RESUMO
BACKGROUND: The increasing burden of atrial fibrillation (AF) emphasizes the need to identify high-risk individuals for enrolment in clinical trials of AF screening and primary prevention. We aimed to develop prediction models to identify individuals at high-risk of AF across prediction horizons from 6-months to 10-years. METHODS: We used secondary-care linked primary care electronic health record data from individuals aged ≥30 years without known AF in the UK Clinical Practice Research Datalink-GOLD dataset between January 2, 1998 and November 30, 2018; randomly divided into derivation (80%) and validation (20%) datasets. Models were derived using logistic regression from known AF risk factors for incident AF in prediction periods of 6 months, 1-year, 2-years, 5-years, and 10-years. Performance was evaluated using in the validation dataset with bootstrap validation with 200 samples, and compared against the CHA2DS2-VASc and C2HEST scores. RESULTS: Of 2,081,139 individuals in the cohort (1,664,911 in the development dataset, 416,228 in the validation dataset), the mean age was 49.9 (SD 15.4), 50.7% were women, and 86.7% were white. New cases of AF were 7,386 (0.4%) within 6 months, 15,349 (0.7%) in 1 year, 38,487 (1.8%) in 5 years, and 79,997 (3.8%) by 10 years. Valvular heart disease and heart failure were the strongest predictors, and association of hypertension with AF increased at longer prediction horizons. The optimal risk models incorporated age, sex, ethnicity, and 8 comorbidities. The models demonstrated good-to-excellent discrimination and strong calibration across prediction horizons (AUROC, 95%CI, calibration slope: 6-months, 0.803, 0.789-0.821, 0.952; 1-year, 0.807, 0.794-0.819, 0.962; 2-years, 0.815, 0.807-0.823, 0.973; 5-years, 0.807, 0.803-0.812, 1.000; 10-years 0.780, 0.777-0.784, 1.010), and superior to the CHA2DS2-VASc and C2HEST scores. The models are available as a web-based FIND-AF calculator. CONCLUSIONS: The FIND-AF models demonstrate high discrimination and calibration across short- and long-term prediction horizons in 2 million individuals. Their utility to inform trial enrolment and clinical decisions for AF screening and primary prevention requires further study.
Assuntos
Fibrilação Atrial , Humanos , Fibrilação Atrial/epidemiologia , Fibrilação Atrial/complicações , Feminino , Masculino , Pessoa de Meia-Idade , Medição de Risco/métodos , Reino Unido/epidemiologia , Incidência , Fatores de Risco , Idoso , AdultoRESUMO
BACKGROUND AND AIMS: Reports of outcomes after atrial fibrillation (AF) diagnosis are conflicting. The aim of this study was to investigate mortality and hospitalization rates following AF diagnosis over time, by cause and by patient features. METHODS: Individuals aged ≥16 years with a first diagnosis of AF were identified from the UK Clinical Practice Research Datalink-GOLD dataset from 1 January 2001, to 31 December 2017. The primary outcomes were all-cause and cause-specific mortality and hospitalization at 1 year following diagnosis. Poisson regression was used to calculate rate ratios (RRs) for mortality and incidence RRs (IRRs) for hospitalization and 95% confidence intervals (CIs) comparing 2001/02 and 2016/17, adjusted for age, sex, region, socio-economic status, and 18 major comorbidities. RESULTS: Of 72 412 participants, mean (standard deviation) age was 75.6 (12.4) years, and 44 762 (61.8%) had ≥3 comorbidities. All-cause mortality declined (RR 2016/17 vs. 2001/02 0.72; 95% CI 0.65-0.80), with large declines for cardiovascular (RR 0.46; 95% CI 0.37-0.58) and cerebrovascular mortality (RR 0.41; 95% CI 0.29-0.60) but not for non-cardio/cerebrovascular causes of death (RR 0.91; 95% CI 0.80-1.04). In 2016/17, deaths caused from dementia (67, 8.0%), outstripped deaths from acute myocardial infarction, heart failure, and acute stroke combined (56, 6.7%, P < .001). Overall hospitalization rates increased (IRR 2016/17 vs. 2001/02 1.17; 95% CI, 1.13-1.22), especially for non-cardio/cerebrovascular causes (IRR 1.42; 95% CI 1.39-1.45). Older, more deprived, and hospital-diagnosed AF patients experienced higher event rates. CONCLUSIONS: After AF diagnosis, cardio/cerebrovascular mortality and hospitalization has declined, whilst hospitalization for non-cardio/cerebrovascular disease has increased.
