1  1124 38 COMPLETE GENOME SEQUENCE OF MORAXELLA CATARRHALIS STRAIN CCRI-195ME, ISOLATED FROM THE MIDDLE EAR. MORAXELLA CATARRHALIS IS AN IMPORTANT BACTERIAL PATHOGEN THAT CAUSES OTITIS MEDIA AND EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE. HERE, WE REPORT THE COMPLETE GENOME SEQUENCE OF M. CATARRHALIS STRAIN CCRI-195ME, WHICH CONTAINS THE PHASE-VARIABLE EPIGENETIC REGULATOR MODM3.	2017	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
2  4395 29 MODM DNA METHYLTRANSFERASE METHYLOME ANALYSIS REVEALS A POTENTIAL ROLE FOR MORAXELLA CATARRHALIS PHASEVARIONS IN OTITIS MEDIA. MORAXELLA CATARRHALIS IS A SIGNIFICANT CAUSE OF OTITIS MEDIA AND EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE. HERE, WE CHARACTERIZE A PHASE-VARIABLE DNA METHYLTRANSFERASE (MODM), WHICH CONTAINS 5'-CAAC-3' REPEATS IN ITS OPEN READING FRAME THAT MEDIATE HIGH-FREQUENCY MUTATION RESULTING IN REVERSIBLE ON/OFF SWITCHING OF MODM EXPRESSION. THREE MODM ALLELES HAVE BEEN IDENTIFIED (MODM1-3), WITH MODM2 BEING THE MOST COMMONLY FOUND ALLELE. USING SINGLE-MOLECULE, REAL-TIME (SMRT) GENOME SEQUENCING AND METHYLOME ANALYSIS, WE HAVE DETERMINED THAT THE MODM2 METHYLATION TARGET IS 5'-GAR(M6)AC-3', AND 100% OF THESE SITES ARE METHYLATED IN THE GENOME OF THE M. CATARRHALIS 25239 MODM2 ON STRAIN. PROTEOMIC ANALYSIS OF MODM2 ON AND OFF VARIANTS REVEALED THAT MODM2 REGULATES EXPRESSION OF MULTIPLE GENES THAT HAVE POTENTIAL ROLES IN COLONIZATION, INFECTION, AND PROTECTION AGAINST HOST DEFENSES. INVESTIGATION OF THE DISTRIBUTION OF MODM ALLELES IN A PANEL OF M. CATARRHALIS STRAINS, ISOLATED FROM THE NASOPHARYNX OF HEALTHY CHILDREN OR MIDDLE EAR EFFUSIONS FROM PATIENTS WITH OTITIS MEDIA, REVEALED A STATISTICALLY SIGNIFICANT ASSOCIATION OF MODM3 WITH OTITIS MEDIA ISOLATES. THE MODULATION OF GENE EXPRESSION VIA THE MODM PHASE-VARIABLE REGULON (PHASEVARION), AND THE SIGNIFICANT ASSOCIATION OF THE MODM3 ALLELE WITH OTITIS MEDIA, SUGGESTS A KEY ROLE FOR MODM PHASEVARIONS IN THE PATHOGENESIS OF THIS ORGANISM.	2014	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
3  6261 31 THE MORAXELLA CATARRHALIS PHASE-VARIABLE DNA METHYLTRANSFERASE MODM3 IS AN EPIGENETIC REGULATOR THAT AFFECTS BACTERIAL SURVIVAL IN AN IN VIVO MODEL OF OTITIS MEDIA. BACKGROUND: MORAXELLA CATARRHALIS IS A LEADING CAUSE OF OTITIS MEDIA (OM) AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD). M. CATARRHALIS CONTAINS A TYPE III DNA ADENINE METHYLTRANSFERASE (MODM) THAT IS PHASE-VARIABLY EXPRESSED (I.E., ITS EXPRESSION IS SUBJECT TO RANDOM, REVERSIBLE ON/OFF SWITCHING). MODM HAS SIX TARGET RECOGNITION DOMAIN ALLELES (MODM1-6), AND WE HAVE PREVIOUSLY SHOWN THAT MODM2 IS THE PREDOMINANT ALLELE, WHILE MODM3 IS ASSOCIATED WITH OM. PHASE-VARIABLE DNA METHYLTRANSFERASES MEDIATE EPIGENETIC REGULATION AND MODULATE PATHOGENESIS IN SEVERAL BACTERIA. MODM2 OF M. CATARRHALIS REGULATES THE EXPRESSION OF A PHASEVARION CONTAINING GENES IMPORTANT FOR COLONIZATION AND INFECTION. HERE WE DESCRIBE THE PHASE-VARIABLE EXPRESSION OF MODM3, THE MODM3 METHYLATION SITE AND THE SUITE OF GENES REGULATED WITHIN THE MODM3 PHASEVARION. RESULTS: PHASE-VARIABLE EXPRESSION OF MODM3, MEDIATED BY VARIATION IN LENGTH OF A 5'-(CAAC)(N)-3' TETRANUCLEOTIDE REPEAT TRACT IN THE OPEN READING FRAME WAS DEMONSTRATED IN M. CATARRHALIS STRAIN CCRI-195ME WITH GENESCAN FRAGMENT LENGTH ANALYSIS AND WESTERN IMMUNOBLOT. WE DETERMINED THAT MODM3 IS AN ACTIVE N6-ADENINE METHYLTRANSFERASE THAT METHYLATES THE SEQUENCE 5'-AC(M6)ATC-3'. METHYLATION WAS DETECTED AT ALL 4446 5'-ACATC-3' SITES IN THE GENOME WHEN MODM3 IS EXPRESSED. RNASEQ ANALYSIS IDENTIFIED 31 GENES THAT ARE DIFFERENTIALLY EXPRESSED BETWEEN MODM3 ON AND OFF VARIANTS, INCLUDING FIVE GENES THAT ARE INVOLVED IN THE RESPONSE TO OXIDATIVE AND NITROSATIVE STRESS, WITH POTENTIAL ROLES IN BIOFILM FORMATION AND SURVIVAL IN ANAEROBIC ENVIRONMENTS. AN IN VIVO CHINCHILLA (CHINCHILLA LANIGERA) MODEL OF OTITIS MEDIA DEMONSTRATED THAT TRANSBULLAR CHALLENGE WITH THE MODM3 OFF VARIANT RESULTED IN AN INCREASED MIDDLE EAR BACTERIAL LOAD COMPARED TO A MODM3 ON VARIANT. IN ADDITION, CO-INFECTION EXPERIMENTS WITH NTHI AND M. CATARRHALIS MODM3 ON OR MODM3 OFF VARIANTS REVEALED THAT PHASE VARIATION OF MODM3 ALTERED SURVIVAL OF NTHI IN THE MIDDLE EAR DURING EARLY AND LATE STAGE INFECTION. CONCLUSIONS: PHASE VARIATION OF MODM3 EPIGENETICALLY REGULATES THE EXPRESSION OF A PHASEVARION CONTAINING MULTIPLE GENES THAT ARE POTENTIALLY IMPORTANT IN THE PROGRESSION OF OTITIS MEDIA.	2019	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
4  1080 29 CLOSED COMPLETE GENOME SEQUENCES OF TWO NONTYPEABLE HAEMOPHILUS INFLUENZAE STRAINS CONTAINING NOVEL MODA ALLELES FROM THE SPUTUM OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE. NONTYPEABLE HAEMOPHILUS INFLUENZAE (NTHI) IS AN IMPORTANT BACTERIAL PATHOGEN THAT CAUSES OTITIS MEDIA AND EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD). HERE, WE REPORT THE COMPLETE GENOME SEQUENCES OF NTHI STRAINS 10P129H1 AND 84P36H1, ISOLATED FROM COPD PATIENTS, WHICH CONTAIN THE PHASE-VARIABLE EPIGENETIC REGULATORS MODA15 AND MODA18, RESPECTIVELY.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
