1 5856 108 SUBSTRATE UTILISATION OF CULTURED SKELETAL MUSCLE CELLS IN PATIENTS WITH CFS. CHRONIC FATIGUE SYNDROME (CFS) PATIENTS OFTEN SUFFER FROM SEVERE MUSCLE PAIN AND AN INABILITY TO EXERCISE DUE TO MUSCLE FATIGUE. IT HAS PREVIOUSLY BEEN SHOWN THAT CFS SKELETAL MUSCLE CELLS HAVE LOWER LEVELS OF ATP AND HAVE AMP-ACTIVATED PROTEIN KINASE DYSFUNCTION. THIS STUDY OUTLINES EXPERIMENTS LOOKING AT THE UTILISATION OF DIFFERENT SUBSTRATES BY SKELETAL MUSCLE CELLS FROM CFS PATIENTS (N = 9) AND HEALTHY CONTROLS (N = 11) USING EXTRACELLULAR FLUX ANALYSIS. RESULTS SHOW THAT CFS SKELETAL MUSCLE CELLS ARE UNABLE TO UTILISE GLUCOSE TO THE SAME EXTENT AS HEALTHY CONTROL CELLS. CFS SKELETAL MUSCLE CELLS WERE SHOWN TO OXIDISE GALACTOSE AND FATTY ACIDS NORMALLY, INDICATING THAT THE BIOENERGETIC DYSFUNCTION LIES UPSTREAM OF THE TCA CYCLE. THE DYSFUNCTION IN GLUCOSE OXIDATION IS SIMILAR TO WHAT HAS PREVIOUSLY BEEN SHOWN IN BLOOD CELLS FROM CFS PATIENTS. THE CONSISTENCY OF CELLULAR BIOENERGETIC DYSFUNCTION IN DIFFERENT CELL TYPES SUPPORTS THE HYPOTHESIS THAT CFS IS A SYSTEMIC DISEASE. THE RETENTION OF BIOENERGETIC DEFECTS IN CULTURED CELLS INDICATES THAT THERE IS A GENETIC OR EPIGENETIC COMPONENT TO THE DISEASE. THIS IS THE FIRST STUDY TO USE CELLS DERIVED FROM SKELETAL MUSCLE BIOPSIES IN CFS PATIENTS AND HEALTHY CONTROLS TO LOOK AT CELLULAR BIOENERGETIC FUNCTION IN WHOLE CELLS. 2020 2 1556 35 DNA METHYLATION MODIFICATIONS ASSOCIATED WITH CHRONIC FATIGUE SYNDROME. CHRONIC FATIGUE SYNDROME (CFS), ALSO KNOWN AS MYALGIC ENCEPHALOMYELITIS, IS A COMPLEX MULTIFACTORIAL DISEASE THAT IS CHARACTERIZED BY THE PERSISTENT PRESENCE OF FATIGUE AND OTHER PARTICULAR SYMPTOMS FOR A MINIMUM OF 6 MONTHS. SYMPTOMS FAIL TO DISSIPATE AFTER SUFFICIENT REST AND HAVE MAJOR EFFECTS ON THE DAILY FUNCTIONING OF CFS SUFFERERS. CFS IS A MULTI-SYSTEM DISEASE WITH A HETEROGENEOUS PATIENT POPULATION SHOWING A WIDE VARIETY OF FUNCTIONAL DISABILITIES AND ITS BIOLOGICAL BASIS REMAINS POORLY UNDERSTOOD. STABLE ALTERATIONS IN GENE FUNCTION IN THE IMMUNE SYSTEM HAVE BEEN REPORTED IN SEVERAL STUDIES OF CFS. EPIGENETIC MODIFICATIONS HAVE BEEN IMPLICATED IN LONG-TERM EFFECTS ON GENE FUNCTION, HOWEVER, TO OUR KNOWLEDGE, GENOME-WIDE EPIGENETIC MODIFICATIONS ASSOCIATED WITH CFS HAVE NOT BEEN EXPLORED. WE EXAMINED THE DNA METHYLOME IN PERIPHERAL BLOOD MONONUCLEAR CELLS ISOLATED FROM CFS PATIENTS AND HEALTHY CONTROLS USING THE ILLUMINA HUMANMETHYLATION450 BEADCHIP ARRAY, CONTROLLING FOR INVARIANT PROBES AND PROBES OVERLAPPING POLYMORPHIC SEQUENCES. GENE ONTOLOGY (GO) AND NETWORK ANALYSIS OF DIFFERENTIALLY METHYLATED GENES WAS PERFORMED TO DETERMINE POTENTIAL BIOLOGICAL PATHWAYS SHOWING CHANGES IN DNA METHYLATION IN CFS. WE FOUND AN INCREASED ABUNDANCE OF DIFFERENTIALLY METHYLATED GENES RELATED TO THE IMMUNE RESPONSE, CELLULAR METABOLISM, AND KINASE ACTIVITY. GENES ASSOCIATED WITH IMMUNE CELL REGULATION, THE LARGEST COORDINATED ENRICHMENT OF DIFFERENTIALLY METHYLATED PATHWAYS, SHOWED HYPOMETHYLATION WITHIN PROMOTERS AND OTHER GENE REGULATORY ELEMENTS IN CFS. THESE DATA ARE CONSISTENT WITH EVIDENCE OF MULTISYSTEM DYSREGULATION IN CFS AND IMPLICATE THE INVOLVEMENT OF DNA MODIFICATIONS IN CFS PATHOLOGY. 2014 3 169 36 ABNORMALITIES OF AMPK ACTIVATION AND GLUCOSE UPTAKE IN CULTURED SKELETAL MUSCLE CELLS FROM INDIVIDUALS WITH CHRONIC FATIGUE SYNDROME. BACKGROUND: POST EXERTIONAL MUSCLE FATIGUE IS A KEY FEATURE IN CHRONIC FATIGUE SYNDROME (CFS). ABNORMALITIES OF SKELETAL MUSCLE FUNCTION HAVE BEEN IDENTIFIED IN SOME BUT NOT ALL PATIENTS WITH CFS. TO TRY TO LIMIT POTENTIAL CONFOUNDERS THAT MIGHT CONTRIBUTE TO THIS CLINICAL HETEROGENEITY, WE DEVELOPED A NOVEL IN VITRO SYSTEM THAT ALLOWS COMPARISON OF AMP KINASE (AMPK) ACTIVATION AND METABOLIC RESPONSES TO EXERCISE IN CULTURED SKELETAL MUSCLE CELLS FROM CFS PATIENTS AND CONTROL SUBJECTS. METHODS: SKELETAL MUSCLE CELL CULTURES WERE ESTABLISHED FROM 10 SUBJECTS WITH CFS AND 7 AGE-MATCHED CONTROLS, SUBJECTED TO ELECTRICAL PULSE STIMULATION (EPS) FOR UP TO 24H AND EXAMINED FOR CHANGES ASSOCIATED WITH EXERCISE. RESULTS: IN THE BASAL STATE, CFS CULTURES SHOWED INCREASED MYOGENIN EXPRESSION BUT DECREASED IL6 SECRETION DURING DIFFERENTIATION COMPARED WITH CONTROL CULTURES. CONTROL CULTURES SUBJECTED TO 16 H EPS SHOWED A SIGNIFICANT INCREASE IN BOTH AMPK PHOSPHORYLATION AND GLUCOSE UPTAKE COMPARED WITH UNSTIMULATED CELLS. IN