Assuntos
Fibrilação Atrial , Insuficiência Cardíaca , Acidente Vascular Cerebral , Humanos , Fibrilação Atrial/epidemiologia , Causas de Morte , Acidente Vascular Cerebral/epidemiologia , Comorbidade , Hospitalização , Fatores de RiscoRESUMO
Antibiotic resistance demands innovative strategies and therapies. The pairs of antimicrobial peptides tested in this work show broad-spectrum synergy and are capable of interacting with diverse bacterial membranes. In most cases, the ATCUN motif enhanced the activity of peptides tested in combination. Our studies also show CP10A to be a multifaceted peptide, displaying both cell membrane and intracellular activity and acting as a chameleon, improving the activity of other peptides as needed. The results of the synergy experiments demonstrate the importance of varied modes of action and how these changes can affect the ability to combat pathogens, while also illustrating the value of the metal-binding domain in enhancing the activity of antimicrobial peptides in combination.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/química , Motivos de Aminoácidos , Cobre/química , Membrana Celular/metabolismoRESUMO
Iron-sulfur cluster proteins play key roles in a multitude of cellular processes. Iron-sulfur cofactors are assembled primarily in mitochondria and are then exported to the cytosol by use of an ABCB7 transporter. It has been shown that the yeast mitochondrial transporter Atm1 can export glutathione-coordinated iron-sulfur clusters, [2Fe-2S](SG)4, providing a source of cluster units for cytosolic iron-sulfur cluster assembly systems. This pathway is consistent with the endosymbiotic model of mitochondrial evolution where homologous bacterial heavy metal transporters, utilizing metal glutathione adducts, were adapted for use in eukaryotic mitochondria. Herein, the basis for endosymbiotic evolution of the human cluster export protein (ABCB7) is developed through a BLAST analysis of transporters from ancient proteobacteria. In addition, a functional comparison of native human protein, versus a disease-causing mutant, demonstrates a key role for residue E433 in promoting cluster transport. Dysfunction in mitochondrial export of Fe-S clusters is a likely cause of the disease condition X-linked sideroblastic anemia.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Evolução Molecular , Mutação , Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/metabolismo , Humanos , Cinética , Modelos Moleculares , Conformação ProteicaRESUMO
Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Sulfurtransferases/metabolismo , Substituição de Aminoácidos , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Técnicas In Vitro , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral , Enxofre/metabolismo , Sulfurtransferases/química , Sulfurtransferases/genética , Ácido Tióctico/biossínteseRESUMO
Mucosal-associated invariant T (MAIT) cells and Vδ2+ γδ T cells are anti-bacterial innate-like lymphocytes (ILLs) that are enriched in blood and mucosa. ILLs have been implicated in control of infection. However, the role of ILLs in community-acquired pneumonia (CAP) is unknown. Using sputum samples from a well-characterized CAP cohort, MAIT cell and Vδ2+ T cell abundance was determined by quantitative polymerase chain reaction (qPCR). Cytokine and chemokine concentrations in sputum were measured. The capacity of bacteria in sputum to produce activating ligands for MAIT cells and Vδ2+ T cells was inferred by 16S rRNA sequencing. MAIT cell abundance in sputum was higher in patients with less severe pneumonia; duration of hospital admission was inversely correlated with both MAIT and Vδ2+ T cell abundance. The abundance of both ILLs was higher in patients with a confirmed bacterial aetiology; however, there was no correlation with total bacterial load or the predicted capacity of bacteria to produce activating ligands. Sputum MAIT cell abundance was associated with interferon (IFN)-α, IFN-γ, and sputum neutrophil abundance, while Vδ2+ T cell abundance was associated with CXCL11 and IFN-γ. Therefore, MAIT and Vδ2+ T cells can be detected in sputum in CAP, where they may contribute to improved clinical outcome.
