1 6703 136 VERSATILE WORKFLOW FOR CELL TYPE-RESOLVED TRANSCRIPTIONAL AND EPIGENETIC PROFILES FROM CRYOPRESERVED HUMAN LUNG. COMPLEXITY OF LUNG MICROENVIRONMENT AND CHANGES IN CELLULAR COMPOSITION DURING DISEASE MAKE IT EXCEPTIONALLY HARD TO UNDERSTAND MOLECULAR MECHANISMS DRIVING DEVELOPMENT OF CHRONIC LUNG DISEASES. ALTHOUGH RECENT ADVANCES IN CELL TYPE-RESOLVED APPROACHES HOLD GREAT PROMISE FOR STUDYING COMPLEX DISEASES, THEIR IMPLEMENTATION RELIES ON LOCAL ACCESS TO FRESH TISSUE, AS TRADITIONAL TISSUE STORAGE METHODS DO NOT ALLOW VIABLE CELL ISOLATION. TO OVERCOME THESE HURDLES, WE DEVELOPED A VERSATILE WORKFLOW THAT ALLOWS STORAGE OF LUNG TISSUE WITH HIGH VIABILITY, PERMITS THOROUGH SAMPLE QUALITY CHECK BEFORE CELL ISOLATION, AND BEFITS SEQUENCING-BASED PROFILING. WE DEMONSTRATE THAT CRYOPRESERVATION ENABLES ISOLATION OF MULTIPLE CELL TYPES FROM BOTH HEALTHY AND DISEASED LUNGS. BASAL CELLS FROM CRYOPRESERVED AIRWAYS RETAIN THEIR DIFFERENTIATION ABILITY, INDICATING THAT CELLULAR IDENTITY IS NOT ALTERED BY CRYOPRESERVATION. IMPORTANTLY, USING RNA SEQUENCING AND EPIC ARRAY, WE SHOW THAT GENE EXPRESSION AND DNA METHYLATION SIGNATURES ARE PRESERVED UPON CRYOPRESERVATION, EMPHASIZING THE SUITABILITY OF OUR WORKFLOW FOR OMICS PROFILING OF LUNG CELLS. MOREOVER, WE OBTAINED HIGH-QUALITY SINGLE-CELL RNA-SEQUENCING DATA OF CELLS FROM CRYOPRESERVED HUMAN LUNGS, DEMONSTRATING THAT CRYOPRESERVATION EMPOWERS SINGLE-CELL APPROACHES. OVERALL, THANKS TO ITS SIMPLICITY, OUR WORKFLOW IS WELL SUITED FOR PROSPECTIVE TISSUE COLLECTION BY ACADEMIC COLLABORATORS AND BIOBANKS, OPENING WORLDWIDE ACCESS TO VIABLE HUMAN TISSUE. 2021 2 5702 30 SINGLE-CELL GENOMICS FOR INVESTIGATING PATHOGENESIS OF INFLAMMATORY DISEASES. RECENT TECHNICAL ADVANCES HAVE ENABLED UNBIASED TRANSCRIPTOMIC AND EPIGENETIC ANALYSIS OF EACH CELL, KNOWN AS "SINGLE-CELL ANALYSIS". SINGLE-CELL ANALYSIS HAS A VARIETY OF TECHNICAL APPROACHES TO INVESTIGATE THE STATE OF EACH CELL, INCLUDING MRNA LEVELS (TRANSCRIPTOME), THE IMMUNE REPERTOIRE (IMMUNE REPERTOIRE ANALYSIS), CELL SURFACE PROTEINS (SURFACE PROTEOME ANALYSIS), CHROMATIN ACCESSIBILITY (EPIGENOME), AND ACCORDANCE WITH GENOME VARIANTS (EQTLS; EXPRESSION QUANTITATIVE TRAIT LOCI). AS AN EFFECTIVE TOOL FOR INVESTIGATING ROBUST IMMUNE RESPONSES IN CORONAVIRUS DISEASE 2019 (COVID-19), MANY RESEARCHERS PERFORMED SINGLE-CELL ANALYSIS TO CAPTURE THE DIVERSE, UNBIASED IMMUNE CELL ACTIVATION AND DIFFERENTIATION. DESPITE CHALLENGES ELUCIDATING THE COMPLICATED IMMUNE MICROENVIRONMENTS OF CHRONIC INFLAMMATORY DISEASES USING EXISTING EXPERIMENTAL METHODS, IT IS NOW POSSIBLE TO CAPTURE THE SIMULTANEOUS IMMUNE FEATURES OF DIFFERENT CELL TYPES ACROSS INFLAMED TISSUES USING VARIOUS SINGLE-CELL TOOLS. IN THIS REVIEW, WE INTRODUCE PATIENT-BASED AND EXPERIMENTAL MOUSE MODEL RESEARCH UTILIZING SINGLE-CELL ANALYSES IN THE FIELD OF CHRONIC INFLAMMATORY DISEASES, AS WELL AS MULTI-ORGAN ATLAS TARGETING IMMUNE CELLS. 2023 3 605 38 BEYOND ENDOMETRIOSIS GENOME-WIDE ASSOCIATION STUDY: FROM GENOMICS TO PHENOMICS TO THE PATIENT. ENDOMETRIOSIS IS A HERITABLE, COMPLEX CHRONIC INFLAMMATORY DISEASE, FOR WHICH MUCH OF THE CAUSAL PATHOGENIC MECHANISM REMAINS UNKNOWN. GENOME-WIDE ASSOCIATION STUDIES (GWAS) TO DATE HAVE IDENTIFIED 12 SINGLE NUCLEOTIDE POLYMORPHISMS AT 10 INDEPENDENT GENETIC LOCI ASSOCIATED WITH ENDOMETRIOSIS. MOST OF THESE WERE MORE STRONGLY ASSOCIATED WITH REVISED AMERICAN FERTILITY SOCIETY STAGE III/IV, RATHER THAN STAGE I/II. THE LOCI ARE ALMOST ALL LOCATED IN INTERGENIC REGIONS