT IDENTIFYING DIFFERENTIALLY EXPRESSED LONG NON-CODING RNAS (LNCRNAS) FUNCTIONALLY CONNECTED TO MODULATED GENES BOTH DIRECTLY AND VIA MICRORNA TARGETING. ONLY TWO LNCRNAS OF THE 298 FOUND MODULATED IN PATIENTS, WERE ABLE TO TARGET THE MOST HIGHLY CONNECTED GENES IN THE FIBROMYALGIA INTERACTOME, SUGGESTING THEIR INVOLVEMENT IN CRUCIAL GENE REGULATION. OUR GENE EXPRESSION DATA WERE CONFIRMED BY REAL TIME PCR, BY AUTOANTIBODY TESTING, DETECTION OF SOLUBLE MEDIATORS AND TH-17 POLARIZATION IN A VALIDATION COHORT OF 50 PATIENTS. OUR RESULTS INDICATE THAT GENETIC AND EPIGENETIC MECHANISMS AS WELL AS AUTOIMMUNITY PLAY A PIVOTAL ROLE IN THE PATHOGENESIS OF FIBROMYALGIA. 2020 6 2119 31 EPIGENETIC HISTONE MODIFICATION REGULATES DEVELOPMENTAL LEAD EXPOSURE INDUCED HYPERACTIVITY IN RATS. LEAD (PB) EXPOSURE WAS COMMONLY CONSIDERED AS A HIGH ENVIRONMENTAL RISK FACTOR FOR THE DEVELOPMENT OF ATTENTION-DEFICIT/HYPERACTIVITY DISORDER (ADHD). HOWEVER, THE MOLECULAR BASIS OF THIS PATHOLOGICAL PROCESS STILL REMAINS ELUSIVE. IN LIGHT OF THE ROLE OF EPIGENETICS IN MODULATING THE NEUROLOGICAL DISEASE AND THE CAUSATIVE ENVIRONMENT, THE ALTERATIONS OF HISTONE MODIFICATIONS IN THE HIPPOCAMPUS OF RATS EXPOSED BY VARIOUS DOSES OF LEAD, ALONG WITH CONCOMITANT BEHAVIORAL DEFICITS, WERE INVESTIGATED IN THIS STUDY. ACCORDING TO THE FREE AND FORCED OPEN FIELD TEST, THERE SHOWED THAT IN A DOSAGE-DEPENDENT MANNER, LEAD EXPOSURE COULD RESULT IN THE INCREASED LOCOMOTOR ACTIVITY OF RATS, THAT IS, HYPERACTIVITY: A SUBTYPE OF ADHD. WESTERN BLOTTING ASSAYS REVEALED THAT THE LEVELS OF HISTONE ACETYLATION INCREASED SIGNIFICANTLY IN THE HIPPOCAMPUS BY CHRONIC LEAD EXPOSURE, WHILE NO DRAMATIC CHANGES WERE DETECTED IN TERMS OF EXPRESSION YIELDS OF ADHD-RELATED DOPAMINERGIC PROTEINS, INDICATING THAT HISTONE ACETYLATION PLAYS ESSENTIAL ROLES IN THIS TOXICANT-INVOLVED PATHOGENESIS. IN ADDITION, THE INCREASED LEVEL OF HISTONE ACETYLATION MIGHT BE ATTRIBUTED TO THE ENZYMATIC ACTIVITY OF P300, A TYPICAL HISTONE ACETYLTRANSFERASE, AS THE TRANSCRIPTIONAL LEVEL OF P300 WAS SIGNIFICANTLY INCREASED UPON HIGHER-DOSE PB EXPOSURE. IN SUMMARY, THIS STUDY FIRST DISCOVERED THE EPIGENETIC MECHANISM BRIDGING THE ENVIRONMENTAL INFLUENCE (PB) AND THE DISEASE ITSELF (ADHD) IN THE HISTONE MODIFICATION LEVEL, PAVING THE WAY FOR THE COMPREHENSIVE UNDERSTANDING OF ADHD'S ETIOLOGY AND IN FURTHER STEPS, ESTABLISHING THE THERAPY STRATEGY OF THIS WIDESPREAD NEUROLOGICAL DISORDER. 