Assuntos
Infecções Comunitárias Adquiridas/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Pneumonia/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Escarro/imunologia , Linfócitos T/imunologia , Infecções Comunitárias Adquiridas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/patologia , Pneumonia/patologia , Linfócitos T/patologiaRESUMO
Degradation of saccharides is relevant to the design of catalytic therapeutics, the production of biofuels, inhibition of biofilms, as well as other applications in chemical biology. Herein, we report the design of multinuclear Cu complexes that enable cleavage of saccharides under physiological conditions. Reactivity studies with para-nitrophenyl (pNP)-conjugated carbohydrates show that dinuclear Cu complexes exhibit a synergistic effect and promote faster and more robust cleavage of saccharide substrates, relative to the mononuclear Cu complex, while no further enhancement is observed for the tetranuclear Cu complex. The use of scavengers for reactive oxygen species confirms that saccharide cleavage is promoted by the formation of superoxide and hydroxyl radicals through CuII/I redox chemistry, similar to that observed for native copper-containing lytic polysaccharide monooxygenases (LMPOs). Differences in selectivity for di- and tetranuclear Cu complexes are modest. However, these are the first reported small multinuclear Cu complexes that show selectivity and reactivity against mono- and disaccharide substrates and form a basis for further development of metalloglycosidases for applications in chemical biology.
Assuntos
Complexos de Coordenação/química , Cobre/química , Glicosídeo Hidrolases/química , Compostos Organometálicos/química , Açúcares/química , Cristalografia por Raios X , Hidrólise , Mimetismo Molecular , Oxirredução , Espécies Reativas de Oxigênio/químicaRESUMO
A new class of mitochondrial disease has been identified and characterized as Multiple Mitochondrial Dysfunctions Syndrome (MMDS). Four different forms of the disease have each been attributed to point mutations in proteins involved in iron-sulfur (Fe-S) biosynthesis; in particular, MMDS2 has been associated with the protein BOLA3. To date, this protein has been characterized in vitro concerning its ability to form heterodimeric complexes with two putative Fe-S cluster-binding partners: GLRX5 and NFU. However, BOLA3 has yet to be characterized in its own discrete holo form. Herein we describe procedures to isolate and characterize the human holo BOLA3 protein in terms of Fe-S cluster binding and trafficking and demonstrate that human BOLA3 can form a functional homodimer capable of engaging in Fe-S cluster transfer.
Assuntos
Ferro/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Enxofre/química , Apoproteínas/química , Apoproteínas/metabolismo , Humanos , Estrutura Quaternária de Proteína , Transporte ProteicoRESUMO
Iron-sulfur (Fe-S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe-S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.