5  4718 24 NON-TYPEABLE HAEMOPHILUS INFLUENZAE ISOLATES FROM PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE CONTAIN NEW PHASE-VARIABLE MODA METHYLTRANSFERASE ALLELES CONTROLLING PHASEVARIONS. PHASEVARIONS (PHASE-VARIABLE REGULONS) ARE EMERGING AS AN IMPORTANT AREA OF BACTERIAL GENE REGULATION. MANY BACTERIAL PATHOGENS CONTAIN PHASEVARIONS, WITH GENE EXPRESSION CONTROLLED BY THE PHASE-VARIABLE EXPRESSION OF DNA METHYLTRANSFERASES VIA EPIGENETIC MECHANISMS. NON-TYPEABLE HAEMOPHILUS INFLUENZAE (NTHI) CONTAINS THE PHASE-VARIABLE METHYLTRANSFERASE MODA, OF WHICH MULTIPLE ALLELIC VARIANTS EXIST (MODA1-21). WE HAVE PREVIOUSLY DEMONSTRATED 5 OF 21 THESE MODA ALLELES ARE OVERREPRESENTED IN NTHI STRAINS ISOLATED FROM CHILDREN WITH MIDDLE EAR INFECTIONS. IN THIS STUDY WE INVESTIGATED THE MODA ALLELE DISTRIBUTION IN NTHI STRAINS ISOLATED FROM PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE, COPD. WE DEMONSTRATE THAT THE DISTRIBUTION OF MODA ALLELES IN A LARGE PANEL OF COPD ISOLATES IS DIFFERENT TO THE DISTRIBUTION SEEN IN MIDDLE EAR INFECTIONS, SUGGESTING DIFFERENT MODA ALLELES MAY PROVIDE DISTINCT ADVANTAGES IN THE DIFFERING NICHES OF THE MIDDLE EAR AND COPD AIRWAYS. WE ALSO IDENTIFIED TWO NEW PHASE-VARIABLE MODA ALLELES - MODA15 AND MODA18 - AND DEMONSTRATE THAT THESE ALLELES METHYLATE DISTINCT DNA SEQUENCES AND CONTROL UNIQUE PHASEVARIONS. THE MODA15 AND MODA18 ALLELES HAVE ONLY BEEN OBSERVED IN COPD ISOLATES, INDICATING THAT THESE TWO ALLELES MAY BE MARKERS FOR ISOLATES LIKELY TO CAUSE EXACERBATIONS OF COPD.	2019	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
6  2273 21 EPIGENETIC REGULATION ALTERS BIOFILM ARCHITECTURE AND COMPOSITION IN MULTIPLE CLINICAL ISOLATES OF NONTYPEABLE HAEMOPHILUS INFLUENZAE. BIOFILMS PLAY A CRITICAL ROLE IN THE COLONIZATION, PERSISTENCE, AND PATHOGENESIS OF MANY HUMAN PATHOGENS. MULTIPLE MUCOSA-ASSOCIATED PATHOGENS HAVE EVOLVED A MECHANISM OF RAPID ADAPTATION, TERMED THE PHASEVARION, WHICH FACILITATES A COORDINATED REGULATION OF NUMEROUS GENES THROUGHOUT THE BACTERIAL GENOME. THIS EPIGENETIC REGULATION OCCURS VIA PHASE VARIATION OF A DNA METHYLTRANSFERASE, MOD. THE PHASEVARION OF NONTYPEABLE HAEMOPHILUS INFLUENZAE (NTHI) SIGNIFICANTLY AFFECTS THE SEVERITY OF EXPERIMENTAL OTITIS MEDIA AND REGULATES SEVERAL DISEASE-RELATED PROCESSES. HOWEVER, THE ROLE OF THE NTHI PHASEVARION IN BIOFILM FORMATION IS UNCLEAR. THE PRESENT STUDY SHOWS THAT THE PHASEVARIONS OF MULTIPLE NTHI CLINICAL ISOLATES REGULATE IN VITRO BIOFILM FORMATION UNDER DISEASE-SPECIFIC MICROENVIRONMENTAL CONDITIONS. THE IMPACT OF PHASEVARION REGULATION WAS GREATEST UNDER ALKALINE CONDITIONS THAT MIMIC THOSE KNOWN TO OCCUR IN THE MIDDLE EAR DURING DISEASE. UNDER ALKALINE CONDITIONS, NTHI STRAINS THAT EXPRESS THE MODA2 METHYLTRANSFERASE FORMED BIOFILMS WITH SIGNIFICANTLY GREATER BIOMASS AND LESS DISTINCT ARCHITECTURE THAN THOSE FORMED BY A MODA2-DEFICIENT POPULATION. THE BIOFILMS FORMED BY NTHI STRAINS THAT EXPRESS MODA2 ALSO CONTAINED LESS EXTRACELLULAR DNA (EDNA) AND SIGNIFICANTLY LESS EXTRACELLULAR HU, A DNABII DNA-BINDING PROTEIN CRITICAL FOR BIOFILM STRUCTURAL STABILITY. STABLE BIOFILM STRUCTURE IS CRITICAL FOR BACTERIAL PATHOGENESIS AND PERSISTENCE IN MULTIPLE EXPERIMENTAL MODELS OF DISEASE. THESE RESULTS IDENTIFY A ROLE FOR THE PHASEVARION IN REGULATION OF BIOFILM FORMATION, A PROCESS INTEGRAL TO THE CHRONIC NATURE OF MANY INFECTIONS. UNDERSTANDING THE ROLE OF THE PHASEVARION IN BIOFILM FORMATION IS CRITICAL TO THE DEVELOPMENT OF PREVENTION AND TREATMENT STRATEGIES FOR THESE CHRONIC DISEASES.IMPORTANCE UPPER RESPIRATORY TRACT INFECTIONS ARE THE NUMBER ONE REASON FOR A CHILD TO VISIT THE EMERGENCY DEPARTMENT, AND OTITIS MEDIA (MIDDLE EAR INFECTION) RANKS THIRD OVERALL. BIOFILMS CONTRIBUTE SIGNIFICANTLY TO THE CHRONIC NATURE OF BACTERIAL RESPIRATORY TRACT INFECTIONS, INCLUDING OTITIS MEDIA, AND MAKE THESE DISEASES PARTICULARLY DIFFICULT TO TREAT. SEVERAL MUCOSA-ASSOCIATED HUMAN PATHOGENS UTILIZE A MECHANISM OF RAPID ADAPTATION TERMED THE PHASEVARION, OR PHASEVARIABLE REGULON, TO RESIST ENVIRONMENTAL AND HOST IMMUNE PRESSURES. IN THIS STUDY, WE ASSESSED THE ROLE OF THE PHASEVARION IN REGULATION OF BIOFILM FORMATION BY NONTYPEABLE HAEMOPHILUS INFLUENZAE (NTHI), WHICH CAUSES NUMEROUS RESPIRATORY TRACT DISEASES. WE FOUND THAT THE NTHI PHASEVARION REGULATES BIOFILM STRUCTURE AND CRITICAL BIOFILM MATRIX COMPONENTS UNDER DISEASE-SPECIFIC CONDITIONS. THE FINDINGS OF THIS WORK COULD BE SIGNIFICANT IN THE DESIGN OF IMPROVED STRATEGIES AGAINST NTHI INFECTIONS, AS WELL AS DISEASES DUE TO OTHER PATHOGENS THAT UTILIZE A PHASEVARION.	2018	

