RESUMO
The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.
Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Homólogo 5 da Proteína Cromobox , Fibroblastos , CamundongosRESUMO
Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.
Assuntos
DNA Ribossômico , Instabilidade Genômica , Mutação , RNA Polimerase I , Processamento Pós-Transcricional do RNA , RNA Ribossômico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerase I/metabolismo , RNA Polimerase I/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Pol1 do Complexo de Iniciação de TranscriçãoRESUMO
OBJECTIVES: Recent insights supporting the safety of live-attenuated vaccines and novel studies on the immunogenicity of vaccinations in the era of biological disease-modifying antirheumatic drugs in paediatric patients with autoimmune/inflammatory rheumatic diseases (pedAIIRD) necessitated updating the EULAR recommendations. METHODS: Recommendations were developed using the EULAR standard operating procedures. Two international expert committees were formed to update the vaccination recommendations for both paediatric and adult patients with AIIRD. After a systematic literature review, separate recommendations were formulated for paediatric and adult patients. For pedAIIRD, six overarching principles and seven recommendations were formulated and provided with the level of evidence, strength of recommendation and Task Force level of agreement. RESULTS: In general, the National Immunisation Programmes (NIP) should be followed and assessed yearly by the treating specialist. If possible, vaccinations should be administered prior to immunosuppressive drugs, but necessary treatment should never be postponed. Non-live vaccines can be safely given to immunosuppressed pedAIIRD patients. Mainly, seroprotection is preserved in patients receiving vaccinations on immunosuppression, except for high-dose glucocorticoids and B-cell depleting therapies. Live-attenuated vaccines should be avoided in immunosuppressed patients. However, it is safe to administer the measles-mumps-rubella booster and varicella zoster virus vaccine to immunosuppressed patients under specific conditions. In addition to the NIP, the non-live seasonal influenza vaccination should be strongly considered for immunosuppressed pedAIIRD patients. CONCLUSIONS: These recommendations are intended for paediatricians, paediatric rheumatologists, national immunisation agencies, general practitioners, patients and national rheumatology societies to attain safe and effective vaccination and optimal infection prevention in immunocompromised pedAIIRD patients.
Assuntos
Antirreumáticos , Doenças Autoimunes , Doenças Reumáticas , Adulto , Humanos , Criança , Vacinas Atenuadas/uso terapêutico , Doenças Reumáticas/tratamento farmacológico , Vacinação/métodos , Imunossupressores/efeitos adversos , Antirreumáticos/uso terapêutico , Doenças Autoimunes/tratamento farmacológicoRESUMO
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , DNA Ribossômico/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/genética , RNA Ribossômico , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.
Assuntos
Cromossomos Fúngicos/química , Modelos Genéticos , Cromatina/química , DNA Fúngico/química , Cinética , Movimento (Física) , Nucleossomos/químicaRESUMO
How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30â nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope.
Assuntos
Ciclo Celular , Forma do Núcleo Celular , Microscopia Confocal/métodos , Saccharomycetales/citologia , Carbono/farmacologia , Ciclo Celular/efeitos dos fármacos , Forma do Núcleo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Imageamento Tridimensional , Interfase/efeitos dos fármacos , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismoRESUMO
The aim of this preliminary study is to analyze genetic specificity of Kosovo Albanians comparing with neighboring populations using new genetic tool - MEDISCOPE gene chip, to investigate the feasibility of this approach. We collected 37 DNA samples (9 Croats, 17 Albanians from Croatia and 11 Albanians from Kosovo) from unrelated males born in Croatia and Kosovo. Additionally, samples were expanded with female individuals and mtDNA analysis included a total of 61 samples (15 Croats, 23 Albanians from Croatia and 23 Albanians from Kosovo). This pilot study suggests that the usage of the MEDISCOPE chip could be recognized as an efficient tool within recognition of the population genetic specificity even within extremely small sample size.