THAT ARE KNOWN TO PLAY A ROLE IN THE REGULATION OF EXPRESSION OF TARGET GENES YET TO BE IDENTIFIED. TO IDENTIFY THE TARGET GENES AND PATHWAYS PERTURBED BY THE IMPLICATED VARIANTS, STUDIES ARE REQUIRED INVOLVING FUNCTIONAL GENOMIC ANNOTATION OF THE SURROUNDING CHROMOSOMAL REGIONS, IN TERMS OF TRANSCRIPTION FACTOR BINDING, EPIGENETIC MODIFICATION (E.G., DNA METHYLATION AND HISTONE MODIFICATION) SITES, AS WELL AS THEIR CORRELATION WITH RNA TRANSCRIPTION. THESE STUDIES NEED TO BE CONDUCTED IN TISSUE TYPES RELEVANT TO ENDOMETRIOSIS-IN PARTICULAR, ENDOMETRIUM. IN ADDITION, TO ALLOW BIOLOGICALLY AND CLINICALLY RELEVANT INTERPRETATION OF MOLECULAR PROFILING DATA, THEY NEED TO BE COMBINED AND CORRELATED WITH DETAILED, SYSTEMATICALLY COLLECTED PHENOTYPIC INFORMATION (SURGICAL AND CLINICAL). THE WERF ENDOMETRIOSIS PHENOME AND BIOBANKING HARMONISATION PROJECT IS A GLOBAL STANDARDIZATION INITIATIVE THAT HAS PRODUCED CONSENSUS DATA AND SAMPLE COLLECTION PROTOCOLS FOR ENDOMETRIOSIS RESEARCH. THESE NOW PAVE THE WAY FOR COLLABORATIVE STUDIES INTEGRATING PHENOMIC WITH GENOMIC DATA, TO IDENTIFY INFORMATIVE SUBTYPES OF ENDOMETRIOSIS THAT WILL ENHANCE UNDERSTANDING OF THE PATHOGENIC MECHANISMS OF THE DISEASE AND DISCOVERY OF NOVEL, TARGETED TREATMENTS. 2016 4 485 25 ARTIFICIAL AIRWAYS FOR THE STUDY OF RESPIRATORY DISEASE. THIS REVIEW WILL FOCUS ON HUMAN CELL-BASED EXPERIMENTAL MODELS TO STUDY RESPIRATORY DISEASES, IN PARTICULAR MODELS OF THE LARGE AIRWAYS RELEVANT TO ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE. SUCH MODELS HAVE THE ADVANTAGE OF INCORPORATING CELLS THAT CAN BE DERIVED FROM DISEASE-RELEVANT TISSUE AND SO HAVE RETAINED IMPORTANT GENETIC AND EPIGENETIC FEATURES THAT CONTRIBUTE TO THE HUMAN DISEASE. THESE MODELS CAN BE USED FOR MECHANISTIC STUDIES, TARGET IDENTIFICATION AND VALIDATION AND TOXICOLOGICAL TESTING. WHILE MANY MODELS HAVE BEEN DEVELOPED TO VARYING DEGREES OF SOPHISTICATION, THE CHALLENGE REMAINS TO DEVELOP AN INTEGRATED SYSTEM THAT RECAPITULATES THE COMPLEX CELL-CELL AND CELL-MATRIX INTERACTIONS THAT OCCUR IN VIVO AND TO PROVIDE THESE WITH A 'CIRCULATION' TO STUDY THE DYNAMICS OF IMMUNE AND INFLAMMATORY CELL INFLUX AND EFFLUX. 2011 5 4228 24 METHYLATION OF INFLAMMATORY CELLS IN LUNG DISEASES. THIS CHAPTER OVERVIEWS ROLES OF DNA METHYLATION IN INFLAMMATORY CELL BIOLOGY WITH THE FOCUSES ON LYMPHOCYTES AND MACROPHAGES/MONOCYTES IN LUNG DISEASES, ALTHOUGH THE MOLECULAR MECHANISMS BY WHICH TARGET GENES ARE METHYLATED AND REGULATED IN LUNG DISEASES REMAIN UNCLEAR. MOST OF EPIGENETIC STUDIES ON DNA METHYLATION OF TARGET GENES IN LUNG DISEASES MAINLY DEMONSTRATED THE CORRELATION OF DNA METHYLATION OF TARGET GENES WITH THE LEVELS OF OTHER CORRESPONDING FACTORS, WITH THE SPECIFICITY OF CLINICAL PHENOMES, AND WITH THE SEVERITY OF LUNG DISEASES. THERE IS AN URGENT NEED TO IDENTIFY AND VALIDATE THE SPECIFICITY AND REGULATORY MECHANISMS OF INFLAMMATORY CELL EPIGENETICS IN DEPTH. THE EPIGENETIC HETEROGENEITY AMONG DIFFERENT SUBSETS OF T CELLS AND AMONG PROMOTERS OR NON-PROMOTERS OF TARGET GENES SHOULD BE FURTHERMORE CLARIFIED IN ACUTE OR CHRONIC LUNG DISEASES AND CANCERS. THE HYPER/HYPO-METHYLATION AND MODIFICATIONS OF CHROMOSOL AND EXTRACHROMOSOMAL DNA MAY RESULT IN ALTERNATIONS IN PROTEINS WITHIN INFLAMMATORY CELLS, WHICH CAN BE IDENTIFIED AS DISEASE-SPECIFIC BIOMARKERS AND THERAPEUTIC TARGETS. 