CONTRAST, CFS CULTURES SHOWED NO INCREASE IN AMPK PHOSPHORYLATION OR GLUCOSE UPTAKE AFTER 16 H EPS. HOWEVER, GLUCOSE UPTAKE REMAINED RESPONSIVE TO INSULIN IN THE CFS CELLS POINTING TO AN EXERCISE-RELATED DEFECT. IL6 SECRETION IN RESPONSE TO EPS WAS SIGNIFICANTLY REDUCED IN CFS COMPARED WITH CONTROL CULTURES AT ALL TIME POINTS MEASURED. CONCLUSION: EPS IS AN EFFECTIVE MODEL FOR ELICITING MUSCLE CONTRACTION AND THE METABOLIC CHANGES ASSOCIATED WITH EXERCISE IN CULTURED SKELETAL MUSCLE CELLS. WE FOUND FOUR MAIN DIFFERENCES IN CULTURED SKELETAL MUSCLE CELLS FROM SUBJECTS WITH CFS; INCREASED MYOGENIN EXPRESSION IN THE BASAL STATE, IMPAIRED ACTIVATION OF AMPK, IMPAIRED STIMULATION OF GLUCOSE UPTAKE AND DIMINISHED RELEASE OF IL6. THE RETENTION OF THESE DIFFERENCES IN CULTURED MUSCLE CELLS FROM CFS SUBJECTS POINTS TO A GENETIC/EPIGENETIC MECHANISM, AND PROVIDES A SYSTEM TO IDENTIFY NOVEL THERAPEUTIC TARGETS. 2015 4 6159 33 THE GENETICS AND EPIGENETICS OF FATIGUE. FATIGUE IS A COMMON SYMPTOM AND INCLUDES BOTH PHYSICAL AND MENTAL COMPONENTS. IT CAN BE ASSOCIATED WITH A VARIETY OF DIFFERENT SYNDROMES AND DISEASES, BUT IN MANY CASES IS NOT ASSOCIATED WITH OTHER COMORBID CONDITIONS. MOST HUMANS HAVE EXPERIENCED ACUTE FATIGUE IN RELATION TO DIFFERENT STRESSORS. ACUTE FATIGUE TYPICALLY DECREASES AS THE EFFECT OF THE TRIGGERING FACTOR IS REDUCED AND A NORMAL HOMEOSTATIC BALANCE IS RESTORED. FATIGUE THAT PERSISTS FOR 6 MONTHS OR MORE IS TERMED CHRONIC FATIGUE. CHRONIC FATIGUE (CF) IN COMBINATION WITH A MINIMUM OF 4 OF 8 SYMPTOMS AND THE ABSENCE OF DISEASES THAT COULD EXPLAIN THESE SYMPTOMS, CONSTITUTE THE CASE DEFINITION FOR CHRONIC FATIGUE SYNDROME. IN SPITE OF ITS PREVALENCE, THE BIOLOGY OF FATIGUE IS RELATIVELY POORLY UNDERSTOOD AND BIOLOGICAL MARKERS HAVE NOT YET BEEN IDENTIFIED. THIS LITERATURE SEARCH WAS PERFORMED IN PUBMED TO IDENTIFY RESEARCH ON THE GENETICS AND EPIGENETICS OF FATIGUE. PUBLICATIONS WERE INCLUDED IF FATIGUE WAS A MAJOR TOPIC AND THE TOPIC WAS COMBINED WITH GENETIC AND/OR EPIGENETIC MEASUREMENTS IN ADULT HUMANS. A TOTAL OF 40 PUBLICATIONS WERE IDENTIFIED. ALTHOUGH ALTERED FUNCTIONING IN THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS, THE SEROTONERGIC SYSTEM, AND ASSOCIATIONS WITH INFECTIOUS AGENTS HAVE BEEN IDENTIFIED, THE SEARCH FOR GENETIC OR EPIGENETIC MARKERS OF FATIGUE, EITHER IN THE CONTEXT OF CF OR CHRONIC FATIGUE SYNDROME (CFS) HAS BEEN RELATIVELY UNPRODUCTIVE OR, IN THE CASE OF EPIGENETICS, NONEXISTENT. ALTHOUGH SEVERAL STUDIES, BOTH HYPOTHESIS-TESTING AND HYPOTHESIS-GENERATING, HAVE BEEN PERFORMED TO SEARCH FOR BIOMARKERS, THEY HAVE MOSTLY BEEN UNDERPOWERED, RESTRICTED BY THE HETEROGENEITY OF THE PHENOTYPE, OR LIMITED BY AN UNSYSTEMATIC STUDY DESIGN. TO BE ABLE TO CONFIRM THE HYPOTHESIS THAT RISK FOR, OR LEVELS OF, FATIGUE ARE INFLUENCED BY THE GENETIC OR EPIGENETIC BACKGROUND OF AN INDIVIDUAL, STUDIES NEED TO BE BASED ON LARGER SAMPLE SIZES WITH A MORE CLEARLY DEFINED PHENOTYPE. STUDIES NEED TO FOCUS NOT ONLY ON THE INFLUENCE OF A SINGLE ASPECT SUCH AS SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) OR DIFFERENTIAL GENE EXPRESSION ON DISEASE RISK OR STATE, BUT ALSO ON THE SYSTEMS BIOLOGY BEHIND THE DISEASE IN COMBINATION WITH INFORMATION ON ENVIRONMENTAL INFLUENCES AND VALIDATION OF FINDINGS IN FUNCTIONAL STUDIES. 2010 5 5894 27 T CELL EPIGENETIC REMODELING AND ACCELERATED EPIGENETIC AGING ARE LINKED TO LONG-TERM IMMUNE ALTERATIONS IN CHILDHOOD CANCER SURVIVORS. BACKGROUND: CANCER TREATMENTS HAVE SUBSTANTIALLY IMPROVED CHILDHOOD CANCER SURVIVAL BUT ARE ACCOMPANIED BY LONG-TERM COMPLICATIONS, NOTABLY CHRONIC INFLAMMATORY DISEASES. WE HYPOTHESIZE THAT CANCER TREATMENTS COULD LEAD TO LONG-TERM EPIGENETIC CHANGES IN IMMUNE CELLS, RESULTING IN INCREASED PREVALENCE OF INFLAMMATORY DISEASES IN CANCER SURVIVORS. RESULTS: TO TEST THIS HYPOTHESIS, WE ESTABLISHED THE EPIGENETIC AND TRANSCRIPTOMIC PROFILES OF IMMUNE CELLS FROM 44 CHILDHOOD CANCER SURVIVORS (CCS, > 16 YEARS OLD) ON FULL REMISSION (> 5 YEARS) WHO HAD RECEIVED CHEMOTHERAPY ALONE OR IN COMBINATION WITH TOTAL BODY IRRADIATION (TBI) AND HEMATOPOIETIC STEM CELL TRANSPLANT (HSCT). WE FOUND THAT MORE THAN 10 YEARS POST-TREATMENT, CCS TREATED WITH TBI/HSCT SHOWED AN ALTERED DNA METHYLATION SIGNATURE IN T CELL, PARTICULARLY AT GENES CONTROLLING IMMUNE AND INFLAMMATORY PROCESSES AND OXIDATIVE STRESS. DNA