Assuntos
Aconitato Hidratase/metabolismo , Citosol/metabolismo , Eucariotos/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Aconitato Hidratase/química , Citosol/química , Espectroscopia de Ressonância de Spin Eletrônica , Eucariotos/química , Humanos , Proteínas Ferro-Enxofre/química , Cinética , Mitocôndrias/químicaRESUMO
Aims: To determine whether changing patterns of anticoagulant use in atrial fibrillation (AF) have impacted on stroke rates in England. Methods and results: English national databases, 2006-2016, were interrogated to assess stroke admissions and oral anticoagulant use. The number of patients with known AF increased linearly from 692 054 to 983 254 (prevalence 1.29% vs. 1.71%). Hospital episodes of AF-related stroke/100 000 AF patients increased from 80/week in 2006 to 98/week in 2011 and declined to 86/week in 2016 (2006-2011 difference 18.0, 95% confidence interval (CI) 17.9-18.1, 2011-2016 difference -12.0, 95% CI -12.1 to -11.9). Anticoagulant use amongst patients with CHA2DS2-VASc ≥2 increased from 48.0% to 78.6% and anti-platelet use declined from 42.9% to 16.1%; the greatest rate of change occurred in the second 5 year period (for anticoagulants 2006-2011 difference 4.8%, 95% CI 4.5-5.1%, 2011-2016 difference 25.8%, 95% CI 25.5-26.1%). After adjustment for AF prevalence, a 1% increase in anticoagulant use was associated with a 0.8% decrease in the weekly rate of AF-related stroke (incidence rate ratio 0.992, 95% CI 0.989-0.994). Had the use of anticoagulants remained at 2009 levels, 4068 (95% CI 4046-4089) more strokes would have been predicted in 2015/2016. Conclusion: Between 2006 and 2016, AF prevalence and anticoagulant use in England increased. From 2011, hospitalized AF-related stroke rates declined and were significantly associated with increased anticoagulant uptake.
Assuntos
Anticoagulantes/uso terapêutico , Fibrilação Atrial/complicações , Acidente Vascular Cerebral/epidemiologia , Administração Oral , Idoso de 80 Anos ou mais , Inglaterra/epidemiologia , Feminino , Hospitalização , Humanos , Estudos Longitudinais , Masculino , Inibidores da Agregação Plaquetária/uso terapêutico , Prevalência , Acidente Vascular Cerebral/prevenção & controleRESUMO
Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.
Assuntos
Glutarredoxinas/química , Glutationa/química , Eletricidade Estática , Dicroísmo Circular , Humanos , Ligação de Hidrogênio , Ligantes , Estereoisomerismo , Compostos de Sulfidrila/químicaRESUMO
Monothiol glutaredoxins (Grx) serve as intermediate cluster carriers in iron-sulfur cluster trafficking. The [2Fe-2S]-bound holo forms of Grx proteins display cysteinyl coordination from exogenous glutathione (GSH), in addition to contact from protein-derived Cys. Herein, we report mechanistic studies that investigate the role of exogenous glutathione in defining cluster chirality, ligand exchange, and the cluster transfer chemistry of Saccharomyces cerevisiae Grx3. Systematic perturbations were introduced to the glutathione-binding site by substitution of conserved charged amino acids that form crucial electrostatic contacts with the glutathione molecule. Native Grx3 could also be reconstituted in the absence of glutathione, with either DTT, BME or free L-cysteine as the source of the exogenous Fe-S ligand contact, while retaining full functional reactivity. The delivery of the [2Fe-2S] cluster to Grx3 from cluster donor proteins such as Isa, Nfu, and a [2Fe-2S](GS)4 complex, revealed that electrostatic contacts are of key importance for positioning the exogenous glutathione that in turn influences the chiral environment of the cluster. All Grx3 derivatives were reconstituted by standard chemical reconstitution protocols and found to transfer cluster to apo ferredoxin 1 (Fdx1) at rates comparable to native protein, even when using DTT, BME or free L-cysteine as a thiol source in place of GSH during reconstitution. Kinetic analysis of cluster transfer from holo derivatives to apo Fdx1 has led to a mechanistic model for cluster transfer chemistry of native holo Grx3, and identification of the likely rate-limiting step for the reaction.