2014 7 405 36 ANALYSIS OF EPIGENETIC MECHANISMS REGULATING OPIOID RECEPTOR GENE TRANSCRIPTION. OPIOID DRUGS ARE GENERALLY USED FOR MODERATE AND SEVERE PAIN REDUCTIONS WHICH ACT THROUGH OPIOID RECEPTORS. STUDIES ON TRANSCRIPTIONAL REGULATION OF OPIOID RECEPTORS ARE STILL INVALUABLE BECAUSE NOT ONLY TRANSCRIPTION IS THE FIRST STEP TO PRODUCE PROTEIN PRODUCTS IN CELLS, BUT THE RECEPTOR TRANSCRIPTION LEVELS ALSO AFFECT THE PAIN REDUCTION BY OPIOIDS, AS OBSERVED IN STUDIES OF HETEROZYGOUS OPIOID RECEPTOR KNOCKOUT MICE.THERE ARE GROWING EVIDENCES THAT EPIGENETIC REGULATION HAS PLAYED SIGNIFICANT ROLES IN TRANSCRIPTIONAL REGULATION OF GENES, INCLUDING OPIOID RECEPTORS. IN GENERAL, EPIGENETIC MECHANISMS INCLUDE THREE MAIN REGULATORY FACTORS: DNA METHYLATION, CHROMATIN MODIFICATION, AND NONCODING RNAS (SUCH AS MICRORNA). FROM PREVIOUS STUDIES OF OURS AND OTHERS ON OPIOID RECEPTORS, THOSE EPIGENETIC FACTORS WERE CLEARLY INVOLVED IN REGULATING OPIOID RECEPTOR EXPRESSION IN VIVO AND IN VITRO. IN THIS CHAPTER, AMONG THOSE THREE TECHNIQUES WE DESCRIBE MORE DETAILS OF DNA METHYLATION METHODS BECAUSE OF EMERGING CONCEPTS OF DNA METHYLATION WITH THE RECENT DISCOVERY OF 5-HYDROXYMETHYLCYTOSINE CONVERTING ENZYME, TET1. ANOTHER ANALYTICAL METHOD OF THE EPIGENETIC FACTORS, CHROMATIN MODIFICATION, WILL BE DESCRIBED BRIEFLY AND INFORMATION OF ANALYZING NONCODING RNAS IS BRIEFLY MENTIONED IN SUBHEADING 1. 2015 8 2598 32 EPIGENETICS OF THE DEPRESSED BRAIN: ROLE OF HISTONE ACETYLATION AND METHYLATION. MAJOR DEPRESSIVE DISORDER IS A CHRONIC, REMITTING SYNDROME INVOLVING WIDELY DISTRIBUTED CIRCUITS IN THE BRAIN. STABLE ALTERATIONS IN GENE EXPRESSION THAT CONTRIBUTE TO STRUCTURAL AND FUNCTIONAL CHANGES IN MULTIPLE BRAIN REGIONS ARE IMPLICATED IN THE HETEROGENEITY AND PATHOGENESIS OF THE ILLNESS. EPIGENETIC EVENTS THAT ALTER CHROMATIN STRUCTURE TO REGULATE PROGRAMS OF GENE EXPRESSION HAVE BEEN ASSOCIATED WITH DEPRESSION-RELATED BEHAVIOR, ANTIDEPRESSANT ACTION, AND RESISTANCE TO DEPRESSION OR 'RESILIENCE' IN ANIMAL MODELS, WITH INCREASING EVIDENCE FOR SIMILAR MECHANISMS OCCURRING IN POSTMORTEM BRAINS OF DEPRESSED HUMANS. IN THIS REVIEW, WE DISCUSS RECENT ADVANCES IN OUR UNDERSTANDING OF EPIGENETIC CONTRIBUTIONS TO DEPRESSION, IN PARTICULAR THE ROLE OF HISTONE ACETYLATION AND METHYLATION, WHICH ARE REVEALING NOVEL MECHANISTIC INSIGHT INTO THE SYNDROME THAT MAY AID IN THE DEVELOPMENT OF NOVEL TARGETS FOR DEPRESSION TREATMENT. 2013 9 4093 31 MATERNAL SEPARATION FOLLOWED BY CHRONIC MILD STRESS IN ADULTHOOD IS ASSOCIATED WITH CONCERTED EPIGENETIC REGULATION OF AP-1 COMPLEX GENES. DEPRESSION IS ONE OF THE MOST PREVALENT MENTAL DISEASES WORLDWIDE. PATIENTS WITH PSYCHIATRIC DISEASES OFTEN HAVE A HISTORY OF CHILDHOOD NEGLECT, INDICATING THAT EARLY-LIFE EXPERIENCES PREDISPOSE TO PSYCHIATRIC DISEASES IN ADULTHOOD. TWO STRONG MODELS WERE USED IN THE PRESENT STUDY: THE MATERNAL SEPARATION/EARLY DEPRIVATION MODEL (MS) AND THE CHRONIC MILD STRESS MODEL (CMS). IN BOTH MODELS, WE FOUND CHANGES IN THE EXPRESSION OF A NUMBER OF GENES SUCH AS CREB AND NPY. STRIKINGLY, THERE WAS A CLEAR REGULATION OF EXPRESSION OF FOUR GENES INVOLVED IN THE AP-1 COMPLEX: C-FOS, C-JUN, FOSB, AND JUN-B. INTERESTINGLY, DIFFERENT EXPRESSION LEVELS WERE OBSERVED DEPENDING ON THE MODEL, WHEREAS THE