METHYLATION REMODELING IN T CELL WAS PARTIALLY ASSOCIATED WITH CHRONIC EXPRESSION CHANGES OF NEARBY GENES, INCREASED FREQUENCY OF TYPE 1 CYTOKINE-PRODUCING T CELL, ELEVATED SYSTEMIC LEVELS OF THESE CYTOKINES, AND OVER-ACTIVATION OF RELATED SIGNALING PATHWAYS. SURVIVORS EXPOSED TO TBI/HSCT WERE FURTHER CHARACTERIZED BY AN EPIGENETIC-AGING-SIGNATURE OF T CELL CONSISTENT WITH ACCELERATED EPIGENETIC AGING. TO INVESTIGATE THE POTENTIAL CONTRIBUTION OF IRRADIATION TO THESE CHANGES, WE ESTABLISHED TWO CELL CULTURE MODELS. WE IDENTIFIED THAT RADIATION PARTIALLY RECAPITULATED THE IMMUNE CHANGES OBSERVED IN SURVIVORS THROUGH A BYSTANDER EFFECT THAT COULD BE MEDIATED BY CIRCULATING FACTORS. CONCLUSION: CANCER TREATMENTS, IN PARTICULAR TBI/HSCT, ARE ASSOCIATED WITH LONG-TERM IMMUNE DISTURBANCES. WE PROPOSE THAT EPIGENETIC REMODELING OF IMMUNE CELLS FOLLOWING CANCER THERAPY AUGMENTS INFLAMMATORY- AND AGE-RELATED DISEASES, INCLUDING METABOLIC COMPLICATIONS, IN CHILDHOOD CANCER SURVIVORS. 2018 6 2408 25 EPIGENETIC REWIRING OF SKELETAL MUSCLE ENHANCERS AFTER EXERCISE TRAINING SUPPORTS A ROLE IN WHOLE-BODY FUNCTION AND HUMAN HEALTH. OBJECTIVES: REGULAR PHYSICAL EXERCISE IMPROVES HEALTH BY REDUCING THE RISK OF A PLETHORA OF CHRONIC DISORDERS. WE HYPOTHESIZED THAT ENDURANCE EXERCISE TRAINING REMODELS THE ACTIVITY OF GENE ENHANCERS IN SKELETAL MUSCLE AND THAT THIS REMODELING CONTRIBUTES TO THE BENEFICIAL EFFECTS OF EXERCISE ON HUMAN HEALTH. METHODS AND RESULTS: BY STUDYING CHANGES IN HISTONE MODIFICATIONS, WE MAPPED THE GENOME-WIDE POSITIONS AND ACTIVITIES OF ENHANCERS IN SKELETAL MUSCLE BIOPSIES COLLECTED FROM YOUNG SEDENTARY MEN BEFORE AND AFTER 6 WEEKS OF ENDURANCE EXERCISE. WE IDENTIFIED EXTENSIVE REMODELING OF ENHANCER ACTIVITIES AFTER EXERCISE TRAINING, WITH A LARGE SUBSET OF THE REMODELED ENHANCERS LOCATED IN THE PROXIMITY OF GENES TRANSCRIPTIONALLY REGULATED AFTER EXERCISE. BY OVERLAPPING THE POSITION OF ENHANCERS WITH GENETIC VARIANTS, WE IDENTIFIED AN ENRICHMENT OF DISEASE-ASSOCIATED GENETIC VARIANTS WITHIN THE EXERCISE-REMODELED ENHANCERS. CONCLUSION: OUR DATA PROVIDE EVIDENCE OF A FUNCTIONAL LINK BETWEEN EPIGENETIC REWIRING OF ENHANCERS TO CONTROL THEIR ACTIVITY AFTER EXERCISE TRAINING AND THE MODULATION OF DISEASE RISK IN HUMANS. 2021 7 2909 31 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 8 5305 29 PROTEOMICS ANALYSIS OF HUMAN OBESITY REVEALS THE EPIGENETIC FACTOR HDAC4 AS A POTENTIAL TARGET FOR OBESITY. SEDENTARY LIFESTYLE AND EXCESSIVE ENERGY INTAKE ARE PROMINENT CONTRIBUTORS TO OBESITY; A MAJOR RISK FACTORS FOR THE DEVELOPMENT OF INSULIN RESISTANCE, TYPE 2 DIABETES AND CARDIOVASCULAR DISEASES. ELUCIDATING THE MOLECULAR MECHANISMS UNDERLYING THESE CHRONIC CONDITIONS IS OF RELEVANT IMPORTANCE AS IT MIGHT LEAD TO THE IDENTIFICATION OF NOVEL ANTI-OBESITY TARGETS. THE PURPOSE OF THE CURRENT STUDY IS TO INVESTIGATE DIFFERENTIALLY EXPRESSED PROTEINS BETWEEN LEAN AND OBESE SUBJECTS THROUGH A SHOT-GUN QUANTITATIVE PROTEOMICS APPROACH USING PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) EXTRACTS AS WELL AS POTENTIAL MODULATION OF THOSE PROTEINS BY PHYSICAL EXERCISE. USING THIS APPROACH, A TOTAL OF 47 PROTEINS SHOWED AT LEAST 1.5 FOLD CHANGE BETWEEN LEAN AND OBESE SUBJECTS. IN OBESE, THE PROTEOMIC PROFILING BEFORE AND AFTER 3 MONTHS OF PHYSICAL EXERCISE SHOWED DIFFERENTIAL EXPRESSION OF 38 PROTEINS. THROMBOSPONDIN 1 (TSP1) WAS AMONG THE PROTEINS THAT WERE UPREGULATED IN OBESE SUBJECTS AND THEN DECREASED BY PHYSICAL EXERCISE. CONVERSELY, THE HISTONE DEACETYLASE 4 (HDAC4) WAS DOWNREGULATED IN OBESE SUBJECTS AND THEN INDUCED BY PHYSICAL EXERCISE. THE PROTEOMIC DATA WAS FURTHER VALIDATED BY QRT-PCR, WESTERN BLOT AND IMMUNOHISTOCHEMISTRY IN BOTH PBMCS AND ADIPOSE TISSUE. WE ALSO SHOWED THAT HDAC4 LEVELS CORRELATED POSITIVELY WITH MAXIMUM OXYGEN CONSUMPTION (VO2 MAX) BUT NEGATIVELY WITH BODY MASS INDEX, PERCENT BODY FAT, AND THE INFLAMMATORY CHEMOKINE RANTES. IN FUNCTIONAL ASSAYS, OUR DATA INDICATED THAT ECTOPIC EXPRESSION OF HDAC4 SIGNIFICANTLY IMPAIRED TNF-ALPHA-DEPENDENT ACTIVATION OF NF-KAPPAB, ESTABLISHING THUS A LINK BETWEEN HDAC4 AND REGULATION OF THE IMMUNE SYSTEM. TOGETHER, THE EXPRESSION PATTERN OF HDAC4 IN OBESE SUBJECTS BEFORE AND AFTER PHYSICAL EXERCISE, ITS CORRELATION WITH VARIOUS PHYSICAL, CLINICAL AND METABOLIC PARAMETERS ALONG WITH ITS INHIBITORY EFFECT ON NF-KAPPAB ARE SUGGESTIVE OF A PROTECTIVE ROLE OF HDAC4 AGAINST OBESITY. HDAC4 COULD THEREFORE REPRESENT A POTENTIAL THERAPEUTIC TARGET FOR THE CONTROL AND MANAGEMENT OF OBESITY AND PRESUMABLY INSULIN RESISTANCE. 