7  4494 28 MORAXELLA CATARRHALIS RESTRICTION-MODIFICATION SYSTEMS ARE ASSOCIATED WITH PHYLOGENETIC LINEAGE AND DISEASE. MORAXELLA CATARRHALIS IS A HUMAN-ADAPTED PATHOGEN, AND A MAJOR CAUSE OF OTITIS MEDIA (OM) AND EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE. THE SPECIES IS COMPRISED OF TWO MAIN PHYLOGENETIC LINEAGES, RB1 AND RB2/3. RESTRICTION-MODIFICATION (R-M) SYSTEMS ARE AMONG THE FEW LINEAGE-ASSOCIATED GENES IDENTIFIED IN OTHER BACTERIAL GENERA AND HAVE MULTIPLE FUNCTIONS INCLUDING DEFENSE AGAINST FOREIGN INVADING DNA, MAINTENANCE OF SPECIATION, AND EPIGENETIC REGULATION OF GENE EXPRESSION. HERE, WE DEFINE THE REPERTOIRE OF R-M SYSTEMS IN 51 PUBLICLY AVAILABLE M. CATARRHALIS GENOMES AND REPORT THEIR DISTRIBUTION AMONG M. CATARRHALIS PHYLOGENETIC LINEAGES. AN ASSOCIATION WITH PHYLOGENETIC LINEAGE (RB1 OR RB2/3) WAS OBSERVED FOR SIX R-M SYSTEMS, WHICH MAY CONTRIBUTE TO THE EVOLUTION OF THE LINEAGES BY RESTRICTING DNA TRANSFORMATION. IN ADDITION, WE OBSERVED A RELATIONSHIP BETWEEN A MUTUALLY EXCLUSIVE TYPE I R-M SYSTEM AND A TYPE III R-M SYSTEM AT A SINGLE LOCUS CONSERVED THROUGHOUT A GEOGRAPHICALLY AND CLINICALLY DIVERSE SET OF M. CATARRHALIS ISOLATES. THE TYPE III R-M SYSTEM AT THIS LOCUS CONTAINS THE PHASE-VARIABLE TYPE III DNA METHYLTRANSFERASE, MODM, WHICH CONTROLS A PHASEVARION (PHASE-VARIABLE REGULON). WE OBSERVED AN ASSOCIATION BETWEEN MODM PRESENCE AND OM-ASSOCIATED MIDDLE EAR ISOLATES, INDICATING A POTENTIAL ROLE FOR MODM-MEDIATED EPIGENETIC REGULATION IN OM PATHOBIOLOGY.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
8  4493 18 MORAXELLA CATARRHALIS INDUCES INFLAMMATORY RESPONSE OF BRONCHIAL EPITHELIAL CELLS VIA MAPK AND NF-KAPPAB ACTIVATION AND HISTONE DEACETYLASE ACTIVITY REDUCTION. MORAXELLA CATARRHALIS IS A MAJOR CAUSE OF INFECTIOUS EXACERBATIONS OF CHRONIC OBSTRUCTIVE LUNG DISEASE (COPD) AND MAY ALSO CONTRIBUTE TO THE PATHOGENESIS OF COPD. LITTLE IS KNOWN ABOUT M. CATARRHALIS-BRONCHIAL EPITHELIUM INTERACTION. WE INVESTIGATED ACTIVATION OF M. CATARRHALIS INFECTED BRONCHIAL EPITHELIAL CELLS AND CHARACTERIZED THE SIGNAL TRANSDUCTION PATHWAYS. MOREOVER, WE TESTED THE HYPOTHESIS THAT THE M. CATARRHALIS-INDUCED CYTOKINE EXPRESSION IS REGULATED BY ACETYLATION OF HISTONE RESIDUES AND CONTROLLED BY HISTONE DEACETYLASE ACTIVITY (HDAC). WE DEMONSTRATED THAT M. CATARRHALIS INDUCED A STRONG TIME- AND DOSE-DEPENDENT INFLAMMATORY RESPONSE IN THE BRONCHIAL EPITHELIAL CELL LINE (BEAS-2B), CHARACTERIZED BY THE RELEASE OF IL-8 AND GM-CSF. FOR THIS CYTOKINE LIBERATION ACTIVATION OF THE ERK AND P38 MITOGEN-ACTIVATED PROTEIN (MAP) KINASES AND TRANSCRIPTION FACTOR NF-KAPPAB WAS REQUIRED. FURTHERMORE, M. CATARRHALIS-INFECTED BRONCHIAL EPITHELIAL CELLS SHOWED AN ENHANCED ACETYLATION OF HISTONE H3 AND H4 GLOBALLY AND AT THE PROMOTER OF THE IL8 GENE. PREVENTING HISTONE DEACETYLATION BY THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A AUGMENTED THE M. CATARRHALIS-INDUCED IL-8 RESPONSE. AFTER EXPOSURE TO M. CATARRHALIS, WE FOUND A DECREASE IN GLOBAL HISTONE DEACETYLASE EXPRESSION AND ACTIVITY. OUR FINDINGS SUGGEST THAT M. CATARRHALIS-INDUCED ACTIVATION OF IL8 GENE TRANSCRIPTION WAS CAUSED BY INTERFERENCE WITH EPIGENETIC MECHANISMS REGULATING IL8 GENE ACCESSIBILITY. OUR FINDINGS PROVIDE INSIGHT INTO IMPORTANT MOLECULAR AND CELLULAR MECHANISMS OF M. CATARRHALIS-INDUCED ACTIVATION OF HUMAN BRONCHIAL EPITHELIUM.	2006	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
9   129 12 A UNIQUE VIRULENCE GENE OCCUPIES A PRINCIPAL POSITION IN IMMUNE EVASION BY THE MALARIA PARASITE PLASMODIUM FALCIPARUM. MUTUALLY EXCLUSIVE GENE EXPRESSION, WHEREBY ONLY ONE MEMBER OF A MULTI-GENE FAMILY IS SELECTED FOR ACTIVATION, IS USED BY THE MALARIA PARASITE PLASMODIUM FALCIPARUM TO ESCAPE THE HUMAN IMMUNE SYSTEM AND PERPETUATE LONG-TERM, CHRONIC INFECTIONS. A FAMILY OF GENES CALLED VAR ENCODES THE CHIEF ANTIGENIC AND VIRULENCE DETERMINANT OF P. FALCIPARUM MALARIA. VAR GENES ARE TRANSCRIBED IN A MUTUALLY EXCLUSIVE MANNER, WITH SWITCHING BETWEEN ACTIVE GENES RESULTING IN ANTIGENIC VARIATION. WHILE RECENT WORK HAS SHED CONSIDERABLE LIGHT ON THE EPIGENETIC BASIS FOR VAR GENE ACTIVATION AND SILENCING, HOW SWITCHING IS CONTROLLED REMAINS A MYSTERY. IN PARTICULAR, SWITCHING SEEMS NOT TO BE RANDOM, BUT INSTEAD APPEARS TO BE COORDINATED TO RESULT IN TIMELY ACTIVATION OF INDIVIDUAL GENES LEADING TO SEQUENTIAL WAVES OF ANTIGENICALLY DISTINCT PARASITE POPULATIONS. THE MOLECULAR BASIS FOR THIS APPARENT COORDINATION IS UNKNOWN. HERE WE SHOW THAT VAR2CSA, AN UNUSUAL AND HIGHLY CONSERVED VAR GENE, OCCUPIES A UNIQUE POSITION WITHIN THE VAR GENE SWITCHING HIERARCHY. INDUCTION OF SWITCHING THROUGH THE DESTABILIZATION OF VAR SPECIFIC CHROMATIN USING BOTH GENETIC AND CHEMICAL METHODS REPEATEDLY LED TO THE RAPID AND EXCLUSIVE ACTIVATION OF VAR2CSA. ADDITIONAL EXPERIMENTS DEMONSTRATED THAT THESE REPRESENT "TRUE" SWITCHING EVENTS AND NOT SIMPLY DE-SILENCING OF THE VAR2CSA PROMOTER, AND THAT ACTIVATION IS LIMITED TO THE UNIQUE LOCUS ON CHROMOSOME 12. COMBINED WITH TRANSLATIONAL REPRESSION OF VAR2CSA TRANSCRIPTS, FREQUENT "DEFAULT" SWITCHING TO THIS LOCUS AND DETECTION OF VAR2CSA UNTRANSLATED TRANSCRIPTS IN NON-PREGNANT INDIVIDUALS, THESE DATA SUGGEST THAT VAR2CSA COULD PLAY A CENTRAL ROLE IN COORDINATING SWITCHING, FULFILLING A PREDICTION MADE BY MATHEMATICAL MODELS DERIVED FROM POPULATION SWITCHING PATTERNS. THESE STUDIES PROVIDE THE FIRST INSIGHTS INTO THE MECHANISMS BY WHICH VAR GENE SWITCHING IS COORDINATED AS WELL AS AN EXAMPLE OF HOW A PHARMACOLOGICAL AGENT CAN DISRUPT ANTIGENIC VARIATION IN PLASMODIUM FALCIPARUM.	