COMBINATION OF THE MODELS RESULTED IN A NORMAL LEVEL OF GENE EXPRESSION. THE EFFECTS OF MS AND CMS ON GENE EXPRESSION WERE ASSOCIATED WITH DISTINCT HISTONE METHYLATION/ACETYLATION PATTERNS OF ALL FOUR GENES. THE EPIGENETIC CHANGES, LIKE GENE EXPRESSION, WERE ALSO DEPENDENT ON THE SPECIFIC STRESSOR OR THEIR COMBINATION. THE OBTAINED RESULTS SUGGEST THAT SINGLE LIFE EVENTS LEAVE A MARK ON GENE EXPRESSION AND THE EPIGENETIC SIGNATURE OF GENE PROMOTERS, BUT A COMBINATION OF DIFFERENT STRESSORS AT DIFFERENT LIFE STAGES CAN FURTHER CHANGE GENE EXPRESSION THROUGH EPIGENETIC FACTORS, POSSIBLY CAUSING THE LONG-LASTING ADVERSE EFFECTS OF STRESS. 2021 10 6895 28 [SYSTEMIC CONTROL OF THE MOLECULAR, CELL, AND EPIGENETIC MECHANISMS OF LONG-LASTING CONSEQUENCES OF STRESS]. BASED ON M.E. LOBASHEV'S VIEWS OF THE SYSTEMIC CONTROL OF GENETIC AND CYTOGENEITC PROCESSES AND A SUBSTANTIAL EFFECT OF EXCITABILITY ON PLASTIC CHANGES IN THE CENTRAL NERVOUS SYSTEM (CNS), THE EFFECT OF PROLONGED EMOTIONAL AND PAIN STRESS (PEPS) ON THE MOLECULAR, CELL, AND EPIGENETIC MECHANISMS OF INJURY MEMORY WAS STUDIED IN RAT STRAINS BRED FOR A CERTAIN EXCITABILITY OF THE NERVOUS SYSTEM. PEPS WAS FOR THE FIRST TIME FOUND TO CAUSE LONG-LASTING (2 MONTHS) MORPHOLOGICAL ALTERATIONS OF THE CA3 REGION OF THE HIPPOCAMPUS AND TO MODIFY THE GENOME ACTIVITY OF ITS PYRAMIDAL NEURONS. THE TWO PHENOMENA WERE POTENTIATED BY A GENETICALLY DETERMINED LOW FUNCTIONAL STATE OF THE CNS. THE POST-STRESS REGULATION OF THE GENOME FUNCTION IN HIPPOCAMPAL NEURONS WAS MEDIATED BY CHANGES IN HETEROCHROMATIN CONFORMATION, ACTIVATION OF METHYL-CPG-BINDING PROTEIN (MECP2) SYNTHESIS, AND SUBSEQUENT CHANGES IN ACETYLATION OF HISTONE H4. GENETICALLY DETERMINED HIGH EXCITABILITY OF THE NERVOUS SYSTEM PROVED TO BE A RISK FACTOR THAT AFFECTS THE SPECIFICS AND TIME COURSE OF THE OBSERVED MOLECULAR, CELL, AND GENETIC TRANSFORMATIONS OF NEURONS. THE RESULTS PROVIDE FOR A BETTER UNDERSTANDING OF THE EPIGENETIC MECHANISMS OF INJURY MEMORY, WHICH FORMS A PATHOGENETIC BASIS FOR POSTTRAUMATIC STRESS DISORDER AND OTHER HUMAN PSYCHOGENIC CONDITIONS CHARACTERIZED BY A PROLONGED DURATION. 2009 11 36 36 A CHROMATIN ASSAY FOR HUMAN BRAIN TISSUE. CHRONIC NEUROPSYCHIATRIC ILLNESSES SUCH AS SCHIZOPHRENIA, BIPOLAR DISEASE AND AUTISM ARE THOUGHT TO RESULT FROM A COMBINATION OF GENETIC AND ENVIRONMENTAL FACTORS THAT MIGHT RESULT IN EPIGENETIC ALTERATIONS OF GENE EXPRESSION AND OTHER MOLECULAR PATHOLOGY. TRADITIONALLY, HOWEVER, EXPRESSION STUDIES IN POSTMORTEM BRAIN WERE CONFINED TO QUANTIFICATION OF MRNA OR PROTEIN. THE LIMITATIONS ENCOUNTERED IN POSTMORTEM BRAIN RESEARCH SUCH AS VARIABILITIES IN AUTOLYSIS TIME AND TISSUE INTEGRITIES ARE ALSO LIKELY TO IMPACT ANY STUDIES OF HIGHER ORDER CHROMATIN STRUCTURES. HOWEVER, THE NUCLEOSOMAL ORGANIZATION OF GENOMIC DNA INCLUDING DNA:CORE HISTONE BINDING - APPEARS TO BE LARGELY