2013 9 271 19 AGE-ASSOCIATED EPIGENETIC MODIFICATIONS IN HUMAN DNA INCREASE ITS IMMUNOGENICITY. CHRONIC INFLAMMATION, INCREASED REACTIVITY TO SELF-ANTIGENS AND INCIDENCES OF CANCER ARE HALLMARKS OF AGING. HOWEVER, THE UNDERLYING MECHANISMS ARE NOT WELL UNDERSTOOD. AGE-ASSOCIATED ALTERATIONS IN THE DNA EITHER DUE TO OXIDATIVE DAMAGE, DEFECTS IN DNA REPAIR OR EPIGENETIC MODIFICATIONS SUCH AS METHYLATION THAT LEAD TO MUTATIONS AND CHANGES IN THE EXPRESSION OF GENES ARE THOUGHT TO BE PARTIALLY RESPONSIBLE. HERE WE REPORT THAT EPIGENETIC MODIFICATIONS IN AGED DNA ALSO INCREASE ITS IMMUNOGENICITY RENDERING IT MORE REACTIVE TO INNATE IMMUNE SYSTEM CELLS SUCH AS THE DENDRITIC CELLS. WE OBSERVED INCREASED UPREGULATION OF COSTIMULATORY MOLECULES AS WELL AS ENHANCED SECRETION OF IFN-ALPHA FROM DENDRITIC CELLS IN RESPONSE TO DNA FROM AGED DONORS AS COMPARED TO DNA FROM YOUNG DONORS WHEN IT WAS DELIVERED INTRACELLULARLY VIA LIPOFECTAMINE. INVESTIGATIONS INTO THE MECHANISMS REVEALED THAT DNA FROM AGED SUBJECTS IS NOT DEGRADED, NEITHER IS IT MORE DAMAGED COMPARED TO DNA FROM YOUNG SUBJECTS. HOWEVER, THERE IS SIGNIFICANTLY DECREASED GLOBAL LEVEL OF METHYLATION SUGGESTING THAT AGE-ASSOCIATED HYPOMETHYLATION OF THE DNA MAY BE THE CAUSE OF ITS INCREASED IMMUNOGENICITY. INCREASED IMMUNOGENICITY OF SELF DNA MAY THUS BE ANOTHER MECHANISM THAT MAY CONTRIBUTE TO THE INCREASE IN AGE-ASSOCIATED CHRONIC INFLAMMATION, AUTOIMMUNITY AND CANCER. 2010 10 1307 32 DEFINING A METHYLATION SIGNATURE ASSOCIATED WITH OPERATIONAL TOLERANCE IN KIDNEY TRANSPLANT RECIPIENTS. OPERATIONAL TOLERANCE AFTER KIDNEY TRANSPLANTATION IS DEFINED AS STABLE GRAFT ACCEPTANCE WITHOUT THE NEED FOR IMMUNOSUPPRESSION THERAPY. HOWEVER, IT IS NOT CLEAR WHICH CELLULAR AND MOLECULAR PATHWAYS ARE DRIVING TOLERANCE IN THESE PATIENTS. WE PERFORMED GENOME-WIDE ANALYSIS OF DNA METHYLATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM KIDNEY TRANSPLANT RECIPIENTS WITH CHRONIC REJECTION AND OPERATIONAL TOLERANCE FROM THE GENETIC ANALYSIS OF MOLECULAR BIOMARKERS OF IMMUNOLOGICAL TOLERANCE (GAMBIT) STUDY. OUR RESULTS SHOWED THAT BOTH CLINICAL STAGES DIVERGE IN 2737 GENES, INDICATING THAT EACH ONE HAS A SPECIFIC METHYLATION SIGNATURE ASSOCIATED WITH TRANSPLANT OUTCOME. WE ALSO OBSERVED THAT TOLERANCE IS ASSOCIATED WITH DEMETHYLATION IN GENES INVOLVED IN IMMUNE FUNCTION, INCLUDING B AND T CELL ACTIVATION AND TH17 DIFFERENTIATION, WHILE IN CHRONIC REJECTION IT IS ASSOCIATED WITH INTRACELLULAR SIGNALING AND UBIQUITINATION PATHWAYS. USING CO-EXPRESSION NETWORK ANALYSIS, WE SELECTED 12 GENOMIC REGIONS THAT ARE SPECIFICALLY HYPOMETHYLATED OR HYPERMETHYLATED IN TOLERANT PATIENTS. ANALYSIS OF THESE GENES IN TRANSPLANTED PATIENTS WITH LOW DOSE OF STEROIDS SHOWED THAT THESE HAVE A SIMILAR METHYLATION SIGNATURE TO THAT OF TOLERANT RECIPIENTS. OVERALL, THESE RESULTS DEMONSTRATE THAT METHYLATION ANALYSIS CAN MIRROR THE IMMUNE STATUS ASSOCIATED WITH TRANSPLANT OUTCOME AND PROVIDES A STARTING POINT FOR UNDERSTANDING THE EPIGENETIC MECHANISMS ASSOCIATED WITH TOLERANCE. 2021 11 3292 29 HIGH FAT DIET AND EXERCISE LEAD TO A DISRUPTED AND PATHOGENIC DNA METHYLOME IN MOUSE LIVER. HIGH-FAT DIET CONSUMPTION AND SEDENTARY LIFESTYLE ELEVATES RISK FOR OBESITY, NON-ALCOHOLIC FATTY LIVER DISEASE, AND CANCER. EXERCISE TRAINING CONVEYS HEALTH BENEFITS IN POPULATIONS WITH OR WITHOUT THESE CHRONIC CONDITIONS. DIET AND EXERCISE REGULATE GENE EXPRESSION BY MEDIATING EPIGENETIC MECHANISMS IN MANY TISSUES; HOWEVER, SUCH EFFECTS ARE POORLY DOCUMENTED IN THE LIVER, A CENTRAL METABOLIC ORGAN. TO DISSECT THE CONSEQUENCES OF DIET AND EXERCISE ON THE LIVER EPIGENOME, WE MEASURED DNA METHYLATION, USING REDUCED REPRESENTATION BISULFITE SEQUENCING, AND TRANSCRIPTION, USING RNA-SEQ, IN MICE MAINTAINED ON A FAST FOOD DIET WITH SEDENTARY LIFESTYLE OR EXERCISE, COMPARED WITH CONTROL DIET WITH AND WITHOUT EXERCISE. OUR ANALYSES REVEAL THAT GENOME-WIDE DIFFERENTIAL DNA METHYLATION AND EXPRESSION OF GENE CLUSTERS ARE INDUCED BY DIET AND/OR EXERCISE. A COMBINATION OF FAST FOOD AND EXERCISE TRIGGERS EXTENSIVE GENE ALTERATIONS, WITH ENRICHMENT OF CARBOHYDRATE/LIPID METABOLIC PATHWAYS AND MUSCLE DEVELOPMENTAL PROCESSES. THROUGH