2020 6 5798 39 STEM CELLS AND LUNG REGENERATION. THE ABILITY TO REPLACE DEFECTIVE CELLS IN AN AIRWAY WITH CELLS THAT CAN ENGRAFT, INTEGRATE, AND RESTORE A FUNCTIONAL EPITHELIUM COULD POTENTIALLY CURE A NUMBER OF LUNG DISEASES. PROGRESS TOWARD THE DEVELOPMENT OF STRATEGIES TO REGENERATE THE ADULT LUNG BY EITHER IN VIVO OR EX VIVO TARGETING OF ENDOGENOUS STEM CELLS OR PLURIPOTENT STEM CELL DERIVATIVES IS LIMITED BY OUR FUNDAMENTAL LACK OF UNDERSTANDING OF THE MECHANISMS CONTROLLING HUMAN LUNG DEVELOPMENT, THE PRECISE IDENTITY AND FUNCTION OF HUMAN LUNG STEM AND PROGENITOR CELL TYPES, AND THE GENETIC AND EPIGENETIC CONTROL OF HUMAN LUNG FATE. IN THIS REVIEW, WE INTEND TO DISCUSS THE KNOWN STEM/PROGENITOR CELL POPULATIONS, THEIR RELATIVE DIFFERENCES BETWEEN RODENTS AND HUMANS, THEIR ROLES IN CHRONIC LUNG DISEASE, AND THEIR THERAPEUTIC PROSPECTS. ADDITIONALLY, WE HIGHLIGHT THE RECENT BREAKTHROUGHS THAT HAVE INCREASED OUR UNDERSTANDING OF THESE CELL TYPES. THESE ADVANCEMENTS INCLUDE NOVEL LINEAGE-TRACED ANIMAL MODELS AND SINGLE-CELL RNA SEQUENCING OF HUMAN AIRWAY CELLS, WHICH HAVE PROVIDED CRITICAL INFORMATION ON THE STEM CELL SUBTYPES, TRANSITION STATES, IDENTIFYING CELL MARKERS, AND INTRICATE PATHWAYS THAT COMMIT A STEM CELL TO DIFFERENTIATE OR TO MAINTAIN PLASTICITY. AS OUR CAPACITY TO MODEL THE HUMAN LUNG EVOLVES, SO WILL OUR UNDERSTANDING OF LUNG REGENERATION AND OUR ABILITY TO TARGET ENDOGENOUS STEM CELLS AS A THERAPEUTIC APPROACH FOR LUNG DISEASE. 2020 7 4531 32 MULTILAYER-OMICS ANALYSES OF HUMAN CANCERS: EXPLORATION OF BIOMARKERS AND DRUG TARGETS BASED ON THE ACTIVITIES OF THE INTERNATIONAL HUMAN EPIGENOME CONSORTIUM. EPIGENETIC ALTERATIONS CONSISTING MAINLY OF DNA METHYLATION ALTERATIONS AND HISTONE MODIFICATION ALTERATIONS ARE FREQUENTLY OBSERVED IN CANCERS ASSOCIATED WITH CHRONIC INFLAMMATION AND/OR PERSISTENT INFECTION WITH VIRUSES OR OTHER PATHOGENIC MICROORGANISMS, OR WITH CIGARETTE SMOKING. ACCUMULATING EVIDENCE SUGGESTS THAT ALTERATIONS OF DNA METHYLATION ARE INVOLVED EVEN IN THE EARLY AND PRECANCEROUS STAGES. ON THE OTHER HAND, IN PATIENTS WITH CANCERS, ABERRANT DNA METHYLATION IS FREQUENTLY ASSOCIATED WITH TUMOR AGGRESSIVENESS AND POOR PATIENT OUTCOME. RECENTLY, EPIGENOME ALTERATIONS HAVE BEEN ATTRACTING A GREAT DEAL OF ATTENTION FROM RESEARCHERS WHO ARE FOCUSING ON NOT ONLY CANCERS BUT ALSO NEURONAL, IMMUNE AND METABOLIC DISORDERS. IN ORDER TO ACCURATELY IDENTIFY DISEASE-SPECIFIC EPIGENOME PROFILES THAT COULD BE POTENTIALLY APPLICABLE FOR DISEASE PREVENTION, DIAGNOSIS AND THERAPY, STRICT COMPARISON WITH STANDARD EPIGENOME PROFILES OF NORMAL TISSUES IS INDISPENSABLE. HOWEVER, EPIGENOME MECHANISMS SHOW HETEROGENEITY AMONG TISSUES AND CELL LINEAGES. THEREFORE, IT IS NOT EASY TO OBTAIN A COMPREHENSIVE PICTURE OF STANDARD EPIGENOME PROFILES OF NORMAL TISSUES. IN 2010, THE INTERNATIONAL HUMAN EPIGENOME CONSORTIUM (IHEC) WAS ESTABLISHED TO COORDINATE THE PRODUCTION OF REFERENCE MAPS OF HUMAN EPIGENOMES FOR KEY CELLULAR STATES. IN ORDER TO GAIN SUBSTANTIAL COVERAGE OF THE HUMAN EPIGENOME, THE IHEC HAS SET AN AMBITIOUS GOAL TO DECIPHER AT LEAST 1000 EPIGENOMES WITHIN THE NEXT 7-10 YEARS. WE CONSIDER THAT PATHWAY ANALYSIS USING GENES SHOWING MULTILAYER-OMICS ABNORMALITIES, INCLUDING GENOME, EPIGENOME, TRANSCRIPTOME, PROTEOME AND METABOLOME ABNORMALITIES, MAY BE USEFUL FOR ELUCIDATING THE MOLECULAR BACKGROUND OF PATHOGENESIS AND FOR EXPLORING POSSIBLE THERAPEUTIC TARGETS FOR EACH DISEASE. 