PRESERVED IN REPRESENTATIVE SAMPLES PROVIDED BY VARIOUS BRAIN BANKS. THEREFORE, IT IS POSSIBLE TO STUDY THE METHYLATION PATTERN AND OTHER COVALENT MODIFICATIONS OF THE CORE HISTONES AT DEFINED GENOMIC LOCI IN POSTMORTEM BRAIN. HERE, WE PRESENT A SIMPLIFIED NATIVE CHROMATIN IMMUNOPRECIPITATION (NCHIP) PROTOCOL FOR FROZEN (NEVER-FIXED) HUMAN BRAIN SPECIMENS. STARTING WITH MICROCOCCAL NUCLEASE DIGESTION OF BRAIN HOMOGENATES, NCHIP FOLLOWED BY QPCR CAN BE COMPLETED WITHIN THREE DAYS. THE METHODOLOGY PRESENTED HERE SHOULD BE USEFUL TO ELUCIDATE EPIGENETIC MECHANISMS OF GENE EXPRESSION IN NORMAL AND DISEASED HUMAN BRAIN. 2008 12 4323 29 MICRORNAS IN PSYCHOLOGICAL STRESS REACTIONS AND THEIR USE AS STRESS-ASSOCIATED BIOMARKERS, ESPECIALLY IN HUMAN SALIVA. MICRORNAS (MIRNAS) PLAY A CENTRAL ROLE IN THE REGULATION OF MANY CELLULAR PROCESSES INCLUDING PHYSIOLOGICAL AND PSYCHOLOGICAL STRESS REACTION PATHWAYS. PSYCHOLOGICAL STRESS IS AN IMPORTANT FACTOR FOR THE GENESIS AND MAINTENANCE OF MANY DISEASES. SEVERAL MIRNAS HAVE ALREADY BEEN DESCRIBED TO BE INVOLVED IN ITS REGULATION. THE PRESENCE OF MIRNAS IN ALL BODY FLUIDS IMPLIES A WIDESPREAD ROLE IN COMMUNICATION THROUGHOUT THE WHOLE ORGANISM AND TOGETHER WITH THEIR STABILITY MAKES THEM FORMIDABLE CANDIDATES AS BIOMARKERS. ALTERATIONS OF STRESS-ASSOCIATED MIRNA EXPRESSION LEVELS HAVE BEEN FOUND IN THE BRAIN AND WHOLE BLOOD OF HUMANS AND ANIMALS. IN THIS PAPER, WE REVIEW THE PARTICIPATION OF MIRNAS IN STRESS-REACTIVE PROCESSES AS WELL AS THEIR USABILITY AS SALIVARY BIOMARKERS OF SUCH PROCESSES. IN CONCLUSION, WE SUGGEST THAT SALIVARY MIRNAS MAY BE USEFUL AS NONINVASIVE BIOMARKERS TO ASSESS EPIGENETIC REGULATION PROCESSES OF CHRONIC OR ACUTE PSYCHOLOGICAL STRESS REACTIONS. 2017 13 6775 33 [ALCOHOL DEPENDENCE MEDIATED BY MONOAMINE NEUROTRANSMITTERS IN THE CENTRAL NERVOUS SYSTEM]. ALCOHOL DEPENDENCE, A CHRONIC RELAPSING BRAIN DISEASE WITH THE CHARACTERISTICS OF DRINKING ALCOHOL OUT OF CONTROL, HAS BECOME A SERIOUS SOCIAL PROBLEM. MONOAMINE NEUROTRANSMITTERS, MAINLY INCLUDING DOPAMINE AND 5-HYDROXYTRYP NOTTAMINE, PLAY IMPORTANT ROLES IN THE OCCURRENCE, DEVELOPMENT AND NEURAL DYSFUNCTION OF ALCOHOL DEPENDENCE SYNDROME. IN THIS REVIEW, THE ROLES OF KEY FACTORS OF THE MONOAMINE SYSTEM (DOPAMINE RECEPTOR GENES, 5-HYDROXYTRYPTAMINE RECEPTOR GENES, TRANSPORTER GENES, TYROSINE HYDROXYLASE GENE, TRYPTOPHANHYDROXYLASE GENE AND MONOAMINE OXIDASE GENE) IN ALCOHOL DEPENDENCE WERE DISCUSSED, AND STRATEGIES FOR FURTHER STUDIES OF MOLECULAR MECHANISMS WERE PROPOSED BASED ON GENE KNOCKOUT MICE MODELS GENERATED IN OUR LABORATORY. THEN, COMBINING WITH STUDIES ON TYROSINE HYDROXYLASE ACTIVATOR CAMKII IN OUR LAB, THERAPEUTIC TARGETS WERE DISCUSSED. BESIDES, EPIGENETIC STRATEGIES FOR PREVENTION AND TREATMENT OF ALCOHOL DEPENDENCE SYNDROME WERE PROPOSED. FURTHERMORE, MANIPULATING METHYLATION LEVELS IN GENE REGULATORY REGIONS AND ALTERNATIVE SPLICING OF PRE-MRNAS MIGHT ALSO HAVE CLINICAL IMPLICATIONS. FINALLY, BASED ON NEW FINDINGS ON GENETIC POLYMORPHISM, IT IS OF GREAT POTENTIAL TO CARRY OUT INDIVIDUAL PREVENTION AND TREATMENT FOR PATIENTS SUFFERING FROM ALCOHOL DEPENDENCE. 