EVALUATION OF PUTATIVE PROTECTIVE EFFECTS OF EXERCISE ON DIET-INDUCED DNA METHYLATION, WE SHOW THAT HYPERMETHYLATION IS EFFECTIVELY PREVENTED, ESPECIALLY AT PROMOTERS AND ENHANCERS, WHEREAS HYPOMETHYLATION IS ONLY PARTIALLY ATTENUATED. WE ASSESSED DIET-INDUCED DNA METHYLATION CHANGES ASSOCIATED WITH LIVER CANCER-RELATED EPIGENETIC MODIFICATIONS AND IDENTIFIED SIGNIFICANT INCREASES AT LIVER-SPECIFIC ENHANCERS IN FAST FOOD GROUPS, SUGGESTING PARTIAL LOSS OF LIVER CELL IDENTITY. HYPERMETHYLATION AT A SUBSET OF GENE PROMOTERS WAS ASSOCIATED WITH INHIBITION OF TISSUE DEVELOPMENT AND PROMOTION OF CARCINOGENIC PROCESSES. OUR STUDY DEMONSTRATES EXTENSIVE REPROGRAMMING OF THE EPIGENOME BY DIET AND EXERCISE, EMPHASIZING THE FUNCTIONAL RELEVANCE OF EPIGENETIC MECHANISMS AS AN INTERFACE BETWEEN LIFESTYLE MODIFICATIONS AND PHENOTYPIC ALTERATIONS. 2017 12 2499 17 EPIGENETICS AND EXERCISE. EPIGENETICS CAN BE DEFINED AS 'THE STRUCTURAL ADAPTATION OF CHROMOSOMAL REGIONS SO AS TO REGISTER, SIGNAL, OR PERPETUATE ALTERED ACTIVITY STATES.' INCREASED TRANSCRIPTION OF KEY REGULATORY, METABOLIC, AND MYOGENIC GENES IS AN EARLY RESPONSE TO EXERCISE AND IS IMPORTANT IN MEDIATING SUBSEQUENT ADAPTATIONS IN SKELETAL MUSCLE. DNA HYPOMETHYLATION AND HISTONE HYPERACETYLATION ARE EMERGING AS IMPORTANT CRUCIAL EVENTS FOR INCREASED TRANSCRIPTION. THE COMPLEX INTERACTIONS BETWEEN MULTIPLE EPIGENETIC MODIFICATIONS AND THEIR REGULATION BY METABOLIC CHANGES AND SIGNALING EVENTS DURING EXERCISE, WITH IMPLICATIONS FOR ENHANCED UNDERSTANDING OF THE ACUTE AND CHRONIC ADAPTATIONS TO EXERCISE, ARE QUESTIONS FOR FURTHER INVESTIGATION. 2019 13 1269 26 CYTOSINE METHYLATION CHANGES IN ENHANCER REGIONS OF CORE PRO-FIBROTIC GENES CHARACTERIZE KIDNEY FIBROSIS DEVELOPMENT. BACKGROUND: ONE IN ELEVEN PEOPLE IS AFFECTED BY CHRONIC KIDNEY DISEASE, A CONDITION CHARACTERIZED BY KIDNEY FIBROSIS AND PROGRESSIVE LOSS OF KIDNEY FUNCTION. EPIDEMIOLOGICAL STUDIES INDICATE THAT ADVERSE INTRAUTERINE AND POSTNATAL ENVIRONMENTS HAVE A LONG-LASTING ROLE IN CHRONIC KIDNEY DISEASE DEVELOPMENT. EPIGENETIC INFORMATION REPRESENTS A PLAUSIBLE CARRIER FOR MEDIATING THIS PROGRAMMING EFFECT. HERE WE DEMONSTRATE THAT GENOME-WIDE CYTOSINE METHYLATION PATTERNS OF HEALTHY AND CHRONIC KIDNEY DISEASE TUBULE SAMPLES OBTAINED FROM PATIENTS SHOW SIGNIFICANT DIFFERENCES. RESULTS: WE IDENTIFY DIFFERENTIALLY METHYLATED REGIONS AND VALIDATE THESE IN A LARGE REPLICATION DATASET. THE DIFFERENTIALLY METHYLATED REGIONS ARE RARELY OBSERVED ON PROMOTERS, BUT MOSTLY OVERLAP WITH PUTATIVE ENHANCER REGIONS, AND THEY ARE ENRICHED IN CONSENSUS BINDING SEQUENCES FOR IMPORTANT RENAL TRANSCRIPTION FACTORS. THIS INDICATES THEIR IMPORTANCE IN GENE EXPRESSION REGULATION. A CORE SET OF GENES THAT ARE KNOWN TO BE RELATED TO KIDNEY FIBROSIS, INCLUDING GENES ENCODING COLLAGENS, SHOW CYTOSINE METHYLATION CHANGES CORRELATING WITH DOWNSTREAM TRANSCRIPT LEVELS. CONCLUSIONS: OUR REPORT RAISES THE POSSIBILITY THAT EPIGENETIC DYSREGULATION PLAYS A ROLE IN CHRONIC KIDNEY DISEASE DEVELOPMENT VIA INFLUENCING CORE PRO-FIBROTIC PATHWAYS AND CAN AID THE DEVELOPMENT OF NOVEL BIOMARKERS AND FUTURE THERAPEUTICS. 2013 14 822 27 CHARACTERIZATION OF BLOOD SURROGATE IMMUNE-METHYLATION BIOMARKERS FOR IMMUNE CELL INFILTRATION IN CHRONIC INFLAMMAGING DISORDERS. ALZHEIMER'S DISEASE (AD) AND ATHEROSCLEROSIS ARE BOTH CHRONIC AGE- AND INFLAMMATION-DEPENDENT DISEASES. IN ADDITION, ATHEROSCLEROSIS IS FREQUENTLY OBSERVED IN AD PATIENTS INDICATING COMMON INVOLVEMENT OF VASCULAR COMPONENTS IN BOTH DISEASE ETIOLOGIES. RECENTLY, EPIGENOME-WIDE ASSOCIATION STUDIES HAVE IDENTIFIED EPIGENETIC ALTERATIONS, AND IN PARTICULARLY DNA METHYLATION CHANGES FOR BOTH DISORDERS. WE HYPOTHESIZED THE EXISTENCE OF A COMMON DNA METHYLATION PROFILE IN ATHEROSCLEROSIS AND AD WHICH MAY BE VALUABLE AS A BLOOD-BASED DNA METHYLATION INFLAMMAGING BIOMARKER. USING PUBLICLY AVAILABLE 450K ILLUMINA METHYLATION DATASETS, WE IDENTIFIED A CO-METHYLATION NETWORK ASSOCIATED WITH BOTH ATHEROSCLEROSIS AND AD IN WHOLE BLOOD SAMPLES. THIS METHYLATION PROFILE APPEARED TO INDICATE SHIFTS IN BLOOD IMMUNE CELL TYPE DISTRIBUTION. REMARKABLY, SIMILAR METHYLATION CHANGES WERE ALSO DETECTED IN DISEASE TISSUES, INCLUDING AD BRAIN TISSUES, ATHEROSCLEROTIC PLAQUES, AND TUMORS AND WERE FOUND TO CORRELATE WITH IMMUNE CELL INFILTRATION. IN ADDITION, THIS IMMUNE-RELATED METHYLATION