2014 8 6013 36 THE APPLICATIONS OF DNA METHYLATION AS A BIOMARKER IN KIDNEY TRANSPLANTATION: A SYSTEMATIC REVIEW. BACKGROUND: ALTHOUGH KIDNEY TRANSPLANTATION IMPROVES PATIENT SURVIVAL AND QUALITY OF LIFE, LONG-TERM RESULTS ARE HAMPERED BY BOTH IMMUNE- AND NON-IMMUNE-MEDIATED COMPLICATIONS. CURRENT BIOMARKERS OF POST-TRANSPLANT COMPLICATIONS, SUCH AS ALLOGRAFT REJECTION, CHRONIC RENAL ALLOGRAFT DYSFUNCTION, AND CUTANEOUS SQUAMOUS CELL CARCINOMA, HAVE A SUBOPTIMAL PREDICTIVE VALUE. DNA METHYLATION IS AN EPIGENETIC MODIFICATION THAT DIRECTLY AFFECTS GENE EXPRESSION AND PLAYS AN IMPORTANT ROLE IN PROCESSES SUCH AS ISCHEMIA/REPERFUSION INJURY, FIBROSIS, AND ALLOREACTIVE IMMUNE RESPONSE. NOVEL TECHNIQUES CAN QUICKLY ASSESS THE DNA METHYLATION STATUS OF MULTIPLE LOCI IN DIFFERENT CELL TYPES, ALLOWING A DEEP AND INTERESTING STUDY OF CELLS' ACTIVITY AND FUNCTION. THEREFORE, DNA METHYLATION HAS THE POTENTIAL TO BECOME AN IMPORTANT BIOMARKER FOR PREDICTION AND MONITORING IN KIDNEY TRANSPLANTATION. PURPOSE OF THE STUDY: THE AIM OF THIS STUDY WAS TO EVALUATE THE ROLE OF DNA METHYLATION AS A POTENTIAL BIOMARKER OF GRAFT SURVIVAL AND COMPLICATIONS DEVELOPMENT IN KIDNEY TRANSPLANTATION. MATERIAL AND METHODS: A SYSTEMATIC REVIEW OF SEVERAL DATABASES HAS BEEN CONDUCTED. THE NEWCASTLE-OTTAWA SCALE AND THE JADAD SCALE HAVE BEEN USED TO ASSESS THE RISK OF BIAS FOR OBSERVATIONAL AND RANDOMIZED STUDIES, RESPECTIVELY. RESULTS: TWENTY ARTICLES REPORTING ON DNA METHYLATION AS A BIOMARKER FOR KIDNEY TRANSPLANTATION WERE INCLUDED, ALL USING DNA METHYLATION FOR PREDICTION AND MONITORING. DNA METHYLATION PATTERN ALTERATIONS IN CELLS ISOLATED FROM DIFFERENT TISSUES, SUCH AS KIDNEY BIOPSIES, URINE, AND BLOOD, HAVE BEEN ASSOCIATED WITH ISCHEMIA-REPERFUSION INJURY AND CHRONIC RENAL ALLOGRAFT DYSFUNCTION. THESE ALTERATIONS OCCURRED IN DIFFERENT AND SPECIFIC LOCI. DNA METHYLATION STATUS HAS ALSO PROVED TO BE IMPORTANT FOR IMMUNE RESPONSE MODULATION, HAVING A CRUCIAL ROLE IN REGULATORY T CELL DEFINITION AND ACTIVITY. RESEARCH ALSO FOCUSED ON A BETTER UNDERSTANDING OF THE ROLE OF THIS EPIGENETIC MODIFICATION ASSESSMENT FOR REGULATORY T CELLS ISOLATION AND EXPANSION FOR FUTURE TOLERANCE INDUCTION-ORIENTED THERAPIES. CONCLUSIONS: STUDIES INCLUDED IN THIS REVIEW ARE HETEROGENEOUS IN STUDY DESIGN, BIOLOGICAL SAMPLES, AND OUTCOME. MORE COORDINATED INVESTIGATIONS ARE NEEDED TO AFFIRM DNA METHYLATION AS A CLINICALLY RELEVANT BIOMARKER IMPORTANT FOR PREVENTION, MONITORING, AND INTERVENTION. 2022 9 6449 38 THERAPEUTIC TARGETING OF TELOMERASE. TELOMERE LENGTH AND CELL FUNCTION CAN BE PRESERVED BY THE HUMAN REVERSE TRANSCRIPTASE TELOMERASE (HTERT), WHICH SYNTHESIZES THE NEW TELOMERIC DNA FROM A RNA TEMPLATE, BUT IS NORMALLY RESTRICTED TO CELLS NEEDING A HIGH PROLIFERATIVE CAPACITY, SUCH AS STEM CELLS. CONSEQUENTLY, TELOMERASE-BASED THERAPIES TO ELONGATE SHORT TELOMERES ARE DEVELOPED, SOME OF WHICH HAVE SUCCESSFULLY REACHED THE STAGE I IN CLINICAL TRIALS. TELOMERASE IS ALSO PERMISSIVE FOR TUMORIGENESIS AND 90% OF ALL MALIGNANT TUMORS USE TELOMERASE TO OBTAIN IMMORTALITY. THUS, REVERSAL OF TELOMERASE UPREGULATION IN TUMOR CELLS IS A POTENTIAL STRATEGY TO TREAT CANCER. NATURAL AND SMALL-MOLECULE TELOMERASE INHIBITORS, IMMUNOTHERAPEUTIC APPROACHES, OLIGONUCLEOTIDE INHIBITORS, AND TELOMERASE-DIRECTED GENE THERAPY ARE USEFUL TREATMENT STRATEGIES. TELOMERASE IS MORE WIDELY EXPRESSED THAN ANY OTHER TUMOR MARKER. THE LOW EXPRESSION IN NORMAL TISSUES, TOGETHER WITH THE LONGER TELOMERES IN NORMAL STEM CELLS VERSUS CANCER CELLS, PROVIDES SOME DEGREE OF SPECIFICITY WITH LOW RISK OF TOXICITY. HOWEVER, LONG TERM TELOMERASE INHIBITION MAY ELICIT NEGATIVE EFFECTS IN HIGHLY-PROLIFERATIVE CELLS WHICH NEED TELOMERASE FOR SURVIVAL, AND IT MAY