Assuntos
Glutarredoxinas/química , Glutationa/química , Oxirredutases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Cinética , Ligantes , Oxirredutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , EstereoisomerismoRESUMO
BACKGROUND: Chronic rhinosinusitis is a very common inflammatory disease that impairs quality of life and is associated with high healthcare spending. Chronic rhinosinusitis treatment commonly involves the use of intranasal corticosteroids, oral antibiotics, and surgery. Macrolides have been identified as a potential treatment option for chronic rhinosinusitis due to their immunomodulatory effects; however, the evidence supporting their use is still conflicting. OBJECTIVE: The purpose of this systematic review was to evaluate new evidence along with previously reported studies of the use of macrolides in the treatment of chronic rhinosinusitis. SEARCH STRATEGY: Medline, EMBASE, Cochrane CENTRAL, LILACS, clinicaltrials.gov, and the International Clinical Trials Registry Platform were all searched (until June 2015 Medline and EMBASE searches were updated January 2016). Randomised controlled trials comparing low-dose macrolide antibiotics versus placebo, as an adjunct to other therapies, or low-dose macrolide therapy alone versus other therapies were included in this review. EVALUATION METHOD: Quality of the evidence was evaluated using the Cochrane risk of bias tool. Continuous outcomes were expressed as mean differences or standardised mean differences with 95% confidence interval. Data were pooled using fixed-effects models. RESULTS: Nine randomised controlled trials met the inclusion criteria. Studies were classified into three distinct comparisons: Low-dose macrolide therapy vs. placebo, low-dose macrolide +/- nasal steroids vs. nasal steroid and low-dose macrolides vs. other therapies. The overall quality of the evidence is low due to limitations in study design, imprecision, and indirectness. CONCLUSIONS: Positive results were seen with the use of macrolide therapy in the postoperative period in patients with nasal polyps. A firm conclusion with respect to the effectiveness of the use of macrolides for the treatment of chronic rhinosinusitis cannot be reached based on the available evidence. Further study using a placebo-controlled design evaluating the use of macrolides in clearly defined chronic rhinosinusitis populations is needed.
Assuntos
Antibacterianos/administração & dosagem , Macrolídeos/administração & dosagem , Rinite/tratamento farmacológico , Sinusite/tratamento farmacológico , Adulto , Doença Crônica , Relação Dose-Resposta a Droga , Seguimentos , Humanos , Rinite/complicações , Sinusite/complicações , Fatores de Tempo , Resultado do TratamentoRESUMO
Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron-sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe-2S](GS)4 cluster complex, and extraction of the Nfu-bound iron-sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe-2S] cluster trafficking and delivery.
Assuntos
Proteínas de Transporte/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Motivos de Aminoácidos , Apoproteínas/metabolismo , Proteínas de Transporte/química , Ferredoxinas/metabolismo , Glutationa/química , Humanos , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS4) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe-S cluster transfer reactions. UV-vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS4) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS4) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe-S cluster proteins, thereby supporting a possible physiological role for such centers.
Assuntos
Glutationa/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Transporte Biológico , Glutarredoxinas/metabolismo , Humanos , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
PURPOSE: We describe the outcomes of patients with low risk localized prostate cancer who were upgraded on a surveillance biopsy while on active surveillance and evaluated whether delayed treatment was associated with adverse outcome. MATERIALS AND METHODS: We included men in the study with lower risk disease managed initially with active surveillance and upgraded to Gleason score 3+4 or greater. Patient demographics and disease characteristics were compared. Kaplan-Meier curve was used to estimate the treatment-free probability stratified by initial upgrade (3+4 vs 4+3 or greater), Cox regression analysis was used to examine factors associated with treatment and multivariate logistic regression analysis was used to evaluate the factors associated with adverse outcome at surgery. RESULTS: The final cohort comprised 219 men, with 150 (68%) upgraded to 3+4 and 69 (32%) to 4+3 or greater. Median time to upgrade was 23 months (IQR 11-49). A total of 163 men (74%) sought treatment, the majority (69%) with radical prostatectomy. The treatment-free survival rate at 5 years was 22% for 3+4 and 10% for 4+3 or greater upgrade. Upgrade to 4+3 or greater, higher prostate specific antigen density at diagnosis and shorter time to initial upgrade were associated with treatment. At surgical pathology 34% of cancers were downgraded while 6% were upgraded. Cancer volume at initial upgrade was associated with adverse pathological outcome at surgery (OR 3.33, 95% CI 1.19-9.29, p=0.02). CONCLUSIONS: After Gleason score upgrade most patients elected treatment with radical prostatectomy. Among men who deferred definitive intervention, few experienced additional upgrading. At radical prostatectomy only 6% of cases were upgraded further and only tumor volume at initial upgrade was significantly associated with adverse pathological outcome.