PROFILE COULD ALSO BE DETECTED IN OTHER INFLAMMAGING DISEASES, INCLUDING PARKINSON'S DISEASE AND OBESITY, BUT NOT IN MULTIPLE SCLEROSIS, SCHIZOPHRENIA, AND OSTEOPOROSIS. IN CONCLUSION, WE IDENTIFIED A BLOOD-BASED IMMUNE-RELATED DNA METHYLATION SIGNATURE IN MULTIPLE INFLAMMAGING DISEASES ASSOCIATED WITH CHANGES IN BLOOD IMMUNE CELL COUNTS AND PREDICTIVE FOR IMMUNE CELL INFILTRATION IN DISEASED TISSUES. IN ADDITION TO EPIGENETIC CLOCK MEASUREMENTS, THIS IMMUNE-METHYLATION SIGNATURE MAY BECOME A VALUABLE BLOOD-BASED BIOMARKER TO PREVENT CHRONIC INFLAMMATORY DISEASE DEVELOPMENT OR MONITOR LIFESTYLE INTERVENTION STRATEGIES WHICH PROMOTE HEALTHY AGING. 2019 15 457 28 APPLYING A MULTISCALE SYSTEMS BIOLOGY APPROACH TO STUDY THE EFFECT OF CHRONIC LOW-DOSE EXPOSURE TO URANIUM IN RAT KIDNEYS. PURPOSE: TO EXAMINE THE EFFECTS OF LOW-DOSE EXPOSURE TO URANIUM WITH A SYSTEMS BIOLOGY APPROACH, A MULTISCALE HIGH-THROUGHPUT MULTI-OMICS ANALYSIS WAS APPLIED WITH A PROTOCOL FOR CHRONIC EXPOSURE TO THE RAT KIDNEY. METHODS: MALE AND FEMALE RATS WERE CONTAMINATED FOR NINE MONTHS THROUGH THEIR DRINKING WATER WITH A NONTOXIC SOLUTION OF URANYL NITRATE. A MULTISCALE APPROACH ENABLED CLINICAL MONITORING ASSOCIATED WITH METABOLOMIC AND TRANSCRIPTOMIC (MRNA AND MICRORNA) ANALYSES. RESULTS: A SEX-INTERACTION EFFECT WAS OBSERVED IN THE KIDNEY, URINE, AND PLASMA METABOLOMES OF CONTAMINATED RATS. MOREOVER, URINE AND KIDNEY METABOLIC PROFILES CORRELATED AND CONFIRMED THAT THE PRIMARY DYSREGULATED METABOLISMS ARE THOSE OF NICOTINATE-NICOTINAMIDE AND OF UNSATURATED FATTY ACID BIOSYNTHESIS. UPSTREAM OF THE METABOLIC PATHWAYS, TRANSCRIPTOMIC PROFILES OF THE KIDNEY REVEAL GENE ACTIVITY FOCUSED ON GENE REGULATION MECHANISMS, CELL SIGNALING, CELL STRUCTURE, DEVELOPMENTAL PROCESSES, AND CELL PROLIFERATION. EXAMINATION OF EPIGENETIC POST-TRANSCRIPTIONAL GENE REGULATION PROCESSES SHOWED SIGNIFICANT DYSREGULATION OF 70 MICRO-RNAS. THE MULTI-OMICS APPROACH HIGHLIGHTED THE ACTIVITIES OF THE CELLS' BIOLOGICAL PROCESSES ON MULTIPLE SCALES THROUGH ANALYSIS OF GENE EXPRESSION, CONFIRMED BY CHANGES OBSERVED IN THE METABOLOME. CONCLUSION: OUR RESULTS SHOWED CHANGES IN MULTI-OMIC PROFILES OF RATS EXPOSED TO LOW DOSES OF URANIUM CONTAMINATION, COMPARED WITH CONTROLS. THESE CHANGES INVOLVED GENE EXPRESSION AS WELL AS MODIFICATIONS IN THE TRANSCRIPTOME AND THE METABOLOME. THE METABOLOMIC PROFILE CONFIRMED THAT THE MAIN MOLECULAR TARGETS OF URANIUM IN KIDNEY CELLS ARE THE METABOLISM OF NICOTINATE-NICOTINAMIDE AND THE BIOSYNTHESIS OF UNSATURATED FATTY ACIDS. ADDITIONALLY, GENE EXPRESSION ANALYSIS SHOWED THAT THE METABOLISM OF FATTY ACIDS IS TARGETED BY PROCESSES ASSOCIATED WITH CELL FUNCTION. THESE RESULTS DEMONSTRATE THAT MULTISCALE SYSTEMS BIOLOGY IS USEFUL IN ELUCIDATING THE MOST DISCRIMINATIVE PATHWAYS FROM GENOMIC TO METABOLOMIC LEVELS FOR ASSESSING THE BIOLOGICAL IMPACT OF THIS LOW-LEVEL ENVIRONMENTAL EXPOSURE, I.E. THE EXPOSOME. 2019 16 1508 30 DNA METHYLATION AND MRNA AND MICRORNA EXPRESSION OF SLE CD4+ T CELLS CORRELATE WITH DISEASE PHENOTYPE. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS AN AUTOIMMUNE DISEASE WELL KNOWN FOR ITS CLINICAL HETEROGENEITY, AND ITS ETIOLOGY SECONDARY TO A CROSS-TALK INVOLVING GENETIC PREDISPOSITION AND ENVIRONMENTAL STIMULI. ALTHOUGH GENOME-WIDE ANALYSIS HAS CONTRIBUTED GREATLY TO OUR UNDERSTANDING OF THE GENETIC BASIS OF SLE, THERE IS INCREASING EVIDENCE FOR A ROLE OF EPIGENETICS. INDEED, RECENT DATA HAVE DEMONSTRATED THAT IN PATIENTS WITH SLE, THERE ARE STRIKING ALTERATIONS OF DNA METHYLATION, HISTONE MODIFICATIONS, AND DEREGULATED MICRORNA EXPRESSION, THE SUM OF WHICH CONTRIBUTE TO OVER-EXPRESSION OF SELECT AUTOIMMUNE-RELATED GENES AND LOSS OF TOLERANCE. TO ADDRESS THIS ISSUE AT THE LEVEL OF CLINICAL PHENOTYPE, WE PERFORMED DNA METHYLATION, MRNA AND MICRORNA EXPRESSION SCREENING USING HIGH-THROUGHPUT SEQUENCING OF PURIFIED CD4+ T CELLS FROM PATIENTS WITH SLE, COMPARED TO AGE AND SEX MATCHED CONTROLS. IN PARTICULAR, WE STUDIED 42 PATIENTS WITH SLE AND DIVIDED THIS GROUP INTO THREE CLINICAL PHENOTYPES: A) THE PRESENCE OF SKIN LESIONS WITHOUT SIGNS OF SYSTEMIC PATHOLOGY; B) SKIN LESIONS BUT ALSO CHRONIC RENAL PATHOLOGY; AND C) SKIN LESIONS, CHRONIC RENAL PATHOLOGY AND POLYARTICULAR DISEASE. INTERESTINGLY, AND AS EXPECTED, SEQUENCING DATA REVEALED CHANGES IN DNA METHYLATION IN SLE COMPARED TO