2014 14 1796 47 EFFECT OF GERM-FREE STATUS ON TRANSCRIPTIONAL PROFILES IN THE NUCLEUS ACCUMBENS AND TRANSCRIPTOMIC RESPONSE TO CHRONIC MORPHINE. OPIOID USE DISORDER IS A PUBLIC HEALTH CRISIS THAT CAUSES TREMENDOUS SUFFERING FOR PATIENTS AS WELL AS SUBSTANTIAL SOCIAL AND ECONOMIC COSTS FOR SOCIETY. THERE ARE CURRENTLY AVAILABLE TREATMENTS FOR PATIENTS WITH OPIOID USE DISORDER, BUT THEY REMAIN INTOLERABLE OR INEFFECTIVE FOR MANY. THUS THE NEED TO DEVELOP NEW AVENUES FOR THERAPEUTICS DEVELOPMENT IN THIS SPACE IS GREAT. SUBSTANTIAL WORK IN MODELS OF SUBSTANCE USE DISORDERS, INCLUDING OPIOID USE DISORDER, DEMONSTRATES THAT PROLONGED EXPOSURE TO DRUGS OF ABUSE LEADS TO MARKED TRANSCRIPTIONAL AND EPIGENETIC DYSREGULATION IN LIMBIC SUBSTRUCTURES. IT IS WIDELY BELIEVED THAT THESE CHANGES IN GENE REGULATION IN RESPONSE TO DRUGS ARE A KEY DRIVING FACTOR IN THE PERPETUATION OF DRUG TAKING AND SEEKING BEHAVIORS. THUS, DEVELOPMENT OF INTERVENTIONS THAT COULD SHAPE TRANSCRIPTIONAL REGULATION IN RESPONSE TO DRUGS OF ABUSE WOULD BE OF HIGH VALUE. OVER THE PAST DECADE THERE HAS BEEN A SURGE IN RESEARCH DEMONSTRATING THAT THE RESIDENT BACTERIA OF THE GASTROINTESTINAL TRACT, COLLECTIVELY THE GUT MICROBIOME, CAN HAVE TREMENDOUS INFLUENCE ON NEUROBIOLOGICAL AND BEHAVIORAL PLASTICITY. PREVIOUS WORK FROM OUR GROUP AND OTHERS HAS DEMONSTRATED THAT ALTERATIONS IN THE GUT MICROBIOME CAN ALTER BEHAVIORAL RESPONSES TO OPIOIDS IN MULTIPLE PARADIGMS. ADDITIONALLY, WE HAVE PREVIOUSLY REPORTED THAT DEPLETION OF THE GUT MICROBIOME WITH ANTIBIOTICS MARKEDLY SHIFTS THE TRANSCRIPTOME OF THE NUCLEUS ACCUMBENS FOLLOWING PROLONGED MORPHINE EXPOSURE. IN THIS MANUSCRIPT WE PRESENT A COMPREHENSIVE ANALYSIS OF THE EFFECTS OF THE GUT MICROBIOME ON TRANSCRIPTIONAL REGULATION OF THE NUCLEUS ACCUMBENS FOLLOWING MORPHINE BY UTILIZING GERM-FREE, ANTIBIOTIC TREATED, AND CONTROL MICE. THIS ALLOWS FOR DETAILED UNDERSTANDING OF THE ROLE OF THE MICROBIOME IN REGULATING BASELINE TRANSCRIPTOMIC CONTROL, AS WELL AS RESPONSE TO MORPHINE. WE FIND THAT GERM-FREE STATUS LEADS TO A MARKED GENE DYSREGULATION IN A MANNER DISTINCT TO ADULT MICE TREATED WITH ANTIBIOTICS, AND THAT ALTERED GENE PATHWAYS ARE HIGHLY RELATED TO CELLULAR METABOLIC PROCESSES. THESE DATA PROVIDE ADDITIONAL INSIGHT INTO THE ROLE OF THE GUT MICROBIOME IN MODULATING BRAIN FUNCTION AND LAY A FOUNDATION FOR FURTHER STUDY IN THIS AREA. 2023 15 5645 36 SEX DEPENDENT ALTERATION OF EPIGENETIC MARKS AFTER CHRONIC MORPHINE TREATMENT IN MICE ORGANS. EPIGENETIC MARKS MAY BE ALSO AFFECTED BY SEVERAL FACTORS, SUCH AS AGE, LIFESTYLE, EARLY LIFE EXPERIENCES