RESUMEN
AIMS: Clinical likelihood (CL) models are designed based on a reference of coronary stenosis in patients with suspected obstructive coronary artery disease. However, a reference standard for myocardial perfusion defects (MPDs) could be more appropriate. We aimed to investigate the ability of the 2019 European Society of Cardiology pre-test probability (ESC-PTP), the risk-factor-weighted (RF-CL) model, and coronary artery calcium score-weighted (CACS-CL) model to diagnose MPDs. METHODS AND RESULTS: Symptomatic stable de novo chest pain patients (n = 3374) underwent coronary computed tomography angiography and subsequent myocardial perfusion imaging by single-photon emission computed tomography, positron emission tomography, or cardiac magnetic resonance. For all modalities, MPD was defined as coronary computed tomography angiography with suspected stenosis and stress-perfusion abnormality in ≥2 segments. The ESC-PTP was calculated based on age, sex, and symptom typicality, and the RF-CL and CACS-CL additionally included a number of risk factors and CACS. In total, 219/3374 (6.5%) patients had an MPD. Both the RF-CL and the CACS-CL classified substantially more patients to low CL (<5%) of obstructive coronary artery disease compared with the ESC-PTP (32.5 and 54.1 vs. 12.0%, P < 0.001) with preserved low prevalences of MPD (<2% for all models). Compared with the ESC-PTP [area under the receiver-operating characteristic curve (AUC) 0.74 (0.71-0.78)], the discrimination of having an MPD was higher for the CACS-CL model [AUC 0.88 (0.86-0.91), P < 0.001], while it was similar for the RF-CL model [AUC 0.73 (0.70-0.76), P = 0.32]. CONCLUSION: Compared with basic CL models, the RF-CL and CACS-CL models improve down classification of patients to a very low-risk group with a low prevalence of MPD.
Asunto(s)
Enfermedad de la Arteria Coronaria , Estenosis Coronaria , Imagen de Perfusión Miocárdica , Humanos , Enfermedad de la Arteria Coronaria/epidemiología , Funciones de Verosimilitud , Angiografía Coronaria/métodos , Estenosis Coronaria/diagnóstico , Tomografía Computarizada de Emisión de Fotón Único/métodos , Angiografía por Tomografía Computarizada , Valor Predictivo de las Pruebas , Imagen de Perfusión Miocárdica/métodosRESUMEN
The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.
Asunto(s)
Geraniltranstransferasa/metabolismo , Isomerasas/metabolismo , Lamiaceae/enzimología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Sesquiterpenos/metabolismo , Biotecnología/métodos , Geraniltranstransferasa/genética , Isomerasas/genética , Lamiaceae/genética , Lamiaceae/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Homologous recombination (HR) is a major DNA repair pathway and therefore essential for maintaining the integrity of the genome. HR is catalyzed by proteins encoded by genes of the RAD52 epistasis group, including the recombinase Rad51 and its mediator Rad52. HR proteins fused with green fluorescent protein form foci at damaged DNA reflecting the assembly of repair centers that harbor a high concentration of repair proteins. Rad52 mediates the recruitment of Rad51 and other HR proteins to DNA damage. To understand the mechanism for the assembly of Rad52-dependent DNA repair centers, we used a mutational strategy to identify a Rad52 domain essential for its recruitment to DNA repair foci. We present evidence to implicate an acidic domain in Rad52 in DNA repair focus formation. Mutations in this domain confer marked DNA damage sensitivity and recombination deficiency. Importantly, these Rad52 mutants are specifically compromised for interaction with the single-stranded DNA-binding factor RPA. Based on these findings, we propose a model where Rad52 displaces RPA from single-stranded DNA using the acidic domain as a molecular lever.
Asunto(s)
Reparación del ADN , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Epistasis Genética , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/química , Recombinación Genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad52 enters the nucleus. In the present study, we have used a combination of mutagenesis and sequence analysis to show that Rad52 from S. cerevisiae contains a single functional pat7 type NLS essential for its nuclear localization. The region containing the NLS seems only to be involved in nuclear transport as it plays no role in repair of MMS-induced DNA damage. The NLS in Rad52 is weak, as monomeric protein species that harbor this NLS are mainly located in the cytosol. In contrast, multimeric protein complexes wherein each subunit contains a single NLS(Rad52) sort efficiently to the nucleus. Based on the results we propose a model where the additive effect of multiple NLS(Rad52) sequences in a Rad52 ring-structure ensures efficient nuclear localization of Rad52.
Asunto(s)
Biopolímeros/metabolismo , Núcleo Celular/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Microscopía Fluorescente , Datos de Secuencia Molecular , Señales de Localización Nuclear , Plásmidos , Proteína Recombinante y Reparadora de ADN Rad52/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
The rapid accumulation of genomic sequences from a large number of eukaryotes, including numerous filamentous fungi, has created a tremendous scientific potential, which can only be realized if precise site-directed genome modifications, like gene deletions, promoter replacements, in-frame GFP fusions and specific point mutations can be made rapidly and reliably. The development of gene-targeting techniques in filamentous fungi and other higher eukaryotes has been hampered because foreign DNA is predominantly integrated randomly into the genome. For Aspergillus nidulans, we have developed a flexible method for gene-targeting employing a bipartite gene-targeting substrate. This substrate is made solely by PCR, which obviates the need for bacterial subcloning steps. The method reduces the number of false positives and can be used to produce virtually any genome alteration. A major advance of the method is that it allows multiple subsequent genome manipulations to be performed as the selectable marker is recycled.
Asunto(s)
Aspergillus nidulans/genética , Marcación de Gen/métodos , Genes Fúngicos/genética , Eliminación de Gen , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 10(6) CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.