INTERFERE WITH TELOMERE-INDEPENDENT PHYSIOLOGICAL FUNCTIONS. MOREOVER, ONLY A FEW HTERT MOLECULES ARE REQUIRED TO OVERCOME SENESCENCE IN CANCER CELLS, AND TELOMERASE INHIBITION REQUIRES PROLIFERATING CELLS OVER A SUFFICIENT NUMBER OF POPULATION DOUBLINGS TO INDUCE TUMOR SUPPRESSIVE SENESCENCE. THESE LIMITATIONS MAY EXPLAIN THE MODERATE SUCCESS RATES IN MANY CLINICAL STUDIES. DESPITE EXTENSIVE STUDIES, ONLY ONE VACCINE AND ONE TELOMERASE ANTAGONIST ARE ROUTINELY USED IN CLINICAL WORK. FOR COMPLETE ERADICATION OF ALL SUBPOPULATIONS OF CANCER CELLS A SIMULTANEOUS TARGETING OF SEVERAL MECHANISMS WILL LIKELY BE NEEDED. POSSIBLE TECHNICAL IMPROVEMENTS HAVE BEEN PROPOSED INCLUDING THE DEVELOPMENT OF MORE SPECIFIC INHIBITORS, METHODS TO INCREASE THE EFFICACY OF VACCINATION METHODS, AND PERSONALIZED APPROACHES. TELOMERASE ACTIVATION AND CELL REJUVENATION IS SUCCESSFULLY USED IN REGENERATIVE MEDICINE FOR TISSUE ENGINEERING AND RECONSTRUCTIVE SURGERY. HOWEVER, THERE ARE ALSO A NUMBER OF PITFALLS IN THE TREATMENT WITH TELOMERASE ACTIVATING PROCEDURES FOR THE WHOLE ORGANISM AND FOR LONGER PERIODS OF TIME. EXTENDED CELL LIFESPAN MAY ACCUMULATE RARE GENETIC AND EPIGENETIC ABERRATIONS THAT CAN CONTRIBUTE TO MALIGNANT TRANSFORMATION. THEREFORE, NOVEL VECTOR SYSTEMS HAVE BEEN DEVELOPED FOR A 'MILD' INTEGRATION OF TELOMERASE INTO THE HOST GENOME AND LOSS OF THE VECTOR IN RAPIDLY-PROLIFERATING CELLS. IT IS CURRENTLY UNCLEAR IF THIS TECHNIQUE CAN ALSO BE USED IN HUMAN BEINGS TO TREAT CHRONIC DISEASES, SUCH AS ATHEROSCLEROSIS. 2016 10 3038 31 GENOME ENGINEERING FOR OSTEOARTHRITIS: FROM DESIGNER CELLS TO DISEASE-MODIFYING DRUGS. BACKGROUND: OSTEOARTHRITIS (OA) IS A HIGHLY PREVALENT DEGENERATIVE JOINT DISEASE INVOLVING JOINT CARTILAGE AND ITS SURROUNDING TISSUES. OA IS THE LEADING CAUSE OF PAIN AND DISABILITY WORLDWIDE. AT PRESENT, THERE ARE NO DISEASE-MODIFYING OA DRUGS, AND THE PRIMARY THERAPIES INCLUDE EXERCISE AND NONSTEROIDAL ANTI-INFLAMMATORY DRUGS UNTIL TOTAL JOINT REPLACEMENT AT THE END-STAGE OF THE DISEASE. METHODS: IN THIS REVIEW, WE SUMMARIZED THE CURRENT STATE OF KNOWLEDGE IN GENETIC AND EPIGENETIC ASSOCIATIONS AND RISK FACTORS FOR OA AND THEIR POTENTIAL DIAGNOSTIC AND THERAPEUTIC APPLICATIONS. RESULTS: GENOME-WIDE ASSOCIATION STUDIES AND ANALYSIS OF EPIGENETIC MODIFICATIONS (SUCH AS MIRNA EXPRESSION, DNA METHYLATION AND HISTONE MODIFICATIONS) CONDUCTED ACROSS VARIOUS POPULATIONS SUPPORT THE NOTION THAT THERE IS A GENETIC BASIS FOR CERTAIN SUBSETS OF OA PATHOGENESIS. CONCLUSION: WITH RECENT ADVANCES IN THE DEVELOPMENT OF GENOME EDITING TECHNOLOGIES SUCH AS THE CRISPR-CAS9 SYSTEM, THESE GENETIC AND EPIGENETIC ALTERNATIONS IN OA CAN BE USED AS PLATFORMS FROM WHICH POTENTIAL BIOMARKERS FOR THE DIAGNOSIS, PROGNOSIS, DRUG RESPONSE, AND DEVELOPMENT OF POTENTIAL PERSONALIZED THERAPEUTIC TARGETS FOR OA CAN BE APPROACHED. FURTHERMORE, GENOME EDITING HAS ALLOWED THE DEVELOPMENT OF "DESIGNER" CELLS, WHEREBY THE RECEPTORS, GENE REGULATORY NETWORKS, OR TRANSGENES CAN BE MODIFIED AS A BASIS FOR NEW CELL-BASED THERAPIES. 