CONTROLS. HOWEVER, AND MORE IMPORTANTLY, ALTHOUGH THERE WERE COMMON METHYLATION CHANGES FOUND IN ALL GROUPS OF SLE COMPARED TO CONTROLS, THERE WAS SPECIFIC DNA METHYLATION CHANGES THAT CORRELATED WITH CLINICAL PHENOTYPE. THESE INCLUDED CHANGES IN THE NOVEL KEY TARGET GENES NLRP2, CD300LB AND S1PR3, AS WELL AS CHANGES IN THE CRITICAL PATHWAYS, INCLUDING THE ADHERENS JUNCTION AND LEUKOCYTE TRANSENDOTHELIAL MIGRATION. WE ALSO NOTED THAT A SIGNIFICANT PROPORTION OF GENES UNDERGOING DNA METHYLATION CHANGES WERE INVERSELY CORRELATED WITH GENE EXPRESSION AND THAT MIRNA SCREENING REVEALED THE EXISTENCE OF SUBSETS WITH CHANGES IN EXPRESSION. INTEGRATED ANALYSIS OF THIS DATA HIGHLIGHTS SPECIFIC SETS OF MIRNAS CONTROLLED BY DNA METHYLATION, AND GENES THAT ARE ALTERED BY METHYLATION AND TARGETED BY MIRNAS. IN CONCLUSION, OUR FINDINGS SUGGEST SELECT EPIGENETIC MECHANISMS THAT CONTRIBUTE TO CLINICAL PHENOTYPES AND FURTHER SHED LIGHT ON A NEW VENUE FOR BASIC SLE RESEARCH. 2014 17 948 17 CHRONIC METABOLIC DERANGEMENT-INDUCED COGNITIVE DEFICITS AND NEUROTOXICITY ARE ASSOCIATED WITH REST INACTIVATION. CHRONIC METABOLIC ALTERATIONS MAY REPRESENT A RISK FACTOR FOR THE DEVELOPMENT OF COGNITIVE IMPAIRMENT, DEMENTIA, OR NEURODEGENERATIVE DISEASES. HYPERGLYCEMIA AND OBESITY ARE KNOWN TO IMPRINT EPIGENETIC MARKERS THAT COMPROMISE THE PROPER EXPRESSION OF CELL SURVIVAL GENES. HERE, WE SHOWED THAT CHRONIC HYPERGLYCEMIA (60 DAYS) INDUCED BY A SINGLE INTRAPERITONEAL INJECTION OF STREPTOZOTOCIN COMPROMISED COGNITION BY REDUCING HIPPOCAMPAL ERK SIGNALING AND BY INDUCING NEUROTOXICITY IN RATS. THE MECHANISMS APPEAR TO BE LINKED TO REDUCED ACTIVE DNA DEMETHYLATION AND DIMINISHED EXPRESSION OF THE NEUROPROTECTIVE TRANSCRIPTION FACTOR REST. THE IMPACT OF THE RELATIONSHIP BETWEEN ADIPOSITY AND DNA HYPERMETHYLATION ON REST EXPRESSION WAS ALSO DEMONSTRATED IN PERIPHERAL BLOOD MONONUCLEAR CELLS IN OBESE CHILDREN WITH REDUCED LEVELS OF BLOOD ASCORBATE. THE REVERSIBLE NATURE OF EPIGENETIC MODIFICATIONS AND THE COGNITIVE IMPAIRMENT REPORTED IN OBESE CHILDREN, ADOLESCENTS, AND ADULTS SUGGEST THAT THE CORRECTION OF THE ANTHROPOMETRY AND THE PERIPHERAL METABOLIC ALTERATIONS WOULD PROTECT BRAIN HOMEOSTASIS AND REDUCE THE RISK OF DEVELOPING NEURODEGENERATIVE DISEASES. 2019 18 1794 31 EFFECT OF DIABETES STATUS AND HYPERGLYCEMIA ON GLOBAL DNA METHYLATION AND HYDROXYMETHYLATION. TYPE 2 DIABETES MELLITUS (T2DM) IS CHARACTERIZED BY OXIDATIVE STRESS THAT COULD LEAD TO CHRONIC MICRO- AND MACROVASCULAR COMPLICATIONS. WE HYPOTHESIZED THAT SOME OF THE TARGET ORGAN DAMAGE IS MEDIATED BY OXIDATIVE ALTERATIONS IN EPIGENETIC MECHANISMS INVOLVING DNA METHYLATION (5MC) AND DNA HYDROXYMETHYLATION (5HMC). WE ANALYZED GLOBAL DNA METHYLATION AND HYDROXYMETHYLATION IN PERIPHERAL BLOOD CELLS IN WELL-CONTROLLED AND POORLY CONTROLLED PATIENTS WITH T2DM AND COMPARED THEM WITH HEALTHY CONTROLS. WE ALSO ANALYZED MICROARRAYS OF DNA METHYLATION AND GENE EXPRESSION OF OTHER IMPORTANT TISSUES IN THE CONTEXT OF DIABETES FROM THE GEO DATABASE REPOSITORY AND THEN COMPARED THESE RESULTS WITH OUR EXPERIMENTAL GENE EXPRESSION DATA. DNA METHYLATION AND, MORE IMPORTANTLY, DNA HYDROXYMETHYLATION LEVELS WERE INCREASED IN POORLY CONTROLLED PATIENTS COMPARED TO WELL-CONTROLLED AND HEALTHY INDIVIDUALS. BOTH 5MC AND 5HMC MEASUREMENTS WERE CORRELATED WITH THE PERCENTAGE OF GLYCATED HEMOGLOBIN, INDICATING A DIRECT IMPACT OF HYPERGLYCEMIA ON CHANGES OVER THE EPIGENOME. THE ANALYSIS OF METHYLATION MICROARRAYS WAS CONCORDANT, AND 5MC LEVELS WERE INCREASED IN THE PERIPHERAL BLOOD OF T2DM PATIENTS. HOWEVER, THE DNA METHYLATION LEVELS WERE THE OPPOSITE OF THOSE IN OTHER TISSUES, SUCH AS THE PANCREAS, ADIPOSE TISSUE AND SKELETAL MUSCLE. WE HYPOTHESIZE THAT A PROCESS OF DNA OXIDATION ASSOCIATED WITH HYPERGLYCEMIA MAY EXPLAIN THE DNA DEMETHYLATION IN WHICH THE ACTIVITY OF TEN-ELEVEN TRANSLOCATION (TET) PROTEINS IS NOT SUFFICIENT TO COMPLETE THE PROCESS. HIGH LEVELS OF GLUCOSE LEAD TO CELLULAR OXIDATION, WHICH TRIGGERS THE PROCESS OF DNA DEMETHYLATION AIDED BY TET ENZYMES, RESULTING IN EPIGENETIC DYSREGULATION OF THE DAMAGED TISSUES. 