Assuntos
Próstata/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Conduta Expectante , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Progressão da Doença , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estudos RetrospectivosRESUMO
Halide salts of alkali and alkaline earth metals were used to probe the contributions of intramolecular salt bridge formation on the stability of glutathione-coordinated [2Fe-2S] cluster toward hydrolysis. The effect of ionic strength on cluster stability was quantitatively investigated by application of Debye-Hückel theory to the rates of hydrolysis. Results from this study demonstrate that ionic strength influences the stability of the cluster, with the rate of cluster degradation depending on the charge density, hydrated ionic radius, and hydration energy. The identity of the salt ions was also observed to be correlated with the binding affinity toward the cluster. Based on the modified Debye-Hückel equation and counterion screening effect, these results suggest that interactions between glutathione molecules in the [2Fe-2S](GS)4 cluster is via salt bridges, in agreement with our previous results where modifications of glutathione carboxylates and amines prevented solution aggregation and cluster formation. These results not only provide a rationale for the stability of such clusters under physiological conditions, but also suggest that the formation of glutathione-complexed [2Fe-2S] cluster from a glutathione tetramer may be facilitated by salt bridge interactions between glutathione molecules prior to cluster assembly, in a manner consistent with Nature's equivalent of dynamic combinatorial chemistry.
Assuntos
Glutationa/química , Proteínas Ferro-Enxofre/química , Cloreto de Sódio/química , Estabilidade de Medicamentos , Íons , Cinética , Estrutura MolecularRESUMO
A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2',3'-cyclic phosphate, 3'-phosphate, 3'-phosphoglycolate, 5'- hydroxyl and 5'- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2'-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.
Assuntos
Clivagem do RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Catálise , Eletroforese em Gel de Poliacrilamida , Genes env , HIV-1/genética , RNA/química , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismoRESUMO
Catalytic metallopeptides that target the membrane-associated sortase A transpeptidase have been developed and evaluated as irreversible inactivators of SrtA∆N59 (sortase A, lacking the initial membrane-binding domain). The copper-binding GGH tripeptide ATCUN motif was linked to amidated forms of the cell wall sorting signal, LPET and LPETG, as sortase-targeting moieties. The resulting metallopeptides were used to determine half maximal inhibitory concentrations (IC50) and rate constants for time-dependent sortase A inactivation. Michaelis-Menten behavior was observed for the catalytic metallopeptides, and k(cat), K(M) and k(cat)/K(M) parameters were obtained as 0.080 ± 0.002 min⻹, 23 ± 2 µM and 0.0035 ± 0.0003 µM⻹ min⻹, respectively. Concentration-dependent inhibition of SrtA∆N59 by the metallopeptides revealed IC50 values ranging from 570 to 700 µM, while Cu-GGH, which lacked a targeting motif, had no measurable IC50 value (>2,000 µM). Time-dependent inactivation of SrtA revealed a range of catalytic activities, with Cu-GGHGLPETG-NH2 demonstrating the fastest rate of inactivation in the presence of ascorbate and hydrogen peroxide coreactants. The active site of the enzyme comprises residues Cys-184, Arg-197 and His-120. LC-MS/MS analysis of the reaction products demonstrated modification of Cys-184 to cysteine sulfonic acid (+48 amu). Results obtained from a DTNB assay support oxidation of the Cys-184 residue. LC-MS/MS also suggested oxidation of the Arg-197 containing peptide. 2D NMR analysis was performed to assess the possible oxidation of His-120, however, none was observed. These compounds possess the potential for irreversible inactivation of SrtA through oxidative modification of essential residues required for substrate binding.