Assuntos
Variação Genética/genética , Genética Populacional/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cromossomos Humanos Y/genética , Croácia , DNA Mitocondrial/genética , Feminino , Marcadores Genéticos/genética , Humanos , Kosovo , Masculino , Projetos Piloto , População Branca/genéticaRESUMO
Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of â¼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.
Assuntos
DNA Ribossômico/metabolismo , Proteínas HMGB/química , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Nucléolo Celular/metabolismo , Sequência Conservada , Proteínas HMGB/genética , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase I/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Relação Estrutura-AtividadeRESUMO
Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC(50) in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.
Assuntos
Elipticinas/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase I/metabolismo , Transcrição Gênica , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Ribossômico/metabolismoRESUMO
BACKGROUND: Juvenile idiopathic arthritis (JIA) refers to a heterogeneous group of rheumatic conditions in children. Novel drugs have greatly improved disease outcomes; however, outcomes are impacted by limited awareness of the importance of early diagnosis and adequate treatment, and by differences in access across health systems. As a result, patients with JIA continue to be at risk for short- and long-term morbidity, as well as impacts on virtually all aspects of life of the child and family. MAIN BODY: Literature on the socioeconomic burden of JIA is largely focused on healthcare costs, and the impact of JIA on patients, families, and communities is not well understood. High quality evidence on the impact of JIA is needed to ensure that patients are receiving necessary support, timely diagnostics, and adequate treatment, and to inform decision making and resource allocation. This commentary introduces the European Joint Programme on Rare Diseases: Producing an Arthritis Value Framework with Economic Evidence: Paving the Way for Rare Childhood Diseases (PAVE) project, which will co-develop a patient-informed value framework to measure the impact of JIA on individuals and on society. With a patient-centered approach, fundamental to PAVE is the involvement of three patient advocacy organizations from Canada, Israel, and Europe, as active research partners co-designing all project phases and ensuring robust patient and family engagement. The framework will build on the findings of projects from six countries: Canada, Germany, Switzerland, Spain, Israel, and Belgium, exploring costs, outcomes (health, well-being), and unmet needs (uveitis, mental health, equity). CONCLUSION: This unique international collaboration will combine evidence on costs (from family to societal), outcomes (clinical, patient and family outcomes), and unmet needs, to co-design and build a framework with patients and families to capture the full impact of JIA. The framework will support the development of high-quality evidence, encompassing economic and clinical considerations, unmet needs, and patient perspectives, to inform equitable resource allocation, health system planning, and quality of care better aligned with the needs of children with JIA, their families, and communities. Knowledge gained from this novel approach may pave the way forward to be applied more broadly to other rare childhood diseases.
Assuntos
Artrite Juvenil , Assistência Centrada no Paciente , Doenças Raras , Humanos , Artrite Juvenil/economia , Assistência Centrada no Paciente/economia , Criança , Doenças Raras/economia , Efeitos Psicossociais da Doença , Europa (Continente)RESUMO
Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Assuntos
Fenômenos Biofísicos , Núcleo Celular/genética , Cromatina/genética , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , Cromossomos/genética , Difusão , Regulação Fúngica da Expressão Gênica , Genoma , Modelos TeóricosRESUMO
In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1-IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1-DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.