2015	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
10  3625 17 IN VIVO GENOME AND METHYLOME ADAPTATION OF CAG-NEGATIVE HELICOBACTER PYLORI DURING EXPERIMENTAL HUMAN INFECTION. MULTIPLE STUDIES HAVE DEMONSTRATED RAPID BACTERIAL GENOME EVOLUTION DURING CHRONIC INFECTION WITH HELICOBACTER PYLORI IN CONTRAST, LITTLE WAS KNOWN ABOUT GENETIC CHANGES DURING THE FIRST STAGES OF INFECTION, WHEN SELECTIVE PRESSURE IS LIKELY TO BE HIGHEST. USING SINGLE-MOLECULE, REAL-TIME (SMRT) AND ILLUMINA SEQUENCING TECHNOLOGIES, WE ANALYZED GENOME AND METHYLOME EVOLUTION DURING THE FIRST 10 WEEKS OF INFECTION BY COMPARING THE CAG PATHOGENICITY ISLAND (CAGPAI)-NEGATIVE H. PYLORI CHALLENGE STRAIN BCS 100 WITH PAIRS OF H. PYLORI REISOLATES FROM GASTRIC ANTRUM AND CORPUS BIOPSY SPECIMENS OF 10 HUMAN VOLUNTEERS WHO HAD BEEN INFECTED WITH THIS STRAIN AS PART OF A VACCINE TRIAL. MOST GENETIC CHANGES DETECTED IN THE REISOLATES AFFECTED GENES WITH A SURFACE-RELATED ROLE OR A PREDICTED FUNCTION IN PEPTIDE UPTAKE. APART FROM PHENOTYPIC CHANGES OF THE BACTERIAL ENVELOPE, A DUPLICATION OF THE CATALASE GENE WAS OBSERVED IN ONE REISOLATE, WHICH RESULTED IN HIGHER CATALASE ACTIVITY AND IMPROVED SURVIVAL UNDER OXIDATIVE STRESS CONDITIONS. THE METHYLOMES ALSO VARIED IN SOME OF THE REISOLATES, MOSTLY BY ACTIVITY SWITCHING OF PHASE-VARIABLE METHYLTRANSFERASE (MTASE) GENES. THE OBSERVED IN VIVO MUTATION SPECTRUM WAS REMARKABLE FOR A VERY HIGH PROPORTION OF NONSYNONYMOUS MUTATIONS. ALTHOUGH THE DATA SHOWED SUBSTANTIAL WITHIN-STRAIN GENOME DIVERSITY IN THE CHALLENGE STRAIN, MOST ANTRUM AND CORPUS REISOLATES FROM THE SAME VOLUNTEERS WERE HIGHLY SIMILAR TO EACH OTHER, INDICATING THAT THE CHALLENGE INFECTION REPRESENTS A MAJOR SELECTIVE BOTTLENECK SHAPING THE TRANSMITTED POPULATION. OUR FINDINGS SUGGEST RAPID IN VIVO SELECTION OF H. PYLORI DURING EARLY-PHASE INFECTION PROVIDING ADAPTATION TO DIFFERENT INDIVIDUALS BY COMMON MECHANISMS OF GENETIC AND EPIGENETIC ALTERATIONS.IMPORTANCE EXCEPTIONAL GENETIC DIVERSITY AND VARIABILITY ARE HALLMARKS OF HELICOBACTER PYLORI, BUT THE BIOLOGICAL ROLE OF THIS PLASTICITY REMAINS INCOMPLETELY UNDERSTOOD. HERE, WE HAD THE RARE OPPORTUNITY TO INVESTIGATE THE MOLECULAR EVOLUTION DURING THE FIRST WEEKS OF H. PYLORI INFECTION BY COMPARING THE GENOMES AND EPIGENOMES OF H. PYLORI STRAIN BCS 100 USED TO CHALLENGE HUMAN VOLUNTEERS IN A VACCINE TRIAL WITH THOSE OF BACTERIA REISOLATED FROM THE VOLUNTEERS 10 WEEKS AFTER THE CHALLENGE. THE DATA PROVIDE MOLECULAR INSIGHTS INTO THE PROCESS OF ESTABLISHMENT OF THIS HIGHLY VERSATILE PATHOGEN IN 10 DIFFERENT HUMAN INDIVIDUAL HOSTS, SHOWING, FOR EXAMPLE, SELECTION FOR CHANGES IN HOST-INTERACTION MOLECULES AS WELL AS CHANGES IN EPIGENETIC METHYLATION PATTERNS. THE DATA PROVIDE IMPORTANT CLUES TO THE EARLY ADAPTATION OF H. PYLORI TO NEW HOST NICHES AFTER TRANSMISSION, WHICH WE BELIEVE IS VITAL TO UNDERSTAND ITS SUCCESS AS A CHRONIC PATHOGEN AND DEVELOP MORE EFFICIENT TREATMENTS AND VACCINES.	2020	
                                                                                                 
11  5016 17 PERSISTENT BACTERIAL COINFECTION OF A COVID-19 PATIENT CAUSED BY A GENETICALLY ADAPTED PSEUDOMONAS AERUGINOSA CHRONIC COLONIZER. PSEUDOMONAS AERUGINOSA IS A BIOFILM-FORMING OPPORTUNISTIC PATHOGEN WHICH CAUSES CHRONIC INFECTIONS IN IMMUNOCOMPROMISED PATIENTS AND LEADS TO HIGH MORTALITY RATE. IT IS IDENTIFIED AS A COMMON COINFECTING PATHOGEN IN COVID-19 PATIENTS CAUSING EXACERBATION OF ILLNESS. IN OUR HOSPITAL, P. AERUGINOSA IS ONE OF THE TOP COINFECTING BACTERIA IDENTIFIED AMONG COVID-19 PATIENTS. WE COLLECTED A STRONG BIOFILM-FORMING P. AERUGINOSA STRAIN DISPLAYING SMALL COLONY VARIANT MORPHOLOGY FROM A SEVERE COVID-19 PATIENT. GENOMIC AND TRANSCRIPTOMIC SEQUENCING ANALYSES WERE PERFORMED WITH PHENOTYPIC VALIDATION TO INVESTIGATE ITS ADAPTATION IN SARS-COV-2 INFECTED ENVIRONMENT. GENOMIC CHARACTERIZATION PREDICTED SPECIFIC GENOMIC ISLANDS HIGHLY ASSOCIATED WITH VIRULENCE, TRANSCRIPTIONAL REGULATION, AND DNA RESTRICTION-MODIFICATION SYSTEMS. EPIGENETIC ANALYSIS REVEALED A SPECIFIC N(6)-METHYL ADENINE (M(6)A) METHYLATING PATTERN INCLUDING METHYLATION OF ALGINATE, FLAGELLAR AND QUORUM SENSING ASSOCIATED GENES. DIFFERENTIAL GENE EXPRESSION ANALYSIS INDICATED THAT THIS ISOLATE FORMED EXCESSIVE BIOFILM BY REDUCING FLAGELLAR FORMATION (7.4 TO 1,624.1 FOLDS) AND OVERPRODUCING EXTRACELLULAR MATRIX COMPONENTS INCLUDING CDRA (4.4 FOLDS), ALGINATE (5.2 TO 29.1 FOLDS) AND PEL (4.8-5.5 FOLDS). IN SUMMARY, WE DEMONSTRATED THAT P. AEUGINOSA CLINICAL ISOLATES WITH NOVEL EPIGENETIC MARKERS COULD FORM EXCESSIVE BIOFILM, WHICH MIGHT ENHANCE ITS ANTIBIOTIC RESISTANCE AND IN VIVO COLONIZATION IN COVID-19 PATIENTS.	2021	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
