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1.
Microbes Infect ; 26(5-6): 105342, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38679229

RESUMO

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.


Assuntos
Glicolipídeos , Síndrome de Guillain-Barré , Mycoplasma pneumoniae , Síndrome de Guillain-Barré/microbiologia , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/imunologia , Humanos , Glicolipídeos/metabolismo , Galactosilceramidas , Reações Cruzadas , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia
2.
Mol Syst Biol ; 19(10): e11301, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37642167

RESUMO

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

3.
Microbiol Spectr ; 11(3): e0485922, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37097155

RESUMO

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.


Assuntos
Cisteína , Infecções por Mycoplasma , Animais , Suínos , Humanos , Proteômica , Mycoplasma pneumoniae , Fosfolipídeos , Vitaminas
4.
Nucleic Acids Res ; 50(22): e127, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36215032

RESUMO

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.


Assuntos
Edição de Genes , Mycoplasma pneumoniae , Sistemas CRISPR-Cas , Mycoplasma pneumoniae/genética , Plasmídeos/genética
5.
iScience ; 24(9): 102985, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34485867

RESUMO

Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.

6.
Mol Syst Biol ; 16(12): e9530, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33320415

RESUMO

Protein degradation is a crucial cellular process in all-living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP-dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA-seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half-life measurements also suggest a role for Lon-modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.


Assuntos
Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Proteólise , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Mutação/genética , Mycoplasma pneumoniae/enzimologia , Peptídeo Hidrolases/metabolismo , Fenótipo , Dobramento de Proteína , Controle de Qualidade , Reprodutibilidade dos Testes , Especificidade por Substrato , Transcrição Gênica
7.
NPJ Syst Biol Appl ; 6(1): 33, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097709

RESUMO

Mycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation challenging and expensive. To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict essential components validated with in vitro serum-free media able to sustain growth. Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae. Altogether, our modelling approach allowed us to optimize medium composition, enabled growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive production.


Assuntos
Meios de Cultura Livres de Soro , Modelos Biológicos , Mycoplasma pneumoniae/crescimento & desenvolvimento , Metabolismo Energético , Glicólise , Mycoplasma pneumoniae/metabolismo
8.
Nucleic Acids Res ; 48(17): e102, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32813015

RESUMO

Transposon sequencing is commonly applied for identifying the minimal set of genes required for cellular life; a major challenge in fields such as evolutionary or synthetic biology. However, the scientific community has no standards at the level of processing, treatment, curation and analysis of this kind data. In addition, we lack knowledge about artifactual signals and the requirements a dataset has to satisfy to allow accurate prediction. Here, we have developed FASTQINS, a pipeline for the detection of transposon insertions, and ANUBIS, a library of functions to evaluate and correct deviating factors known and uncharacterized until now. ANUBIS implements previously defined essentiality estimate models in addition to new approaches with advantages like not requiring a training set of genes to predict general essentiality. To highlight the applicability of these tools, and provide a set of recommendations on how to analyze transposon sequencing data, we performed a comprehensive study on artifacts corrections and essentiality estimation at a 1.5-bp resolution, in the genome-reduced bacterium Mycoplasma pneumoniae. We envision FASTQINS and ANUBIS to aid in the analysis of Tn-seq procedures and lead to the development of accurate genome essentiality estimates to guide applications such as designing live vaccines or growth optimization.


Assuntos
Elementos de DNA Transponíveis , Genômica/métodos , Análise de Sequência de DNA/métodos , Software , Genômica/normas , Mycoplasma pneumoniae , Recombinação Genética , Análise de Sequência de DNA/normas
9.
Mol Syst Biol ; 16(5): e9208, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32449593

RESUMO

The C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C-terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.


Assuntos
Aminoácidos/química , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Aminoácidos/metabolismo , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/metabolismo , Arginina/química , Arginina/metabolismo , Bactérias/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Análise por Conglomerados , Uso do Códon/genética , Códon de Terminação/genética , Biologia Computacional , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Lisina/metabolismo , Mycoplasma pneumoniae/química , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Filogenia , Domínios Proteicos , Processamento de Proteína Pós-Traducional/genética
10.
Disaster Med Public Health Prep ; 13(1): 82-89, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30841955