2019 11 3768 28 INTEGRATIVE EPIGENOMICS IN CHRONIC LYMPHOCYTIC LEUKAEMIA: BIOLOGICAL INSIGHTS AND CLINICAL APPLICATIONS. CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) IS NOT ONLY CHARACTERISED BY DRIVER GENETIC ALTERATIONS BUT BY EXTENSIVE EPIGENETIC CHANGES. OVER THE LAST DECADE, EPIGENOMIC STUDIES HAVE DESCRIBED THE DNA METHYLOME, CHROMATIN ACCESSIBILITY, HISTONE MODIFICATIONS AND THE THREE-DIMENSIONAL (3D) GENOME ARCHITECTURE OF CLL. BEYOND ITS REGULATORY ROLE, THE DNA METHYLOME CONTAINS IMPRINTS OF THE CELLULAR ORIGIN AND PROLIFERATIVE HISTORY OF CLL CELLS. THESE TWO ASPECTS ARE STRONG INDEPENDENT PROGNOSTIC FACTORS. INTEGRATIVE ANALYSES OF CHROMATIN MARKS HAVE UNCOVERED NOVEL REGULATORY ELEMENTS AND ALTERED TRANSCRIPTION FACTOR NETWORKS AS NON-GENETIC MEANS MEDIATING GENE DEREGULATION IN CLL. ADDITIONALLY, CLL CELLS DISPLAY A DISEASE-SPECIFIC PATTERN OF 3D GENOME INTERACTIONS. FROM THE TECHNOLOGICAL PERSPECTIVE, WE ARE CURRENTLY WITNESSING A TRANSITION FROM BULK OMICS TO SINGLE-CELL ANALYSES. THIS REVIEW AIMS AT SUMMARISING THE MAJOR FINDINGS FROM THE EPIGENOMICS FIELD AS WELL AS PROVIDING A PROSPECT OF THE PRESENT AND FUTURE OF SINGLE-CELL ANALYSES IN CLL. 2023 12 3100 31 GENOMIC IMPACT OF CIGARETTE SMOKE, WITH APPLICATION TO THREE SMOKING-RELATED DISEASES. THERE IS CONSIDERABLE EVIDENCE THAT INHALED TOXICANTS SUCH AS CIGARETTE SMOKE CAN CAUSE BOTH IRREVERSIBLE CHANGES TO THE GENETIC MATERIAL (DNA MUTATIONS) AND PUTATIVELY REVERSIBLE CHANGES TO THE EPIGENETIC LANDSCAPE (CHANGES IN THE DNA METHYLATION AND CHROMATIN MODIFICATION STATE). THE DISEASES THAT ARE BELIEVED TO INVOLVE GENETIC AND EPIGENETIC PERTURBATIONS INCLUDE LUNG CANCER, CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD), AND CARDIOVASCULAR DISEASE (CVD), ALL OF WHICH ARE STRONGLY LINKED EPIDEMIOLOGICALLY TO CIGARETTE SMOKING. IN THIS REVIEW, WE HIGHLIGHT THE SIGNIFICANCE OF GENOMICS AND EPIGENOMICS IN THESE MAJOR SMOKING-RELATED DISEASES. WE ALSO SUMMARIZE THE IN VITRO AND IN VIVO FINDINGS ON THE SPECIFIC PERTURBATIONS THAT SMOKE AND ITS CONSTITUENT COMPOUNDS CAN INFLICT UPON THE GENOME, PARTICULARLY ON THE PULMONARY SYSTEM. FINALLY, WE REVIEW STATE-OF-THE-ART GENOMICS AND NEW TECHNIQUES SUCH AS HIGH-THROUGHPUT SEQUENCING AND GENOME-WIDE CHROMATIN ASSAYS, RAPIDLY EVOLVING TECHNIQUES WHICH HAVE ALLOWED EPIGENETIC CHANGES TO BE CHARACTERIZED AT THE GENOME LEVEL. THESE TECHNIQUES HAVE THE POTENTIAL TO SIGNIFICANTLY IMPROVE OUR UNDERSTANDING OF THE SPECIFIC MECHANISMS BY WHICH EXPOSURE TO ENVIRONMENTAL CHEMICALS CAUSES DISEASE. SUCH MECHANISTIC KNOWLEDGE PROVIDES A VARIETY OF OPPORTUNITIES FOR ENHANCED PRODUCT SAFETY ASSESSMENT AND THE DISCOVERY OF NOVEL THERAPEUTIC INTERVENTIONS. 2012 13 6031 22 THE CANCER EPIGENOME: ITS ORIGINS, CONTRIBUTIONS TO TUMORIGENESIS, AND TRANSLATIONAL IMPLICATIONS. EPIGENETIC ABNORMALITIES IN LUNG AND OTHER CANCERS CONTINUE TO BE DEFINED AT A RAPID PACE. WE ARE COMING TO APPRECIATE THAT CANCERS HAVE AN "EPIGENETIC LANDSCAPE" WHEREIN GENES VULNERABLE TO ABNORMALITIES, SUCH AS PROMOTER DNA HYPERMETHYLATION AND ASSOCIATED GENE SILENCING, TEND TO RESIDE IN DEFINED NUCLEAR POSITIONS AND CHROMOSOME DOMAINS AND RELATIONSHIPS TO CHROMATIN REGULATION, WHICH FACILITATES STATES OF STEM CELL RENEWAL. THESE SAME GENES AND DOMAINS ARE ALSO VULNERABLE TO EPIGENETIC ABNORMALITIES INDUCED BY FACTORS TO WHICH CELLS ARE EXPOSED DURING CANCER RISK STATES, SUCH AS CHRONIC INFLAMMATION. WE CAN USE ALL OF THIS BASIC INFORMATION FOR TRANSLATIONAL PURPOSES IN TERMS OF DERIVING BIOMARKERS FOR CANCER RISK STATES AND DETECTION AND THERAPEUTIC STRATEGIES. 