12  6179 11 THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM CAN SENSE ENVIRONMENTAL CHANGES AND RESPOND BY ANTIGENIC SWITCHING. THE PRIMARY ANTIGENIC AND VIRULENCE DETERMINANT OF THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM IS A VARIANT SURFACE PROTEIN CALLED PFEMP1. DIFFERENT FORMS OF PFEMP1 ARE ENCODED BY A MULTICOPY GENE FAMILY CALLED VAR, AND SWITCHING BETWEEN ACTIVE GENES ENABLES THE PARASITES TO EVADE THE ANTIBODY RESPONSE OF THEIR HUMAN HOSTS. VAR GENE SWITCHING IS KEY FOR THE MAINTENANCE OF CHRONIC INFECTIONS; HOWEVER, WHAT CONTROLS SWITCHING IS UNKNOWN, ALTHOUGH IT HAS BEEN SUGGESTED TO OCCUR AT A CONSTANT FREQUENCY WITH LITTLE OR NO ENVIRONMENTAL INFLUENCE. VAR GENE TRANSCRIPTION IS CONTROLLED EPIGENETICALLY THROUGH THE ACTIVITY OF HISTONE METHYLTRANSFERASES (HMTS). STUDIES IN MODEL SYSTEMS HAVE SHOWN THAT METABOLISM AND EPIGENETIC CONTROL OF GENE EXPRESSION ARE LINKED THROUGH THE AVAILABILITY OF INTRACELLULAR S-ADENOSYLMETHIONINE (SAM), THE PRINCIPAL METHYL DONOR IN BIOLOGICAL METHYLATION MODIFICATIONS, WHICH CAN FLUCTUATE BASED ON NUTRIENT AVAILABILITY. TO DETERMINE WHETHER ENVIRONMENTAL CONDITIONS AND CHANGES IN METABOLISM CAN INFLUENCE VAR GENE EXPRESSION, P. FALCIPARUM WAS CULTURED IN MEDIA WITH ALTERED CONCENTRATIONS OF NUTRIENTS INVOLVED IN SAM METABOLISM. WE FOUND THAT CONDITIONS THAT INFLUENCE LIPID METABOLISM INDUCE VAR GENE SWITCHING, INDICATING THAT PARASITES CAN RESPOND TO CHANGES IN THEIR ENVIRONMENT BY ALTERING VAR GENE EXPRESSION PATTERNS. GENETIC MODIFICATIONS THAT DIRECTLY MODIFIED EXPRESSION OF THE ENZYMES THAT CONTROL SAM LEVELS SIMILARLY LED TO PROFOUND CHANGES IN VAR GENE EXPRESSION, CONFIRMING THAT CHANGES IN SAM AVAILABILITY MODULATE VAR GENE SWITCHING. THESE OBSERVATIONS DIRECTLY CHALLENGE THE PARADIGM THAT ANTIGENIC VARIATION IN P. FALCIPARUM FOLLOWS AN INTRINSIC, PROGRAMED SWITCHING RATE, WHICH OPERATES INDEPENDENTLY OF ANY EXTERNAL STIMULI.	2023	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
13  3037 15 GENOME AND METHYLOME VARIATION IN HELICOBACTER PYLORI WITH A CAG PATHOGENICITY ISLAND DURING EARLY STAGES OF HUMAN INFECTION. BACKGROUND & AIMS: HELICOBACTER PYLORI IS REMARKABLE FOR ITS GENETIC VARIATION; YET, LITTLE IS KNOWN ABOUT ITS GENETIC CHANGES DURING EARLY STAGES OF HUMAN INFECTION, AS THE BACTERIA ADAPT TO THEIR NEW ENVIRONMENT. WE ANALYZED GENOME AND METHYLOME VARIATIONS IN A FULLY VIRULENT STRAIN OF H PYLORI DURING EXPERIMENTAL INFECTION. METHODS: WE PERFORMED A RANDOMIZED PHASE I/II, OBSERVER-BLIND, PLACEBO-CONTROLLED STUDY OF 12 HEALTHY, H PYLORI-NEGATIVE ADULTS IN GERMANY FROM OCTOBER 2008 THROUGH MARCH 2010. THE VOLUNTEERS WERE GIVEN A PROPHYLACTIC VACCINE CANDIDATE (N = 7) OR PLACEBO (N = 5) AND THEN CHALLENGED WITH H PYLORI STRAIN BCM-300. BIOPSY SAMPLES WERE COLLECTED AND H PYLORI WERE ISOLATED. GENOMES OF THE CHALLENGE STRAIN AND 12 REISOLATES, OBTAINED 12 WEEKS AFTER (OR IN 1 CASE, 62 WEEKS AFTER) INFECTION WERE SEQUENCED BY SINGLE-MOLECULE, REAL-TIME TECHNOLOGY, WHICH, IN PARALLEL, PERMITTED DETERMINATION OF GENOME-WIDE METHYLATION PATTERNS FOR ALL STRAINS. FUNCTIONAL EFFECTS OF GENETIC CHANGES OBSERVED IN H PYLORI STRAINS DURING HUMAN INFECTION WERE ASSESSED BY MEASURING RELEASE OF INTERLEUKIN 8 FROM AGS CELLS (TO DETECT CAG PATHOGENICITY ISLAND FUNCTION), NEUTRAL RED UPTAKE (TO DETECT VACUOLATING CYTOTOXIN ACTIVITY), AND ADHESION ASSAYS. RESULTS: THE OBSERVED MUTATION RATE WAS IN AGREEMENT WITH RATES PREVIOUSLY DETERMINED FROM PATIENTS WITH CHRONIC H PYLORI INFECTIONS, WITHOUT EVIDENCE OF A MUTATION BURST. A LOSS OF CAG PATHOGENICITY ISLAND FUNCTION WAS OBSERVED IN 3 REISOLATES. IN ADDITION, 3 REISOLATES FROM THE VACCINE GROUP ACQUIRED MUTATIONS IN THE VACUOLATING CYTOTOXIN GENE VACA, RESULTING IN LOSS OF VACUOLIZATION ACTIVITY. WE OBSERVED INTERSTRAIN VARIATION IN METHYLOMES DUE TO PHASE VARIATION IN GENES ENCODING METHYLTRANSFERASES. CONCLUSIONS: WE ANALYZED ADAPTATION OF A FULLY VIRULENT STRAIN OF H PYLORI TO 12 DIFFERENT VOLUNTEERS TO OBTAIN A ROBUST ESTIMATE OF THE FREQUENCY OF GENETIC AND EPIGENETIC CHANGES IN THE ABSENCE OF INTERSTRAIN RECOMBINATION. OUR FINDINGS INDICATE THAT THE LARGE AMOUNT OF GENETIC VARIATION IN H PYLORI POSES A CHALLENGE TO VACCINE DEVELOPMENT. CLINICALTRIALS.GOV NO: NCT00736476.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