AND EXPOSURE TO CHEMICALS OR DRUGS, SUCH AS OPIOIDS. PREVIOUS STUDIES HAVE FOCUSED ON HOW MORPHINE EPIGENETICALLY REGULATES DIFFERENT REGIONS OF THE BRAIN THAT ARE IMPLICATED IN TOLERANCE, DEPENDENCE AND OTHER PSYCHIATRIC DISORDERS MORE RELATED TO THE PHYSIO-PATHOLOGICAL EFFECTS OF OPIOIDS. NEVERTHELESS, A SIGNIFICANT KNOWLEDGE GAP REMAINS REGARDING THE EFFECT OF CHRONIC TREATMENT ON OTHER ORGANS AND BIOLOGICAL SYSTEMS. THEREFORE, THE AIM OF THIS WORK IS TO INCREASE OUR KNOWLEDGE ABOUT THE IMPACT OF CHRONIC MORPHINE EXPOSURE ON DNA METHYLATION AND HISTONE MODIFICATION LEVELS IN EACH OF THE ORGANS OF MALE AND FEMALE MODEL MICE IN VIVO. OUR RESULTS REVEAL, FOR THE FIRST TIME, THAT CHRONIC MORPHINE TREATMENT INDUCED CHANGES IN DNA METHYLATION/HYDROXYMETHYLATION AND HISTONE MODIFICATION IN-VIVO AT THE SYSTEMIC LEVEL, REVEALING A POTENTIAL PHYSIOLOGICAL EFFECT ON THE REGULATION OF GENE EXPRESSION. NOTABLY, MORPHINE-INDUCED EPIGENETIC MODIFICATION OCCURS IN A SEX-DEPENDENT MANNER, REVEALING THE EXISTENCE OF DIFFERENT UNDERLYING MECHANISMS OF EPIGENETIC MODIFICATION IN MALE AND FEMALE MICE. 2021 16 2523 35 EPIGENETICS AND THE TRANSITION FROM ACUTE TO CHRONIC PAIN. OBJECTIVE: THE OBJECTIVE OF THIS STUDY WAS TO REVIEW THE EPIGENETIC MODIFICATIONS INVOLVED IN THE TRANSITION FROM ACUTE TO CHRONIC PAIN AND TO IDENTIFY POTENTIAL TARGETS FOR THE DEVELOPMENT OF NOVEL, INDIVIDUALIZED PAIN THERAPEUTICS. BACKGROUND: EPIGENETICS IS THE STUDY OF HERITABLE MODIFICATIONS IN GENE EXPRESSION AND PHENOTYPE THAT DO NOT REQUIRE A CHANGE IN GENETIC SEQUENCE TO MANIFEST THEIR EFFECTS. ENVIRONMENTAL TOXINS, MEDICATIONS, DIET, AND PSYCHOLOGICAL STRESSES CAN ALTER EPIGENETIC PROCESSES SUCH AS DNA METHYLATION, HISTONE ACETYLATION, AND RNA INTERFERENCE. AS EPIGENETIC MODIFICATIONS POTENTIALLY PLAY AN IMPORTANT ROLE IN INFLAMMATORY CYTOKINE METABOLISM, STEROID RESPONSIVENESS, AND OPIOID SENSITIVITY, THEY ARE LIKELY KEY FACTORS IN THE DEVELOPMENT OF CHRONIC PAIN. ALTHOUGH OUR KNOWLEDGE OF THE HUMAN GENETIC CODE AND DISEASE-ASSOCIATED POLYMORPHISMS HAS GROWN SIGNIFICANTLY IN THE PAST DECADE, WE HAVE NOT YET BEEN ABLE TO ELUCIDATE THE MECHANISMS THAT LEAD TO THE DEVELOPMENT OF PERSISTENT PAIN AFTER NERVE INJURY OR SURGERY. DESIGN: THIS IS A FOCUSED LITERATURE REVIEW OF EPIGENETIC SCIENCE AND ITS RELATIONSHIP TO CHRONIC PAIN. RESULTS: SIGNIFICANT LABORATORY AND CLINICAL DATA SUPPORT THE NOTION THAT EPIGENETIC MODIFICATIONS ARE AFFECTED BY THE ENVIRONMENT AND LEAD TO DIFFERENTIAL GENE EXPRESSION. SIMILAR TO MECHANISMS INVOLVED IN THE DEVELOPMENT OF CANCER, NEURODEGENERATIVE DISEASE, AND INFLAMMATORY DISORDERS, THE LITERATURE ENDORSES AN IMPORTANT POTENTIAL ROLE FOR EPIGENETICS IN CHRONIC PAIN. CONCLUSIONS: EPIGENETIC ANALYSIS MAY IDENTIFY MECHANISMS CRITICAL TO THE DEVELOPMENT OF CHRONIC PAIN AFTER INJURY, AND MAY PROVIDE NEW PATHWAYS AND TARGET MECHANISMS FOR FUTURE DRUG DEVELOPMENT AND INDIVIDUALIZED MEDICINE. 