RESUMO

OBJECTIVE: The aim of this study was the construction and validation of a novel research instrument to quantify the degree of post-hurricane trauma and distress in an affected population. The Post-Hurricane Distress Scale (PHDS) has quantitative measures of both acute and prolonged distress, attributable to meteorological and hydrological disasters. METHODS: A careful evaluation of existing questionnaires, as well as extensive canvasing of the post-Maria population of Puerto Rico, availed the construction of the PHDS. The PHDS consists of 20 items, organized into 4 subscales. The PHDS was pre-validated (n=79), revised, and then distributed to a broad sampling of the post-Hurricane Maria Puerto Rican population (n=597). Validation, including factor analysis, analyses of concurrent validity, discriminant validity, and internal reliability, was performed. RESULTS: After comparing various scales, factor loading profiles, concurrent validities, and models of fit, we show that the PHDS is best scored as a single 0-6 distress scale. When compared with the Traumatic Exposure Severity Scale, the PHDS shows superior concurrent validity, more accurately predicting scores for the Peritraumatic Distress Inventory, Impact of Event Scale - Revised, and Generalized Anxiety Disorder 7 Scale. The PHDS shows good internal reliability and discriminant validity. CONCLUSIONS: The PHDS represents a novel, useful instrument for disaster first-responders and researchers. The prompt identification of high-risk populations is possible using this instrument. (Disaster Med Public Health Preparedness. 2019;13:82-89).


Assuntos
Tempestades Ciclônicas/estatística & dados numéricos , Socorristas/psicologia , Psicometria/normas , Pesquisadores/psicologia , Estresse Psicológico/classificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Socorristas/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Psicometria/instrumentação , Psicometria/métodos , Porto Rico/epidemiologia , Reprodutibilidade dos Testes , Pesquisadores/estatística & dados numéricos , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Estresse Psicológico/complicações , Estresse Psicológico/psicologia , Inquéritos e Questionários
11.
Infect Immun ; 86(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29358335

RESUMO

Antigenic variation of the immunodominant MgpB and MgpC proteins has been suggested to be a mechanism of immune evasion of the human pathogen Mycoplasma genitalium, a cause of several reproductive tract disease syndromes. Phase variation resulting in the loss of adherence has also been documented, but the molecular mechanisms underlying this process and its role in pathogenesis are still poorly understood. In this study, we isolated and characterized 40 spontaneous, nonadherent phase variants from in vitro-passaged M. genitalium cultures. In all cases, nonadherence was associated with the loss of MgpBC protein expression, attributable to sequence changes in the mgpBC expression site. Phase variants were grouped into seven classes on the basis of the nature of the mutation. Consistent with the established role of RecA in phase variation, 31 (79.5%) variants arose via recombination with MgPa repeat regions that contain mgpBC variable sequences. The remaining mutants arose via nonsense or frameshift mutations. As expected, revertants were obtained for phase variants that were predicted to be reversible but not for those that arose via an irreversible mechanism. Furthermore, phase variants were enriched in M. genitalium cultures exposed to antibodies reacting to the extracellular, conserved C terminus of MgpB but not in cultures exposed to antibodies reacting to an intracellular domain of MgpB or the cytoplasmic HU protein. Genetic characterization of the antibody-selected phase variants confirmed that they arose via reversible and irreversible recombination and point mutations within mgpBC These phase variants resisted antibody-mediated growth inhibition, suggesting that phase variation promotes immune evasion.


Assuntos
Anticorpos Antibacterianos/imunologia , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Deleção de Genes , Expressão Gênica , Marcação de Genes , Variação Genética , Humanos , Mutação , Mycoplasma genitalium/genética , Recombinação Genética
12.
Mol Microbiol ; 94(2): 290-306, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25138908

RESUMO

The human pathogen Mycoplasma genitalium employs homologous recombination to generate antigenic diversity in the immunodominant MgpB and MgpC proteins. Only recently, some of the molecular factors involved in this process have been characterized, but nothing is known about its regulation. Here, we show that M. genitalium expresses N-terminally truncated RecA isoforms via alternative translation initiation, but only the full-length protein is essential for gene variation. We also demonstrate that overexpression of MG428 positively regulates the expression of recombination genes, including recA, ruvA, ruvB and ORF2, a gene of unknown function co-transcribed with ruvAB. The co-ordinated induction of these genes correlated with an increase of mgpBC gene variation. In contrast, cells lacking MG428 were unable to generate variants despite expressing normal levels of RecA. Similarly, deletion analyses of the recA upstream region defined sequences required for gene variation without abolishing RecA expression. The requirement of these sequences is consistent with the presence of promoter elements associated with MG428-dependent recA induction. Sequences upstream of recA also influence the relative abundance of RecA isoforms, possibly through translational regulation. Overall, these results suggest that MG428 is a positive regulator of recombination and that precise control of recA expression is required to initiate mgpBC variation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycoplasma genitalium/genética , Recombinação Genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Deleção de Genes , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Recombinases/biossíntese , Alinhamento de Sequência , Fatores de Transcrição/genética
13.
J Bacteriol ; 196(8): 1608-18, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24532771