14  3235 15 HEMIN AVAILABILITY INDUCES COORDINATED DNA METHYLATION AND GENE EXPRESSION CHANGES IN PORPHYROMONAS GINGIVALIS. PERIODONTAL DISEASE IS A CHRONIC INFLAMMATORY DISEASE IN WHICH THE ORAL PATHOGEN PORPHYROMONAS GINGIVALIS PLAYS AN IMPORTANT ROLE. PORPHYROMONAS GINGIVALIS EXPRESSES VIRULENCE DETERMINANTS IN RESPONSE TO HIGHER HEMIN CONCENTRATIONS, BUT THE UNDERLYING REGULATORY PROCESSES REMAIN UNCLEAR. BACTERIAL DNA METHYLATION HAS THE POTENTIAL TO FULFIL THIS MECHANISTIC ROLE. WE CHARACTERIZED THE METHYLOME OF P. GINGIVALIS, AND COMPARED ITS VARIATION TO TRANSCRIPTOME CHANGES IN RESPONSE TO HEMIN AVAILABILITY. PORPHYROMONAS GINGIVALIS W50 WAS GROWN IN CHEMOSTAT CONTINUOUS CULTURE WITH EXCESS OR LIMITED HEMIN, PRIOR TO WHOLE-METHYLOME AND TRANSCRIPTOME PROFILING USING NANOPORE AND ILLUMINA RNA-SEQ. DNA METHYLATION WAS QUANTIFIED FOR DAM/DCM MOTIFS AND ALL-CONTEXT N6-METHYLADENINE (6MA) AND 5-METHYLCYTOSINE (5MC). OF ALL 1,992 GENES ANALYZED, 161 AND 268 WERE RESPECTIVELY OVER- AND UNDER-EXPRESSED WITH EXCESS HEMIN. NOTABLY, WE DETECTED DIFFERENTIAL DNA METHYLATION SIGNATURES FOR THE DAM "GATC" MOTIF AND BOTH ALL-CONTEXT 6MA AND 5MC IN RESPONSE TO HEMIN AVAILABILITY. JOINT ANALYSES IDENTIFIED A SUBSET OF COORDINATED CHANGES IN GENE EXPRESSION, 6MA, AND 5MC METHYLATION THAT TARGET GENES INVOLVED IN LACTATE UTILIZATION AND ABC TRANSPORTERS. THE RESULTS IDENTIFY ALTERED METHYLATION AND EXPRESSION RESPONSES TO HEMIN AVAILABILITY IN P. GINGIVALIS, WITH INSIGHTS INTO MECHANISMS REGULATING ITS VIRULENCE IN PERIODONTAL DISEASE. IMPORTANCE DNA METHYLATION HAS IMPORTANT ROLES IN BACTERIA, INCLUDING IN THE REGULATION OF TRANSCRIPTION. PORPHYROMONAS GINGIVALIS, AN ORAL PATHOGEN IN PERIODONTITIS, EXHIBITS WELL-ESTABLISHED GENE EXPRESSION CHANGES IN RESPONSE TO HEMIN AVAILABILITY. HOWEVER, THE REGULATORY PROCESSES UNDERLYING THESE EFFECTS REMAIN UNKNOWN. WE PROFILED THE NOVEL P. GINGIVALIS EPIGENOME, AND ASSESSED EPIGENETIC AND TRANSCRIPTOME VARIATION UNDER LIMITED AND EXCESS HEMIN CONDITIONS. AS EXPECTED, MULTIPLE GENE EXPRESSION CHANGES WERE DETECTED IN RESPONSE TO LIMITED AND EXCESS HEMIN THAT REFLECT HEALTH AND DISEASE, RESPECTIVELY. NOTABLY, WE ALSO DETECTED DIFFERENTIAL DNA METHYLATION SIGNATURES FOR THE DAM "GATC" MOTIF AND BOTH ALL-CONTEXT 6MA AND 5MC IN RESPONSE TO HEMIN. JOINT ANALYSES IDENTIFIED COORDINATED CHANGES IN GENE EXPRESSION, 6MA, AND 5MC METHYLATION THAT TARGET GENES INVOLVED IN LACTATE UTILIZATION AND ABC TRANSPORTERS. THE RESULTS IDENTIFY NOVEL REGULATORY PROCESSES UNDERLYING THE MECHANISM OF HEMIN REGULATED GENE EXPRESSION IN P. GINGIVALIS, WITH PHENOTYPIC IMPACTS ON ITS VIRULENCE IN PERIODONTAL DISEASE.	2023	
                                                                                                                                                                                                                                                                                                                                                  
15   414 13 ANALYSIS OF PROMOTER METHYLATION IN STOOL: A NOVEL METHOD FOR THE DETECTION OF COLORECTAL CANCER. BACKGROUND & AIMS: DETECTION OF TUMOR-DERIVED DNA ALTERATIONS IN STOOL IS AN INTRIGUING NEW APPROACH WITH HIGH POTENTIAL FOR THE NONINVASIVE DETECTION OF COLORECTAL CANCER (CRC). BECAUSE OF HETEROGENEITY OF TUMORS, USUALLY MULTIPLE MARKERS DISTRIBUTED THROUGHOUT THE HUMAN GENOME NEED TO BE ANALYZED. THIS IS LABOR INTENSIVE AND DOES NOT ALLOW FOR HIGH THROUGH-PUT SCREENING. THEREFORE, MARKERS WITH HIGH SENSITIVITY AND GOOD SPECIFICITY ARE NEEDED. WE EXPLORED THE POTENTIAL OF A SINGLE EPIGENETIC MARKER IN COMPARISON WITH FECAL OCCULT BLOOD TESTING (FOBT) FOR THE DISCRIMINATION OF PATIENTS WITH CRCS AND ADENOMAS FROM THOSE WITHOUT. METHODS: METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (PCR) WAS PERFORMED TO ANALYZE HYPERMETHYLATED IN CANCER 1 (HIC1) PROMOTER METHYLATION STATUS IN A BLINDED FASHION IN STOOL SAMPLES FROM 26 PATIENTS WITH CRC, 13 WITH ADENOMA > OR =1 CM, 9 WITH HYPERPLASTIC POLYPS, 9 WITH CHRONIC INFLAMMATORY BOWEL DISEASE, AND 32 WITH ENDOSCOPICALLY NORMAL COLON. RESULTS: NINETY-SEVEN PERCENT OF THE STOOL SAMPLES CONTAINED AMPLIFIABLE DNA. FORTY-TWO PERCENT OF THE SAMPLES FROM PATIENTS WITH CRC AND 31% OF THE SAMPLES FROM PATIENTS WITH COLORECTAL ADENOMA > OR =1 CM WERE POSITIVE FOR HIC1 PROMOTER METHYLATION. NO METHYLATED HIC1 PROMOTER DNA WAS DETECTED IN THE FECAL DNA FROM PATIENTS WITH ENDOSCOPICALLY NORMAL COLON OR HYPERPLASTIC POLYPS. CONCLUSIONS: THE EPIGENETIC MARKER HIC1 PROMOTER METHYLATION CARRIES HIGH POTENTIAL FOR THE REMOTE DETECTION OF CRCS. WE POSTULATE THAT A PANEL OF MERELY A FEW GENETIC AND EPIGENETIC MARKERS WILL BE REQUIRED FOR THE HIGHLY SENSITIVE AND SPECIFIC DETECTION OF CRCS AND ADENOMAS IN FECAL SAMPLES FROM AFFECTED PATIENTS.	2005	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