2012 17 2625 34 EPIGENOME-WIDE ASSOCIATION STUDY AND MULTI-TISSUE REPLICATION OF INDIVIDUALS WITH ALCOHOL USE DISORDER: EVIDENCE FOR ABNORMAL GLUCOCORTICOID SIGNALING PATHWAY GENE REGULATION. ALCOHOL USE DISORDER (AUD) IS A CHRONIC DEBILITATING DISORDER WITH LIMITED TREATMENT OPTIONS AND POORLY DEFINED PATHOPHYSIOLOGY. THERE ARE SUBSTANTIAL GENETIC AND EPIGENETIC COMPONENTS; HOWEVER, THE UNDERLYING MECHANISMS CONTRIBUTING TO AUD REMAIN LARGELY UNKNOWN. WE CONDUCTED THE LARGEST DNA METHYLATION EPIGENOME-WIDE ASSOCIATION STUDY (EWAS) ANALYSES CURRENTLY AVAILABLE FOR AUD (TOTAL N = 625) AND EMPLOYED A TOP HIT REPLICATION (N = 4798) USING A CROSS-TISSUE/CROSS-PHENOTYPIC APPROACH WITH THE GOAL OF IDENTIFYING NOVEL EPIGENETIC TARGETS RELEVANT TO AUD. RESULTS SHOW THAT A NETWORK OF DIFFERENTIALLY METHYLATED REGIONS IN GLUCOCORTICOID SIGNALING AND INFLAMMATION-RELATED GENES WERE ASSOCIATED WITH ALCOHOL USE BEHAVIORS. A TOP PROBE CONSISTENTLY ASSOCIATED ACROSS ALL COHORTS WAS LOCATED IN THE LONG NON-CODING RNA GROWTH ARREST SPECIFIC FIVE GENE (GAS5) (P < 10(-24)). GAS5 HAS BEEN IMPLICATED IN REGULATING TRANSCRIPTIONAL ACTIVITY OF THE GLUCOCORTICOID RECEPTOR AND HAS MULTIPLE FUNCTIONS RELATED TO APOPTOSIS, IMMUNE FUNCTION AND VARIOUS CANCERS. ENDOPHENOTYPIC ANALYSES USING PERIPHERAL CORTISOL LEVELS AND NEUROIMAGING PARADIGMS SHOWED THAT METHYLOMIC VARIATION IN GAS5 NETWORK-RELATED PROBES WERE ASSOCIATED WITH STRESS PHENOTYPES. POSTMORTEM BRAIN ANALYSES DOCUMENTED INCREASED GAS5 EXPRESSION IN THE AMYGDALA OF INDIVIDUALS WITH AUD. OUR DATA SUGGEST THAT ALCOHOL USE IS ASSOCIATED WITH DIFFERENTIAL METHYLATION IN THE GLUCOCORTICOID SYSTEM THAT MIGHT INFLUENCE STRESS AND INFLAMMATORY REACTIVITY AND SUBSEQUENTLY RISK FOR AUD. 2021 18 2139 33 EPIGENETIC INSIGHTS INTO MULTIPLE SCLEROSIS DISEASE PROGRESSION. MULTIPLE SCLEROSIS (MS), A CHRONIC INFLAMMATORY DEMYELINATING AND NEURODEGENERATIVE DISEASE OF THE CENTRAL NERVOUS SYSTEM, IS TODAY A LEADING CAUSE OF UNPREDICTABLE LIFELONG DISABILITY IN YOUNG ADULTS. THE TREATMENT OF PATIENTS IN PROGRESSIVE STAGES REMAINS HIGHLY CHALLENGING, ALLUDING TO OUR LIMITED UNDERSTANDING OF THE UNDERLYING PATHOLOGICAL PROCESSES. IN THIS REVIEW, WE PROVIDE INSIGHTS INTO THE MECHANISMS UNDERPINNING MS PROGRESSION FROM A PERSPECTIVE OF EPIGENETICS, THAT REFERS TO STABLE AND MITOTICALLY HERITABLE, YET REVERSIBLE, CHANGES IN THE GENOME ACTIVITY AND GENE EXPRESSION. WE FIRST RECAPITULATE FINDINGS FROM EPIGENETIC STUDIES EXAMINING THE BRAIN TISSUE OF PROGRESSIVE MS PATIENTS, WHICH SUPPORT A CONTRIBUTION OF DNA AND HISTONE MODIFICATIONS IN IMPAIRED OLIGODENDROCYTE DIFFERENTIATION, DEFECTIVE MYELINATION/REMYELINATION AND SUSTAINED NEURO-AXONAL VULNERABILITY. WE NEXT EXPLORE POSSIBILITIES FOR IDENTIFYING FACTORS AFFECTING PROGRESSION USING EASILY ACCESSIBLE