RESUMO

Mycoplasma genitalium is an emerging sexually transmitted pathogen associated with reproductive tract disease in men and women, and it can persist for months to years despite the development of a robust antibody response. Mechanisms that may contribute to persistence in vivo include phase and antigenic variation of the MgpB and MgpC adhesins. These processes occur by segmental recombination between discrete variable regions within mgpB and mgpC and multiple archived donor sequences termed MgPa repeats (MgPars). The molecular factors governing mgpB and mgpC variation are poorly understood and obscured by the paucity of recombination genes conserved in the M. genitalium genome. Recently, we demonstrated the requirement for RecA using a quantitative PCR (qPCR) assay developed to measure recombination between the mgpB and mgpC genes and MgPars. Here, we expand these studies by examining the roles of M. genitalium ruvA and ruvB homologs. Deletion of ruvA and ruvB impaired the ability to generate mgpB and mgpC phase and sequence variants, and these deficiencies could be complemented with wild-type copies, including the ruvA gene from Mycoplasma pneumoniae. In contrast, ruvA and ruvB deletions did not affect the sensitivity to UV irradiation, reinforcing our previous findings that the recombinational repair pathway plays a minor role in M. genitalium. Reverse transcription-PCR (RT-PCR) and primer extension analyses also revealed a complex transcriptional organization of the RuvAB system of M. genitalium, which is cotranscribed with two novel open reading frames (ORFs) (termed ORF1 and ORF2 herein) conserved only in M. pneumoniae. These findings suggest that these novel ORFs may play a role in recombination in these two closely related bacteria.


Assuntos
Adesinas Bacterianas/genética , Variação Antigênica , DNA Helicases/genética , DNA Helicases/metabolismo , Mycoplasma genitalium/enzimologia , Mycoplasma genitalium/genética , Recombinação Genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Pneumonia por Mycoplasma/genética , Transcrição Gênica
14.
Rev. esp. cardiol. (Ed. impr.) ; 66(10): 797-802, oct. 2013.
Artigo em Espanhol | IBECS | ID: ibc-115595

RESUMO

Introducción y objetivos. Los receptores de trasplante cardiaco que sobreviven más de 20 años están aumentando. Poco se conoce de su seguimiento, sus comorbilidades y su mortalidad. Identificar predictores de larga supervivencia puede guiar la selección de candidatos para los donantes disponibles. Métodos. Se revisó la información sobre la clase funcional, las comorbilidades y la mortalidad de pacientes trasplantados antes de 1992. Para identificar los predictores de supervivencia > 20 años, se construyó un modelo de regresión logística utilizando las variables asociadas a supervivencia en el análisis univariable. Resultados. Se comparó a 39 supervivientes con seguimiento > 20 años (el 26% del total) con 90 pacientes que sobrevivieron entre 1 y 20 años. Las principales complicaciones fueron hipertensión, disfunción renal, infecciones y neoplasias. Tras 30 meses de seguimiento, 6 murieron, lo que implica una mortalidad del 6%/año (frente a un 2,5-3% en los años 1 a 19). Las principales causas de muerte fueron infección (50%), cáncer (33%) y vasculopatía del injerto (17%). Los supervivientes eran más jóvenes y delgados, y tenían cardiopatía no isquémica y menos isquemia en cirugía. La regresión logística identificó la edad del receptor < 45 años (odds ratio = 3,9; intervalo de confianza del 95%, 1,6-9,7; p = 0,002) y la miocardiopatía idiopática (odds ratio = 3; intervalo de confianza del 95%, 1,4-7,8; p = 0,012) como predictores independientes de supervivencia > 20 años. Conclusiones. En nuestra serie, más del 25% sobrevive más de 20 años con el mismo injerto y lleva vida independiente a pesar de las comorbilidades. La edad del receptor < 45 años y la miocardiopatía idiopática se asociaron a larga supervivencia. Estos datos pueden ayudar a la asignación de donantes (AU)