16   972 12 CHRONIC OBSTRUCTIVE PULMONARY DISEASE IS ASSOCIATED WITH EPIGENOME-WIDE DIFFERENTIAL METHYLATION IN BAL LUNG CELLS. DNA METHYLATION PATTERNS IN CHRONIC PULMONARY OBSTRUCTIVE DISEASE (COPD) MIGHT OFFER NEW INSIGHTS INTO DISEASE PATHOGENESIS. TO ASSESS METHYLATION PROFILES IN THE MAIN COPD TARGET ORGAN, WE PERFORMED AN EPIGENOME-WIDE ASSOCIATION STUDY ON BAL CELLS. BRONCHOSCOPIES WERE PERFORMED IN 18 SUBJECTS WITH COPD AND 15 CONTROL SUBJECTS (EX- AND CURRENT SMOKERS). DNA METHYLATION WAS MEASURED USING THE ILLUMINA METHYLATIONEPIC BEADCHIP KIT, COVERING MORE THAN 850,000 CPGS. DIFFERENTIALLY METHYLATED POSITIONS (DMPS) WERE EXAMINED FOR 1) ENRICHMENT IN PATHWAYS AND FUNCTIONAL GENE RELATIONSHIPS USING THE KYOTO ENCYCLOPEDIA OF GENES AND GENOMES AND GENE ONTOLOGY, 2) ACCELERATED AGING USING HORVATH'S EPIGENETIC CLOCK, 3) CORRELATION WITH GENE EXPRESSION, AND 4) COLOCALIZATION WITH GENETIC VARIATION. WE FOUND 1,155 BONFERRONI-SIGNIFICANT (P < 6.74 X 10(-8)) DMPS ASSOCIATED WITH COPD, MANY WITH LARGE EFFECT SIZES. FUNCTIONAL ANALYSIS IDENTIFIED BIOLOGICALLY PLAUSIBLE PATHWAYS AND GENE RELATIONSHIPS, INCLUDING ENRICHMENT FOR TRANSCRIPTION FACTOR ACTIVITY. STRONG CORRELATION WAS FOUND BETWEEN DNA METHYLATION AND CHRONOLOGICAL AGE BUT NOT BETWEEN COPD AND ACCELERATED AGING. FOR 79 UNIQUE DMPS, DNA METHYLATION CORRELATED SIGNIFICANTLY WITH GENE EXPRESSION IN BAL CELLS. THIRTY-NINE PERCENT OF DMPS WERE COLOCALIZED WITH COPD-ASSOCIATED SNPS. TO THE BEST OF OUR KNOWLEDGE, THIS IS THE FIRST EPIGENOME-WIDE ASSOCIATION STUDY OF COPD ON BAL CELLS, AND OUR ANALYSES REVEALED MANY DIFFERENTIAL METHYLATION SITES. INTEGRATION WITH MRNA DATA SHOWED A STRONG FUNCTIONAL READOUT FOR RELEVANT GENES, IDENTIFYING SITES WHERE DNA METHYLATION MIGHT DIRECTLY AFFECT EXPRESSION. ALMOST HALF OF DMPS WERE COLOCATED WITH SNPS IDENTIFIED IN PREVIOUS GENOME-WIDE ASSOCIATION STUDIES OF COPD, SUGGESTING JOINT GENETIC AND EPIGENETIC PATHWAYS RELATED TO DISEASE.	2022	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
17  2091 12 EPIGENETIC DYSREGULATION OF VIRULENCE GENE EXPRESSION IN SEVERE PLASMODIUM FALCIPARUM MALARIA. CHRONIC INFECTIONS WITH THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM DEPEND ON ANTIGENIC VARIATION. P. FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1), THE MAJOR ERYTHROCYTE SURFACE ANTIGEN MEDIATING PARASITE SEQUESTRATION IN THE MICROVASCULATURE, IS ENCODED IN PARASITES BY A HIGHLY DIVERSE FAMILY OF VAR GENES. ANTIGENIC SWITCHING IS MEDIATED BY CLONAL VARIATION IN VAR EXPRESSION, AND RECENT IN VITRO STUDIES HAVE DEMONSTRATED A ROLE FOR EPIGENETIC PROCESSES IN VAR REGULATION. EXPRESSION OF PARTICULAR PFEMP1 VARIANTS MAY RESULT IN PARASITE ENRICHMENT IN DIFFERENT TISSUES, A FACTOR IN THE DEVELOPMENT OF SEVERE DISEASE. HERE, WE STUDY IN VIVO HUMAN INFECTIONS AND PROVIDE EVIDENCE THAT INFECTION-INDUCED STRESS RESPONSES IN THE HOST CAN MODIFY PFEMP1 EXPRESSION VIA THE PERTURBATION OF EPIGENETIC MECHANISMS. OUR WORK SUGGESTS THAT SEVERE DISEASE MAY NOT BE THE DIRECT RESULT OF AN ADAPTIVE VIRULENCE STRATEGY TO MAXIMIZE PARASITE SURVIVAL BUT THAT IT MAY INDICATE A LOSS OF CONTROL OF THE CAREFULLY REGULATED PROCESS OF ANTIGENIC SWITCHING THAT MAINTAINS CHRONIC INFECTIONS.	2012	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
18  1501  7 DNA METHYLATION ANALYSIS OF HUMAN TISSUE-SPECIFIC CONNEXIN GENES. CONNEXINS ARE THE STRUCTURAL PROTEINS OF GAP JUNCTIONS AND THEIR FUNCTIONING AS TUMOR SUPPRESSORS IS WELL KNOWN. EPIGENETIC MODIFICATIONS, SUCH AS METHYLATION OF CONNEXIN GENES, PLAY IMPORTANT ROLES IN REGULATING GENE EXPRESSION. OVER THE PAST DECADE, SEVERAL METHODS HAVE BEEN APPLIED TO CHARACTERIZE DNA METHYLATION-SPECIFIC LOCI OF CONNEXIN GENES. THIS CHAPTER DESCRIBES ANALYSIS OF SELECTIVE CONNEXIN32 AND CONNEXIN43 GENE DNA METHYLATION IN HUMAN GASTRIC TISSUES USING METHYLATION-SPECIFIC PCR, BISULFITE-SPECIFIC PCR SEQUENCING AS WELL AS MASSARRAY TECHNIQUES.	2016	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
19  1219 14 CRISPR/CAS9 GENOME EDITING REVEALS THAT THE INTRON IS NOT ESSENTIAL FOR VAR2CSA GENE ACTIVATION OR SILENCING IN PLASMODIUM FALCIPARUM. PLASMODIUM FALCIPARUM RELIES ON MONOALLELIC EXPRESSION OF 1 OF 60 VAR VIRULENCE GENES FOR ANTIGENIC VARIATION AND HOST IMMUNE EVASION. EACH VAR GENE CONTAINS A CONSERVED INTRON WHICH HAS BEEN IMPLICATED IN PREVIOUS STUDIES IN BOTH ACTIVATION AND REPRESSION OF TRANSCRIPTION VIA SEVERAL EPIGENETIC MECHANISMS, INCLUDING INTERACTION WITH THE VAR PROMOTER, PRODUCTION OF LONG NONCODING RNAS (LNCRNAS), AND LOCALIZATION TO REPRESSIVE PERINUCLEAR SITES. HOWEVER, FUNCTIONAL STUDIES HAVE RELIED PRIMARILY ON ARTIFICIAL EXPRESSION CONSTRUCTS. USING THE RECENTLY DEVELOPED P. FALCIPARUM CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/CAS9 SYSTEM, WE DIRECTLY DELETED THE VAR2CSA