Assuntos
DNA Nucleotidiltransferases/química , Recombinases/química , Motivos de Aminoácidos , Biocatálise , Clivagem do DNA , DNA Nucleotidiltransferases/metabolismo , Modelos Moleculares , Recombinases/metabolismo , Recombinação Genética , Sequências Repetitivas de AminoácidosRESUMO
Background: In 2011, the first European League Against Rheumatism (EULAR) vaccination recommendations for pediatric patients with autoimmune inflammatory rheumatic diseases (pedAIIRD) were published. The past decade numerous new studies were performed to assess the safety, efficacy and immunogenicity of vaccinations in pedAIIRD. A systematic literature review (SLR) was therefore performed to serve as the basis for the updated 2021 EULAR/PRES recommendations. Methods: An SLR was performed according to the standard operating procedures for EULAR-endorsed recommendations. Primary outcomes were efficacy, immunogenicity and safety of vaccination in pedAIIRD. The search was performed in Medline, Embase and the Cochrane Library and included studies published from November 2010 until July 2020. Results: The SLR yielded 57 studies which were included for critical appraisal and data extraction. Only 8 studies described the occurrence of vaccine-preventable infections after vaccination (efficacy), none of these studies were powered to assess efficacy. The majority of studies assessed (humoral) immune responses as surrogate endpoint for vaccine efficacy. Studies on non-live vaccines showed that these were safe and in general immunogenic. Biologic disease-modifying antirheumatic drugs (bDMARDs) in general did not significantly reduce seroprotection rates, except for B-cell depleting therapies which severely hampered humoral responses. Four new studies on human papilloma virus vaccination showed that this vaccine was safe and immunogenic in pedAIIRD. Regarding live-attenuated vaccinations, level 1 evidence of the measles mumps rubella (MMR) booster vaccination became available which showed the safety of this booster for patients treated with methotrexate. In addition, level 3 evidence became available that suggested that the MMR and varicella zoster virus (VZV) vaccination for patients on low dose glucocorticosteroids and bDMARDs might be safe as well. Conclusions: The past decade, knowledge on the safety and immunogenicity of (live-attenuated) vaccines in pedAIIRD significantly increased. Data on efficacy (infection prevention) remains scarce. The results from this SLR are the basis for the updated EULAR/PRES vaccination recommendations in pedAIIRD.
RESUMO
Branched chain amino acids (BCAA) are essential elements of the human diet, which display increased plasma levels in obesity and regained particular interest as potential biomarkers for development of diabetes. To define determinants of insulin resistance (IR) we investigated 73 genes involved in BCAA metabolism or regulation by fine-scale haplotype mapping in two European populations with metabolic syndrome. French and Romanians (n = 465) were genotyped for SNPs (Affymetrix) and enriched by imputation (BEAGLE 4.1) at 1000 genome project density. Initial association hits detected by sliding window were refined (HAPLOVIEW 3.1 and PHASE 2.1) and correlated to homeostasis model assessment (HOMAIR) index, in vivo insulin sensitivity (SI) and BCAA plasma levels (ANOVA). Four genomic regions were associated with IR located downstream of MUT, AACS, SLC6A15 and PRKCA genes (P between 9.3 and 3.7 x 10-5). Inferred haplotypes up to 13 SNPs length were associated with IR (e.g. MUT gene with P < 4.9 x 10-5; Bonferroni 1.3 x 10-3) and synergistic to HOMAIR. SNPs in the same regions were also associated with one order of magnitude lower P values (e.g. rs20167284 in the MUT gene with P < 1.27 x 10-4) and replicated in Mediterranean samples (n = 832). In French, influential haplotypes (OR > 2.0) were correlated with in vivo insulin sensitivity (1/SI) except for SLC6A15 gene. Association of these genes with BCAA levels was variable, but influential haplotypes confirmed implication of MUT from BCAA metabolism as well as a role of regulatory genes (AACS and PRKCA) and suggested potential changes in transcriptional activity. These data drive attention towards new regulatory regions involved in IR in relation with BCAA and show the ability of haplotypes in phased DNA to detect signals complimentary to SNPs, which may be useful in designing genetic markers for clinical applications in ethnic populations.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos de Cadeia Ramificada/genética , Haplótipos , Resistência à Insulina/genética , Síndrome Metabólica/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Proteína Quinase C-alfa/genética , Adulto , Aminoácidos de Cadeia Ramificada/metabolismo , Feminino , Humanos , Masculino , Síndrome Metabólica/sangue , Pessoa de Meia-IdadeRESUMO
Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d'Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27â»65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Bases de Dados Factuais/estatística & dados numéricos , Análise de Alimentos/métodos , Avaliação Nutricional , Software , Adulto , Idoso , Feminino , França , Grécia , Humanos , Masculino , Região do Mediterrâneo , Pessoa de Meia-Idade , Inquéritos NutricionaisRESUMO
AIM OF THE STUDY: APOA5 has been linked to metabolic syndrome (MetS) or its traits in several populations. In North Africa, only the Moroccan population was investigated. Our aim is to assess the association between APOA5 gene polymorphisms with the susceptibility to MetS and its components in the Tunisian population. MATERIALS AND METHODS: A total of 594 participants from the Tunisian population were genotyped for two polymorphisms rs3135506 and rs651821 located in APOA5 gene using KASPar technology. Statistical analyses were performed using R software. RESULTS: The SNP rs651821 increased the risk of MetS under the dominant model (OR=1.91 [1.17-3.12], P=0.008) whereas the variant rs3135506 was not associated with MetS. After stratification of the cohort following the sex, only the variant rs651821 showed a significant association with MetS among the women group. The influence of the geographic origin of the studied population on the genotype distribution of APOA5 variants showed that the variant rs651821 was significantly associated with MetS only for the Northern population. The association analyses of the variants rs651821 and rs3135506 with different quantitative traits of MetS showed a significant association only between the variant rs3135506 and triglycerides levels. CONCLUSION: This is the first study reporting the association of APOA5 gene variants with MetS in Tunisia. Our study emphasizes the role of APOA5 variants in the regulation of the triglycerides blood levels. Further studies are needed to confirm the clinical relevance of these associations and to better understand the physiopathology of the MetS.
Assuntos
Apolipoproteína A-V/genética , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/epidemiologia , Pessoa de Meia-Idade , Fenótipo , Triglicerídeos/sangue , Tunísia/epidemiologiaRESUMO
Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.
Assuntos
Nucléolo Celular/genética , Cromossomos Fúngicos , Ensaios de Triagem em Larga Escala , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Genes Reporter , Loci Gênicos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Imagem com Lapso de TempoRESUMO
Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging.
Assuntos
Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Transcrição Gênica , Microscopia Eletrônica de Transmissão , RNA Polimerase I/genética , RNA Ribossômico/genéticaRESUMO
The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization.
Assuntos
Nucléolo Celular/metabolismo , Centrômero/metabolismo , RNA Polimerase III/genética , Nucléolo Celular/fisiologia , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromossomos/fisiologia , Imagem Óptica/métodos , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Telômero/fisiologia , Transcrição GênicaRESUMO
The FTO (fat mass and obesity associated) gene was associated with different metabolic disorders in populations from different origins but with great difference between African and non-African populations. North-African populations combine many genetic backgrounds, among which African, Berber and Caucasian components, which makes North-Africans a good model for studying the genetic association of FTO. In the present investigation we explored the association of FTO gene with polycystic ovary syndrome (PCOS) in a population from Tunisia (n=278). Single nucleotide polymorphisms (SNPs) used in this study were previously associated in non-African populations: rs8050136 (A/C), rs9939609 (A/T), rs9930506 (G/A), or in both African and non-African populations: rs8057044 (A/G). Genotyping was performed by allelic discrimination method on StepOne real-time PCR system or KASPar technology. Linkage disequilibrium (LD) pattern was assessed by HAPLOVIEW and reconstruction of haplotypes was performed by PHASE, while statistical analyses were performed using StatView and GoldenHelix programs. Among the 13 haplotypes in the population, three (h1, h7 and h13) were strongly associated with PCOS notably h13 (P<0.0001, OR95%CI=0.040 [0.005-0.294]) while SNPs display weaker association. Moreover the LD pattern in FTO in the Tunisian population (r(2) index) was intermediary between those of Caucasian and Africans. This highlights the need for studying the genetics of complex disorders in the North-African populations taking into-account the haplotype structure of candidate loci more than SNPs taken alone.