2017 19 2914 32 GENE REGULATORY NETWORK UNDERLYING THE IMMORTALIZATION OF EPITHELIAL CELLS. BACKGROUND: TUMORIGENIC TRANSFORMATION OF HUMAN EPITHELIAL CELLS IN VITRO HAS BEEN DESCRIBED EXPERIMENTALLY AS THE POTENTIAL RESULT OF SPONTANEOUS IMMORTALIZATION. THIS PROCESS IS CHARACTERIZED BY A SERIES OF CELL-STATE TRANSITIONS, IN WHICH NORMAL EPITHELIAL CELLS ACQUIRE FIRST A SENESCENT STATE WHICH IS LATER SURPASSED TO ATTAIN A MESENCHYMAL STEM-LIKE PHENOTYPE WITH A POTENTIALLY TUMORIGENIC BEHAVIOR. IN THIS PAPER WE AIM TO PROVIDE A SYSTEM-LEVEL MECHANISTIC EXPLANATION TO THE EMERGENCE OF THESE CELL TYPES, AND TO THE TIME-ORDERED TRANSITION PATTERNS THAT ARE COMMON TO NEOPLASIAS OF EPITHELIAL ORIGIN. TO THIS END, WE FIRST INTEGRATE PUBLISHED FUNCTIONAL AND WELL-CURATED MOLECULAR DATA OF THE COMPONENTS AND INTERACTIONS THAT HAVE BEEN FOUND TO BE INVOLVED IN SUCH CELL STATES AND TRANSITIONS INTO A NETWORK OF 41 MOLECULAR COMPONENTS. WE THEN REDUCE THIS INITIAL NETWORK BY REMOVING SIMPLE MEDIATORS (I.E., LINEAR PATHWAYS), AND FORMALIZE THE RESULTING REGULATORY CORE INTO LOGICAL RULES THAT GOVERN THE DYNAMICS OF EACH OF THE NETWORK COMPONENTS AS A FUNCTION OF THE STATES OF ITS REGULATORS. RESULTS: COMPUTATIONAL DYNAMIC ANALYSIS SHOWS THAT OUR PROPOSED GENE REGULATORY NETWORK MODEL RECOVERS EXACTLY THREE ATTRACTORS, EACH OF THEM DEFINED BY A SPECIFIC GENE EXPRESSION PROFILE THAT CORRESPONDS TO THE EPITHELIAL, SENESCENT, AND MESENCHYMAL STEM-LIKE CELLULAR PHENOTYPES, RESPECTIVELY. WE SHOW THAT ALTHOUGH A MESENCHYMAL STEM-LIKE STATE CAN BE ATTAINED EVEN UNDER UNPERTURBED PHYSIOLOGICAL CONDITIONS, THE LIKELIHOOD OF CONVERGING TO THIS STATE IS INCREASED WHEN PRO-INFLAMMATORY CONDITIONS ARE SIMULATED, PROVIDING A SYSTEMS-LEVEL MECHANISTIC EXPLANATION FOR THE CARCINOGENIC ROLE OF CHRONIC INFLAMMATORY CONDITIONS OBSERVED IN THE CLINIC. WE ALSO FOUND THAT THE REGULATORY CORE YIELDS AN EPIGENETIC LANDSCAPE THAT RESTRICTS TEMPORAL PATTERNS OF PROGRESSION BETWEEN THE STEADY STATES, SUCH THAT RECOVERED PATTERNS RESEMBLE THE TIME-ORDERED TRANSITIONS OBSERVED DURING THE SPONTANEOUS IMMORTALIZATION OF EPITHELIAL CELLS, BOTH IN VIVO AND IN VITRO. CONCLUSION: OUR STUDY STRONGLY SUGGESTS THAT THE IN VITRO TUMORIGENIC TRANSFORMATION OF EPITHELIAL CELLS, WHICH STRONGLY CORRELATES WITH THE PATTERNS OBSERVED DURING THE PATHOLOGICAL PROGRESSION OF EPITHELIAL CARCINOGENESIS IN VIVO, EMERGES FROM UNDERLYING REGULATORY NETWORKS INVOLVED IN EPITHELIAL TRANS-DIFFERENTIATION DURING DEVELOPMENT. 2017 20 2022 24 EPIGENETIC CHANGES ASSOCIATED WITH DISEASE PROGRESSION IN A MOUSE MODEL OF CHILDHOOD ALLERGIC ASTHMA. DEVELOPMENT OF ASTHMA IN CHILDHOOD IS LINKED TO VIRAL INFECTIONS OF THE LOWER RESPIRATORY TRACT IN EARLY LIFE, WITH SUBSEQUENT CHRONIC EXPOSURE TO ALLERGENS. PROGRESSION TO PERSISTENT ASTHMA IS ASSOCIATED WITH A TH2-BIASED IMMUNOLOGICAL RESPONSE AND STRUCTURAL REMODELLING OF THE AIRWAYS. THE UNDERLYING MECHANISMS ARE UNCLEAR, BUT COULD INVOLVE EPIGENETIC CHANGES. TO INVESTIGATE THIS, WE EMPLOYED A RECENTLY DEVELOPED MOUSE MODEL IN WHICH SELF-LIMITED NEONATAL INFECTION WITH A PNEUMOVIRUS, FOLLOWED BY SENSITISATION TO OVALBUMIN VIA THE RESPIRATORY TRACT AND LOW-LEVEL CHRONIC CHALLENGE WITH AEROSOLISED ANTIGEN, LEADS TO DEVELOPMENT OF AN ASTHMATIC PHENOTYPE. WE ASSESSED EXPRESSION OF MICRORNA BY CELLS IN THE PROXIMAL AIRWAYS, COMPARING CHANGES OVER THE PERIOD OF DISEASE PROGRESSION, AND USED TARGET PREDICTION DATABASES TO IDENTIFY GENES LIKELY TO BE UP- OR DOWNREGULATED AS A CONSEQUENCE OF ALTERED REGULATION OF MICRORNA. IN PARALLEL, WE ASSESSED DNA METHYLATION IN PULMONARY CD4(+) T CELLS. WE FOUND THAT A LIMITED NUMBER OF MICRORNAS EXHIBITED MARKED UP- OR DOWNREGULATION FOLLOWING EARLY-LIFE INFECTION AND SENSITISATION, FOR MANY OF WHICH THE LEVELS OF EXPRESSION WERE FURTHER CHANGED FOLLOWING CHRONIC CHALLENGE WITH THE SENSITIZING ANTIGEN. TARGETS OF THESE MICRORNAS INCLUDED GENES INVOLVED IN IMMUNE OR INFLAMMATORY RESPONSES (E.G. GATA3, KITL) AND IN TISSUE REMODELLING (E.G. IGF1, TGFBR1), AS WELL AS GENES FOR VARIOUS TRANSCRIPTION FACTORS AND SIGNALLING PROTEINS. IN PULMONARY CD4(+) T CELLS, THERE WAS SIGNIFICANT DEMETHYLATION AT PROMOTER SITES FOR INTERLEUKIN-4 AND INTERFERON-GAMMA, THE LATTER INCREASING FOLLOWING CHRONIC CHALLENGE. WE CONCLUDE THAT, IN THIS MODEL, PROGRESSION TO AN ASTHMATIC PHENOTYPE IS LINKED TO EPIGENETIC REGULATION OF GENES ASSOCIATED WITH INFLAMMATION AND STRUCTURAL REMODELLING, AND WITH T-CELL COMMITMENT TO A TH2 IMMUNOLOGICAL RESPONSE. EPIGENETIC CHANGES ASSOCIATED WITH THIS PATTERN OF GENE ACTIVATION MIGHT PLAY A ROLE IN THE DEVELOPMENT OF CHILDHOOD ASTHMA. 2013