P. FALCIPARUM 3D7_1200600 (PF3D7_1200600) ENDOGENOUS INTRON, RESULTING IN AN INTRONLESS VAR GENE IN A NATURAL, MARKER-FREE CHROMOSOMAL CONTEXT. DELETION OF THE VAR2CSA INTRON RESULTED IN AN UPREGULATION OF TRANSCRIPTION OF THE VAR2CSA GENE IN RING-STAGE PARASITES AND SUBSEQUENT EXPRESSION OF THE PFEMP1 PROTEIN IN LATE-STAGE PARASITES. INTRON DELETION DID NOT AFFECT THE NORMAL TEMPORAL REGULATION AND SUBSEQUENT TRANSCRIPTIONAL SILENCING OF THE VAR GENE IN TROPHOZOITES BUT DID RESULT IN INCREASED RATES OF VAR GENE SWITCHING IN SOME MUTANT CLONES. TRANSCRIPTIONAL REPRESSION OF THE INTRONLESS VAR2CSA GENE COULD BE ACHIEVED VIA LONG-TERM CULTURE OR PANNING WITH THE CD36 RECEPTOR, AFTER WHICH REACTIVATION WAS POSSIBLE WITH CHONDROITIN SULFATE A (CSA) PANNING. THESE DATA SUGGEST THAT THE VAR2CSA INTRON IS NOT REQUIRED FOR SILENCING OR ACTIVATION IN RING-STAGE PARASITES BUT POINT TO A SUBTLE ROLE IN REGULATION OF SWITCHING WITHIN THE VAR GENE FAMILY.IMPORTANCEPLASMODIUM FALCIPARUM IS THE MOST VIRULENT SPECIES OF MALARIA PARASITE, CAUSING HIGH RATES OF MORBIDITY AND MORTALITY IN THOSE INFECTED. CHRONIC INFECTION DEPENDS ON AN IMMUNE EVASION MECHANISM TERMED ANTIGENIC VARIATION, WHICH IN TURN RELIES ON MONOALLELIC EXPRESSION OF 1 OF ~60 VAR GENES. UNDERSTANDING ANTIGENIC VARIATION AND THE TRANSCRIPTIONAL REGULATION OF MONOALLELIC EXPRESSION IS IMPORTANT FOR DEVELOPING DRUGS AND/OR VACCINES. THE VAR GENE FAMILY ENCODES THE ANTIGENIC SURFACE PROTEINS THAT DECORATE INFECTED ERYTHROCYTES. UNTIL RECENTLY, STUDYING THE UNDERLYING GENETIC ELEMENTS THAT REGULATE MONOALLELIC EXPRESSION IN P. FALCIPARUM WAS DIFFICULT, AND MOST STUDIES RELIED ON ARTIFICIAL SYSTEMS SUCH AS EPISOMAL REPORTER GENES. OUR STUDY WAS THE FIRST TO USE CRISPR/CAS9 GENOME EDITING FOR THE FUNCTIONAL STUDY OF AN IMPORTANT, CONSERVED GENETIC ELEMENT OF VAR GENES-THE INTRON-IN AN ENDOGENOUS, EPISOME-FREE MANNER. OUR FINDINGS SHED LIGHT ON THE ROLE OF THE VAR GENE INTRON IN TRANSCRIPTIONAL REGULATION OF MONOALLELIC EXPRESSION.	2017	
                                                                                                                                                              
20   348 14 ALTERED DNA METHYLATION IS ASSOCIATED WITH ABERRANT GENE EXPRESSION IN PARENCHYMAL BUT NOT AIRWAY FIBROBLASTS ISOLATED FROM INDIVIDUALS WITH COPD. BACKGROUND: CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS A HETEROGENEOUS DISEASE OF THE LUNGS THAT IS CURRENTLY THE FOURTH LEADING CAUSE OF DEATH WORLDWIDE. GENETIC FACTORS ACCOUNT FOR ONLY A SMALL AMOUNT OF COPD RISK, BUT EPIGENETIC MECHANISMS, INCLUDING DNA METHYLATION, HAVE THE POTENTIAL TO MEDIATE THE INTERACTIONS BETWEEN AN INDIVIDUAL'S GENETICS AND ENVIRONMENTAL EXPOSURE. DNA METHYLATION IS HIGHLY CELL TYPE-SPECIFIC, AND INDIVIDUAL CELL TYPE STUDIES OF DNA METHYLATION IN COPD ARE SPARSE. FIBROBLASTS ARE PRESENT WITHIN THE AIRWAY AND PARENCHYMA OF THE LUNG AND CONTRIBUTE TO THE ABERRANT DEPOSITION OF EXTRACELLULAR MATRIX IN COPD. NO ASSESSMENT OR COMPARISON OF GENOME-WIDE DNA METHYLATION PROFILES IN THE AIRWAY AND PARENCHYMAL FIBROBLASTS FROM INDIVIDUALS WITH AND WITHOUT COPD HAS BEEN UNDERTAKEN. THESE DATA PROVIDE VALUABLE INSIGHT INTO THE MOLECULAR MECHANISMS CONTRIBUTING TO COPD AND THE DIFFERING PATHOLOGIES OF SMALL AIRWAYS DISEASE AND EMPHYSEMA IN COPD. METHODS: GENOME-WIDE DNA METHYLATION WAS EVALUATED AT OVER 485,000 CPG SITES USING THE ILLUMINA INFINIUM HUMANMETHYLATION450 BEADCHIP ARRAY IN THE AIRWAY (NON-COPD N = 8, COPD N = 7) AND PARENCHYMAL FIBROBLASTS (NON-COPD N = 17, COPD N = 29) ISOLATED FROM INDIVIDUALS WITH AND WITHOUT COPD. TARGETED GENE EXPRESSION WAS ASSESSED BY QPCR IN MATCHED RNA SAMPLES. RESULTS: DIFFERENTIALLY METHYLATED DNA REGIONS WERE IDENTIFIED BETWEEN CELLS ISOLATED FROM INDIVIDUALS WITH AND WITHOUT COPD IN BOTH AIRWAY AND PARENCHYMAL FIBROBLASTS. ONLY IN PARENCHYMAL FIBROBLASTS WAS DIFFERENTIAL DNA METHYLATION ASSOCIATED WITH DIFFERENTIAL GENE EXPRESSION. A SECOND ANALYSIS OF DIFFERENTIAL DNA METHYLATION VARIABILITY IDENTIFIED 359 INDIVIDUAL DIFFERENTIALLY VARIABLE CPG SITES IN PARENCHYMAL FIBROBLASTS. NO DIFFERENTIALLY VARIABLE CPG SITES WERE IDENTIFIED IN THE AIRWAY FIBROBLASTS. FIVE DIFFERENTIALLY VARIABLE-METHYLATED CPG SITES, ASSOCIATED WITH THREE GENES, WERE SUBSEQUENTLY ASSESSED FOR GENE EXPRESSION DIFFERENCES. TWO GENES (OAT AND GRIK2) DISPLAYED SIGNIFICANTLY INCREASED GENE EXPRESSION IN CELLS ISOLATED FROM INDIVIDUALS WITH COPD. CONCLUSIONS: DIFFERENTIAL AND VARIABLE DNA METHYLATION WAS ASSOCIATED WITH COPD STATUS IN THE PARENCHYMAL FIBROBLASTS BUT NOT AIRWAY FIBROBLASTS. ABERRANT DNA METHYLATION WAS ASSOCIATED WITH ALTERED GENE EXPRESSION IMPARTING BIOLOGICAL FUNCTION TO DNA METHYLATION CHANGES. CHANGES IN DNA METHYLATION ARE THEREFORE IMPLICATED IN THE MOLECULAR MECHANISMS UNDERLYING COPD PATHOGENESIS AND MAY REPRESENT NOVEL THERAPEUTIC TARGETS.	2018