Introduction and objectives. The number of heart-transplant recipients exceeding 20 years of follow-up is steadily increasing. However, little is known about their functional status, comorbidities, and mortality. Identifying the predictors of prolonged survival could guide the selection of candidates for the low number of available donors. Methods. Functional status, morbidities, and mortality of heart-transplant patients between 1984 and 1992 were analyzed. To identify predictors of 20-year survival, a logistic regression model was constructed using the covariates associated with survival in the univariate analysis. Results. A total of 39 patients who survived 20 years (26% of patients transplanted before 1992) were compared to 90 recipients from the same period who died between 1 and 20 years post-transplantation. Major complications were hypertension, renal dysfunction, infections, and cancer. After a mean follow-up of 30 months, 6 survivors had died, yielding a mortality rate of 6% per year (vs 2.5%-3% in years 1-19). Causes of mortality were infection (50%), malignancy (33%), and allograft vasculopathy (17%). Long-term survivors were younger and leaner, and had nonischemic cardiomyopathy and lower ischemic time. Logistic regression identified recipient age <45 years (odds ratio=3.9; 95% confidence interval, 1.6-9.7; P=.002) and idiopathic cardiomyopathy (odds ratio=3; 95% confidence interval, 1.4-7.8; P=.012) as independent predictors for 20-year survival. Conclusions. One fourth of all heart-transplant patients in our series survived >20 years with the same graft, and most enjoy independent lives despite significant comorbidities. Recipient age <45 years and idiopathic cardiomyopathy were associated with survival beyond 2 decades. These data may help decide donor allocation (AU)


Assuntos
Adulto , Pessoa de Meia-Idade , Humanos , Transplante de Coração/métodos , Transplante de Coração , Qualidade de Vida , Sobrevivência de Enxerto/fisiologia , Cardiomiopatia Hipertrófica/complicações , Terapia de Imunossupressão/instrumentação , Terapia de Imunossupressão/métodos , Fatores de Risco , Transplante de Coração/reabilitação , Transplante de Coração/tendências , Comorbidade , Razão de Chances , Taxa de Sobrevida , Intervalos de Confiança , Índice de Massa Corporal , Análise Multivariada
16.
Rev Esp Cardiol (Engl Ed) ; 66(10): 797-802, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24773860

RESUMO

INTRODUCTION AND OBJECTIVES: The number of heart-transplant recipients exceeding 20 years of follow-up is steadily increasing. However, little is known about their functional status, comorbidities, and mortality. Identifying the predictors of prolonged survival could guide the selection of candidates for the low number of available donors. METHODS: Functional status, morbidities, and mortality of heart-transplant patients between 1984 and 1992 were analyzed. To identify predictors of 20-year survival, a logistic regression model was constructed using the covariates associated with survival in the univariate analysis. RESULTS: A total of 39 patients who survived 20 years (26% of patients transplanted before 1992) were compared to 90 recipients from the same period who died between 1 and 20 years post-transplantation. Major complications were hypertension, renal dysfunction, infections, and cancer. After a mean follow-up of 30 months, 6 survivors had died, yielding a mortality rate of 6% per year (vs 2.5%-3% in years 1-19). Causes of mortality were infection (50%), malignancy (33%), and allograft vasculopathy (17%). Long-term survivors were younger and leaner, and had nonischemic cardiomyopathy and lower ischemic time. Logistic regression identified recipient age <45 years (odds ratio=3.9; 95% confidence interval, 1.6-9.7; P=.002) and idiopathic cardiomyopathy (odds ratio=3; 95% confidence interval, 1.4-7.8; P=.012) as independent predictors for 20-year survival. CONCLUSIONS: One fourth of all heart-transplant patients in our series survived >20 years with the same graft, and most enjoy independent lives despite significant comorbidities. Recipient age <45 years and idiopathic cardiomyopathy were associated with survival beyond 2 decades. These data may help decide donor allocation.


Assuntos
Causas de Morte , Transplante de Coração/mortalidade , Transplante de Coração/métodos , Qualidade de Vida , Adulto , Fatores Etários , Análise de Variância , Bases de Dados Factuais , Feminino , Seguimentos , Rejeição de Enxerto , Sobrevivência de Enxerto , Transplante de Coração/psicologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/fisiopatologia , Estudos Retrospectivos , Fatores Sexuais , Espanha , Análise de Sobrevida , Sobreviventes/estatística & dados numéricos , Fatores de Tempo , Doadores de Tecidos , Adulto Jovem
17.
Mol Microbiol ; 85(4): 669-83, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22686427

RESUMO

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived 'MgPar' donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10(-4) events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption-deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C-MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.


Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Variação Antigênica , Reparo do DNA , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismo , Recombinases Rec A/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Teste de Complementação Genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mitomicina/toxicidade , Dados de Sequência Molecular , Taxa de Mutação , Mycoplasma genitalium/fisiologia , Reação em Cadeia da Polimerase , Recombinases Rec A/genética , Recombinação Genética , Análise de Sequência de DNA , Raios Ultravioleta
18.
J Heart Lung Transplant ; 30(6): 644-51, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21470878

RESUMO

BACKGROUND: Primary graft failure (PGF) is the leading cause of early mortality after heart transplantation (HT). Our aim is to propose a working definition of PGF and to develop a predictive risk score. METHODS: PGF was defined by four criteria reflecting significant myocardial dysfunction, severe hemodynamic impairment, early onset after HT, and absence of secondary causes of graft dysfunction. We identified independent risk factors for PGF in a derivation series of 621 HTs and constructed a predictive model. After proving its internal consistency we tested the model in a prospective validation series. RESULTS: The incidence and lethality of PGF in our series were 9% and 80%, respectively. We identified 6 multivariate risk factors for PGF (Right atrial pressure ≥ 10 mm Hg, recipient Age ≥ 60 years, Diabetes mellitus, Inotrope dependence, donor Age ≥ 30 years, Length of ischemic time ≥ 240 minutes--i.e., RADIAL). Analysis of isolated right ventricular failure showed similar predictors. The RADIAL score was obtained by adding 1 point for each of these factors present in a given HT. PGF incidence increased significantly as the RADIAL score increased (p < 0.001 for trend). Rates of actual and predicted PGF incidence for RADIAL subgroups showed a good correlation (C-statistic = 0.74). In a prospective validation cohort, RADIAL score kept its predictive ability. CONCLUSIONS: PGF as defined by these criteria showed a high impact on early post-HT mortality in our series. The RADIAL score showed good ability to predict the development of PGF, and could be useful in the prevention and early treatment of this complication.


Assuntos
Rejeição de Enxerto/etiologia , Transplante de Coração/efeitos adversos , Adolescente , Adulto , Idoso , Criança , Feminino , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/mortalidade , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de Risco , Terminologia como Assunto , Adulto Jovem
19.
Physiol Meas ; 31(12): 1553-65, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20980717

RESUMO

In recent times, significant effort has been made to understand the mechanical behaviour of the arterial wall and how it is affected by the different vascular pathologies. However, to be able to interpret the results correctly, it is essential that the influence of other factors, such as aging or anisotropy, be understood. Knowledge of mechanical behaviour of the aorta has been customarily constrained by lack of data on fresh aortic tissue, especially from healthy young individuals. In addition, information regarding the point of rupture is also very limited. In this study, the mechanical behaviour of the descending thoracic aorta of 28 organ donors with no apparent disease, whose ages vary from 17 to 60 years, is evaluated. Tensile tests up to rupture are carried out to evaluate the influence of age and wall anisotropy. Results reveal that the tensile strength and stretch at failure of healthy descending aortas show a significant reduction with age, falling abruptly beyond the age of 30. This fact places age as a key factor when mechanical properties of descending aorta are considered.


Assuntos
Aorta Torácica/fisiologia , Saúde , Adolescente , Adulto , Fenômenos Biomecânicos/fisiologia , Cotovelo/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Resistência à Tração/fisiologia , Adulto Jovem
20.
Rev Esp Cardiol ; 63(5): 598-601, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20450854

RESUMO

Intravascular ultrasound (IVUS) has been successfully used to guide the implantation of stents in the thoracic aorta. However, its accuracy in measuring the diameter of the aortic lumen has not been clearly established. Thirteen patients with thoracic aortic disease underwent IVUS, and lumen diameter measurements were compared with those obtained by CT or magnetic resonance imaging. A total of 31 comparable measurements were obtained. The correlation was good (r=0.98; P< .001), with IVUS tending to give a larger minimum diameter than CT (systematic error, 0.59+/-1.8 mm; P=.077). Given that the aorta is often not circular, the diameter obtained by IVUS was also compared to the mean diameter obtained by CT, and it was found that these two measurements were more closely related (P=.425), except in aortic segments with significant eccentricity (i.e., >10%). In conclusion, IVUS was a reliable tool for measuring the diameter of the aorta, particularly in concentric segments where stents are normally placed. Consequently, IVUS could supplement conventional imaging techniques.


Assuntos
Aorta Torácica/diagnóstico por imagem , Ultrassonografia de Intervenção/métodos , Adulto , Idoso , Aorta Torácica/cirurgia , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/cirurgia , Procedimentos Cirúrgicos Cardíacos , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X , Adulto Jovem
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