2012 14 2914 35 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 15 6809 29 [EPIGENETICS IN INFLAMMATORY SYSTEMIC DISEASES]. IN ADDITION TO ANALYSIS OF THE GENETIC CODE, IN RECENT YEARS MORE AND MORE STUDIES HAVE CONCENTRATED ON CHANGES IN THE EPIGENETIC CODE. EPIGENETIC MECHANISMS DETERMINE WHICH GENES IN A CELL ARE TRANSCRIBED AND THUS FORM THE PHENOTYPE OF A CELL. THE EPIGENETIC CODE CAN BE CHANGED BY ENVIRONMENTAL INFLUENCES, WHICH ALLOWS CELLS TO ADAPT TO LONGSTANDING CHANGES IN THE ENVIRONMENT. THEREFORE, IT IS FEASIBLE TO ASSUME THAT EPIGENETIC CHANGES ARE THE MOLECULAR BASIS FOR LONG-TERM EFFECTS OF THE ENVIRONMENT ON DISEASE DEVELOPMENT. IN PARTICULAR IN TUMORS AND CHRONIC INFLAMMATORY DISEASES EPIGENETIC CHANGES WERE FOUND TO CORRELATE WITH DISEASE SEVERITY AND PROGRESSION. KNOWLEDGE ABOUT THESE EPIGENETIC CHANGES MIGHT HELP THAT EPIGENETIC MODIFICATIONS CAN BE USED IN THE FUTURE AS BIOMARKERS, PROGNOSTIC FACTORS AND THERAPEUTIC TARGETS. 2014 16 5742 24 SMOKING MOLECULAR DAMAGE IN BRONCHIAL EPITHELIUM. OUR UNDERSTANDING OF THE MOLECULAR PATHOLOGY OF LUNG CANCER IS ADVANCING RAPIDLY WITH SEVERAL SPECIFIC GENES AND CHROMOSOMAL REGIONS BEING IDENTIFIED. LUNG CANCER APPEARS TO REQUIRE MANY MUTATIONS IN BOTH DOMINANT AND RECESSIVE ONCOGENES TO POSSESS MALIGNANT PHENOTYPES. SEVERAL GENETIC AND EPIGENETIC CHANGES ARE COMMON TO ALL LUNG CANCER HISTOLOGIC TYPES, WHILE OTHERS APPEAR TO BE CELL TYPE SPECIFIC. HOWEVER, SPECIFIC ROLES OF THE GENES UNDERGOING MUTATIONS AND THE ORDER OF CUMULATIVE MOLECULAR CHANGES THAT LEAD TO THE DEVELOPMENT OF EACH LUNG TUMOR HISTOLOGIC TYPE REMAIN TO BE FULLY ELUCIDATED. RECENT FINDINGS OF MOLECULAR ABNORMALITIES IN NORMAL APPEARING AND PRENEOPLASTIC BRONCHIAL EPITHELIUM FROM PATIENTS WITH LUNG CANCER AND CHRONIC SMOKERS SUGGEST THAT GENETIC CHANGES MAY SERVE AS BIOMARKERS FOR EARLY DIAGNOSIS, RISK ASSESSMENT AND MONITORING RESPONSE TO CHEMOPREVENTION. 2002 17 4028 28 LUNG TUMORS, COPD AND IMMUNE RESPONSE: IS EPIGENETICS THE BOTTOM LINE? NSCLC IS A HETEROGENEOUS DISORDER CONSISTING OF DISTINCT MOLECULAR SUBTYPES WHICH CAN BE TREATED BY USING SPECIFIC DRUGS TARGETED TO DISTINCT GENETIC LESIONS. IT IS WELL KNOWN THAT NSCLS INCIDENCE IS HIGHER IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) PATIENTS BECAUSE THEY SHARE A COMMON RISK FACTOR (CIGARETTE SMOKING) AND IT IS BELIEVED THAT THE TYPICAL INFLAMMATORY MICROENVIRONMENT OBSERVED IN COPD MAY INFLUENCE THE MOLECULAR MECHANISMS RESPONSIBLE OF CARCINOGENESIS. IN THE LAST YEARS, THE ROLE OF EPIGENETIC PROCESSES IN CELL BIOLOGY AND TISSUE PATHOLOGY HAS BEEN EXTENSIVELY STUDIED BOTH IN COPD AND NSCLC. THE RECENT PAPER BY WAUTERS ET AL. SHOWED A SPECIFIC PATTERN OF DRIVER MUTATIONS AND MOLECULAR FEATURES IN NSCLC RAISING IN THE CONTEXT OF COPD. ALL THESE FINDINGS HAVE SHOWN FOR THE FIRST TIME THAT LUNG TUMORS FOUND IN COPD PATIENTS DIFFER FROM THOSE OBSERVED IN PATIENT WITHOUT COPD DUE TO THE PRESENCE OF A SPECIFIC TUMOR MICROENVIRONMENT WHICH IS CHARACTERIZED BY REDUCED CD4+ TREG CELLS. ON THIS BASIS, THE PRESENT WORK AIMS AT DISCUSSING AND ANALYZING THE CONTEXT-SPECIFIC MECHANISMS OF CLONAL SELECTION AND EVOLUTION MAINLY FOCUSING ON THE EPIGENETIC ALTERATIONS AND AT POINTING OUT THE POTENTIAL THERAPEUTIC IMPLICATIONS. 2016 18 127 26 A TRANSCRIPTIONAL PERSPECTIVE ON HUMAN MACROPHAGE BIOLOGY. MACROPHAGES ARE A MAJOR CELL TYPE IN TISSUE HOMEOSTASIS AND CONTRIBUTE TO BOTH PATHOLOGY AND RESOLUTION IN ALL ACUTE AND CHRONIC INFLAMMATORY DISEASES RANGING FROM INFECTIONS, CANCER, OBESITY, ATHEROSCLEROSIS, AUTOIMMUNE DISORDERS TO NEURODEGENERATIVE DISEASES SUCH AS ALZHEIMER'S DISEASE. THE CELLULAR AND FUNCTIONAL DIVERSITY OF MACROPHAGES DEPENDS UPON TIGHTLY REGULATED TRANSCRIPTION. THE INNATE IMMUNE SYSTEM IS UNDER PROFOUND EVOLUTIONARY SELECTION. THERE IS INCREASING RECOGNITION THAT HUMAN MACROPHAGE BIOLOGY DIFFERS VERY SIGNIFICANTLY FROM THAT OF COMMONLY STUDIED ANIMAL MODELS, WHICH THEREFORE CAN HAVE A LIMITED PREDICTIVE VALUE. HERE WE REPORT ON THE NEWEST FINDINGS ON TRANSCRIPTIONAL CONTROL OF MACROPHAGE ACTIVATION, AND HOW WE ENVISION INTEGRATING STUDIES ON TRANSCRIPTIONAL AND EPIGENETIC REGULATION, AND MORE CLASSICAL APPROACHES IN MURINE MODELS. MOREOVER, WE PROVIDE NEW INSIGHTS INTO HOW WE CAN LEARN ABOUT TRANSCRIPTIONAL REGULATION IN THE HUMAN SYSTEM FROM LARGER EFFORTS SUCH AS THE FANTOM (FUNCTIONAL ANNOTATION OF THE MAMMALIAN GENOME) CONSORTIUM. 