TISSUES SUCH AS BLOOD BY COMPARING EPIGENETIC SIGNATURES IN PERIPHERAL IMMUNE CELLS AND BRAIN TISSUE. DESPITE MINOR OVERLAP AT INDIVIDUAL METHYLATION SITES, NEARLY 30% OF ALTERED GENES REPORTED IN PERIPHERAL IMMUNE CELLS OF PROGRESSIVE MS PATIENTS WERE FOUND IN BRAIN TISSUE, JOINTLY CONVERGING ON ALTERATIONS OF NEURONAL FUNCTIONS. WE FURTHER SPECULATE ABOUT THE MECHANISMS UNDERLYING SHARED EPIGENETIC PATTERNS BETWEEN BLOOD AND BRAIN, WHICH LIKELY IMPLY THE INFLUENCE OF INTERNAL (GENETIC CONTROL) AND/OR EXTERNAL (E.G. SMOKING AND AGEING) FACTORS IMPRINTING A COMMON SIGNATURE IN BOTH COMPARTMENTS. OVERALL, WE PROPOSE THAT EPIGENETICS MIGHT SHED LIGHT ON CLINICALLY RELEVANT MECHANISMS INVOLVED IN DISEASE PROGRESSION AND OPEN NEW AVENUES FOR THE TREATMENT OF PROGRESSIVE MS PATIENTS IN THE FUTURE. 2020 19 6226 19 THE LINK BETWEEN EPIGENETICS, PAIN SENSITIVITY AND CHRONIC PAIN. INCREASING EVIDENCE SUGGESTS AN ASSOCIATION BETWEEN GENE EXPRESSION AND CLINICAL PAIN. EPIGENETIC MODIFICATIONS ARE THE MAIN MODULATORS OF GENE EXPRESSION OR PROTEIN TRANSLATION IN RESPONSE TO ENVIRONMENTAL STIMULI AND PATHOPHYSIOLOGICAL CONDITIONS. PRECLINICAL AND CLINICAL STUDIES INDICATE THAT EPIGENETIC MODIFICATIONS COULD ALSO IMPACT THE DEVELOPMENT OF PAIN, THE TRANSITION FROM ACUTE TO CHRONIC PAIN, AND THE MAINTENANCE HEREOF. 2022 20 5828 33 STRESS, EPIGENETICS, AND ALCOHOLISM. ACUTE AND CHRONIC STRESSORS HAVE BEEN ASSOCIATED WITH ALTERATIONS IN MOOD AND INCREASED ANXIETY THAT MAY EVENTUALLY RESULT IN THE DEVELOPMENT OF STRESS-RELATED PSYCHIATRIC DISORDERS. STRESS AND ASSOCIATED DISORDERS, INCLUDING ANXIETY, ARE KEY FACTORS IN THE DEVELOPMENT OF ALCOHOLISM BECAUSE ALCOHOL CONSUMPTION CAN TEMPORARILY REDUCE THE DRINKER'S DYSPHORIA. ONE MOLECULE THAT MAY HELP MEDIATE THE RELATIONSHIP BETWEEN STRESS AND ALCOHOL CONSUMPTION IS BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF), A PROTEIN THAT REGULATES THE STRUCTURE AND FUNCTION OF THE SITES WHERE TWO NERVE CELLS INTERACT AND EXCHANGE NERVE SIGNALS (I.E., SYNAPSES) AND WHICH IS INVOLVED IN NUMEROUS PHYSIOLOGICAL PROCESSES. ABERRANT REGULATION OF BDNF SIGNALING AND ALTERATIONS IN SYNAPSE ACTIVITY (I.E., SYNAPTIC PLASTICITY) HAVE BEEN ASSOCIATED WITH THE PATHOPHYSIOLOGY OF STRESS-RELATED DISORDERS AND ALCOHOLISM. MECHANISMS THAT CONTRIBUTE TO THE REGULATION OF GENETIC INFORMATION WITHOUT MODIFICATION OF THE DNA SEQUENCE (I.E., EPIGENETIC MECHANISMS) MAY PLAY A ROLE IN THE COMPLEX CONTROL OF BDNF SIGNALING AND SYNAPTIC PLASTICITY-FOR EXAMPLE, BY MODIFYING THE STRUCTURE OF THE DNA-PROTEIN COMPLEXES (I.E., CHROMATIN) THAT MAKE UP THE CHROMOSOMES AND THEREBY MODULATING THE EXPRESSION OF CERTAIN GENES. STUDIES REGARDING THE EPIGENETIC CONTROL OF BDNF SIGNALING AND SYNAPTIC PLASTICITY PROVIDE A PROMISING DIRECTION TO UNDERSTAND THE MECHANISMS MEDIATING THE INTERACTION BETWEEN STRESS AND ALCOHOLISM. 2012