2015 19 4429 30 MOLECULAR BIOLOGY AS A TOOL FOR THE TREATMENT OF CANCER. CANCER IS A GENETIC DISEASE CHARACTERIZED BY UNCONTROLLED CELL GROWTH AND METASTASIS. CANCER CAN HAVE A NUMBER OF CAUSES, SUCH THE ACTIVATION OF ONCOGENES, THE INACTIVATION OF TUMOR-SUPPRESSING GENES, MUTAGENESIS PROVOKED BY EXTERNAL FACTORS, AND EPIGENETIC MODIFICATIONS. THE DEVELOPMENT OF DIAGNOSTIC TOOLS AND TREATMENTS USING A MOLECULAR BIOLOGICAL APPROACH PERMITS THE USE OF SENSITIVE, LOW-COST, NONINVASIVE TESTS FOR CANCER PATIENTS. BIOMARKERS CAN BE USED TO PROVIDE RAPID, PERSONALIZED ONCOLOGY, IN PARTICULAR THE MOLECULAR DIAGNOSIS OF CHRONIC MYELOID LEUKEMIA, AND GASTRIC, COLON, AND BREAST CANCERS. MOLECULAR TESTS BASED ON DNA METHYLATION CAN ALSO BE USED TO DIRECT TREATMENTS OR EVALUATE THE TOXIC EFFECTS OF CHEMOTHERAPY. THE ADEQUATE DIAGNOSIS, PROGNOSIS, AND PREDICTION OF THE RESPONSE OF CANCER PATIENTS TO TREATMENT ARE ESSENTIAL TO ENSURE THE MOST EFFECTIVE THERAPY, REDUCE THE DAMAGING EFFECTS OF TREATMENT, AND DIRECT THE THERAPY TO SPECIFIC TARGETS, AND IN THIS CONTEXT, MOLECULAR BIOLOGY HAS BECOME INCREASINGLY IMPORTANT IN ONCOLOGY. IN THIS BRIEF REVIEW, WE WILL DEMONSTRATE THE FUNDAMENTAL IMPORTANCE OF MOLECULAR BIOLOGY FOR THE TREATMENT OF THREE TYPES OF CANCER-CHRONIC MYELOID LEUKEMIA, HEREDITARY DIFFUSE GASTRIC CANCER, AND ASTROCYTOMAS (SPORADIC TUMORS OF THE CENTRAL NERVOUS SYSTEM). IN EACH OF THESE THREE MODELS, DISTINCT BIOLOGICAL MECHANISMS ARE INVOLVED IN THE TRANSFORMATION OF THE CELLS, BUT IN ALL CASES, MOLECULAR BIOLOGY IS FUNDAMENTAL TO THE DEVELOPMENT OF PERSONALIZED ANALYSES FOR EACH PATIENT AND EACH TYPE OF NEOPLASIA, AND TO GUARANTEE THE SUCCESS OF THE TREATMENT. 2018 20 6284 32 THE POTENTIAL OF HUMAN INDUCED PLURIPOTENT STEM CELLS FOR MODELLING DIABETIC WOUND HEALING IN VITRO. IMPAIRED WOUND HEALING AND ULCERATION CAUSED BY DIABETES MELLITUS, IS A SIGNIFICANT HEALTHCARE BURDEN, MARKEDLY IMPAIRS QUALITY OF LIFE FOR PATIENTS, AND IS THE MAJOR CAUSE OF AMPUTATION WORLDWIDE. CURRENT EXPERIMENTAL APPROACHES USED TO INVESTIGATE THE COMPLEX WOUND HEALING PROCESS OFTEN INVOLVE CULTURES OF FIBROBLASTS AND/OR KERATINOCYTES IN VITRO, WHICH CAN BE LIMITED IN TERMS OF COMPLEXITY AND CAPACITY, OR UTILISATION OF RODENT MODELS IN WHICH THE MECHANISMS OF WOUND REPAIR DIFFER SUBSTANTIVELY FROM THAT IN HUMANS. HOWEVER, ADVANCES IN TISSUE ENGINEERING, AND THE DISCOVERY OF STRATEGIES TO REPROGRAMME ADULT SOMATIC CELLS TO PLURIPOTENCY, HAS LED TO THE POSSIBILITY OF DEVELOPING MODELS OF HUMAN SKIN ON A LARGE SCALE. GENERATION OF INDUCED PLURIPOTENT STEM CELLS (IPSCS) FROM TISSUES DONATED BY DIABETIC PATIENTS ALLOWS THE (EPI)GENETIC BACKGROUND OF THIS DISEASE TO BE STUDIED, AND THE ABILITY TO DIFFERENTIATE IPSCS TO MULTIPLE CELL TYPES FOUND WITHIN SKIN MAY FACILITATE THE DEVELOPMENT OF MORE COMPLEX SKIN MODELS; THESE ADVANCES OFFER KEY OPPORTUNITIES FOR IMPROVING MODELLING OF WOUND HEALING IN DIABETES, AND THE DEVELOPMENT OF EFFECTIVE THERAPEUTICS FOR TREATMENT OF CHRONIC WOUNDS. 2018