RESUMO
DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as â¼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates.
Assuntos
Benzaldeídos , DNA Catalítico , Oligonucleotídeos , DNA Catalítico/química , DNA Catalítico/metabolismo , Aminação , Alquilação , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Benzaldeídos/química , DNA/química , DNA/metabolismo , Oxirredução , Catálise , RNA/química , RNA/metabolismo , Aminas/químicaRESUMO
Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here, we present a method, epigenetic MRI (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene-expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C-labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.
Assuntos
Encéfalo/metabolismo , Metilação de DNA , Epigênese Genética , Imageamento por Ressonância Magnética/métodos , Animais , Encéfalo/diagnóstico por imagem , Isótopos de Carbono/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Metionina/administração & dosagem , Reprodutibilidade dos Testes , SuínosRESUMO
We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5'-carboxyl oligonucleotide. Yields are as high as >95 % in 6â h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.
Assuntos
DNA Catalítico , DNA Catalítico/genética , Oligonucleotídeos , DNA , RNA , CatáliseRESUMO
Amines can be alkylated using various reactions, such as reductive amination of aldehydes. In this study, we sought DNAzymes as catalytic DNA sequences that promote reductive amination with aliphatic amines, including DNA-anchored peptide substrates with lysine residues. By in vitro selection starting with either N40 or N20 random DNA pools, we identified many DNAzymes that catalyze reductive amination between the DNA oligonucleotide-anchored aliphatic amino group of DNA-C3-NH2 (C3 = short three-carbon tether) and a DNA-anchored benzaldehyde group in the presence of NaCNBH3 as reducing agent. At pH 5.2, 6.0, 7.5, or 9.0 in the presence of various divalent metal ion cofactors including Mg2+, Mn2+, Zn2+ and Ni2+, the DNAzymes have kobs up to 0.12 h-1 and up to 130-fold rate enhancement relative to the DNA-splinted but uncatalyzed background reaction. However, analogous selection experiments did not lead to any DNAzymes that function with DNA-HEG-NH2 [HEG = long hexa(ethylene glycol) tether], or with short- and long-tethered DNA-AAAKAA and DNA-HEG-AAAKAA lysine-containing hexapeptide substrates (A = alanine, K = lysine). Including a variety of other amino acids in place of the neighboring alanines also did not lead to DNAzymes. These findings establish a practical limit on the substrate scope of DNAzyme catalysis for N-alkylation of aliphatic amines by reductive amination. The lack of DNAzymes for reductive amination with any substrate more structurally complex than DNA-C3-NH2 is likely related to the challenge in binding and spatially organizing those other substrates. Because other reactions such as aliphatic amine N-acylation are feasible for DNAzymes with DNA-anchored peptides, our findings show that the ability to identify DNAzymes depends strongly on both the investigated reaction and the composition of the substrate.
Assuntos
DNA Catalítico , Aminação , DNA Catalítico/metabolismo , Lisina/metabolismo , Aminas/química , DNA/metabolismo , CatáliseRESUMO
OBJECTIVES: Early recurrence of cerebral ischemia in acutely symptomatic carotid artery stenosis can precede revascularization. The optimal antithrombotic regimen for this high-risk population is not well established. Although antiplatelet agents are commonly used, there is limited evidence for the use of anticoagulants. We sought to understand the safety and efficacy of short-term preoperative anticoagulants in secondary prevention of recurrent cerebral ischemic events from acutely symptomatic carotid stenosis in patients awaiting carotid endarterectomy (CEA). MATERIALS AND METHODS: A retrospective query of a prospective single institution registry of carotid revascularization was performed. Patients who presented with acute ischemic stroke or transient ischemic attack (TIA) attributable to an ipsilateral internal carotid artery stenosis (ICA) were included. Antiplatelet (AP) only and anticoagulation (AC) treatment arms were compared. The primary outcome was a composite of preoperative recurrent ischemic stroke or TIA. The primary safety outcome was symptomatic intracranial hemorrhage. RESULTS: Out of 443 CEA patients, 342 were in the AC group and 101 in the AP group. Baseline characteristics between groups (AC vs AP) were similar apart from age (71±10.5 vs 73±9.5, p=0.04), premorbid modified Rankin scale (mRS) score (1.0±1.2 vs 1.4±1.3, p=0.03) and stroke as presenting symptom (65.8 vs 53.5%, p=0.02). Patients in the AC group had a lower incidence of recurrent stroke/TIA (3.8 vs 10.9%, p=0.006). One patient had symptomatic intracranial hemorrhage in the AC group, and none in the AP group. In multivariate analysis controlling for age, premorbid mRS, stroke severity, degree of stenosis, presence of intraluminal thrombus (ILT) and time to surgery, AC was protective (OR 0.30, p=0.007). This effect persisted in the cohort exclusively without ILT (OR 0.23, p=0.002). CONCLUSIONS: Short term preoperative anticoagulation in patients with acutely symptomatic carotid stenosis appears safe and effective compared to antiplatelet agents alone in the prevention of recurrent cerebral ischemic events while awaiting CEA.
Assuntos
Estenose das Carótidas , Endarterectomia das Carótidas , Ataque Isquêmico Transitório , AVC Isquêmico , Acidente Vascular Cerebral , Anticoagulantes/efeitos adversos , Estenose das Carótidas/complicações , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/tratamento farmacológico , Endarterectomia das Carótidas/efeitos adversos , Fibrinolíticos/efeitos adversos , Humanos , Hemorragias Intracranianas/complicações , Ataque Isquêmico Transitório/diagnóstico , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/etiologia , Inibidores da Agregação Plaquetária/efeitos adversos , Estudos Prospectivos , Estudos Retrospectivos , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Resultado do TratamentoAssuntos
Discinesias , Inconsciência , Idoso , Humanos , Masculino , Discinesias/etiologia , Inconsciência/etiologiaRESUMO
DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However, finding DNAzymes for peptide lysine acylation is a substantial challenge. By using suitably reactive aryl ester acyl donors as the electrophiles, here we used in vitro selection to identify DNAzymes that acylate amines, including lysine side chains of DNA-anchored peptides. Some of the DNAzymes can transfer a small glutaryl group to an amino group. These results expand the scope of DNAzyme catalysis and suggest the future broader applicability of DNAzymes for sequence-selective lysine acylation of peptide and protein substrates.
Assuntos
Aminas/química , Biocatálise , DNA Catalítico/metabolismo , Lisina/química , Peptídeos/química , AcilaçãoRESUMO
OBJECTIVES: Luminal-based imaging have identified different risk factors for extracranial and intracranial atherosclerosis (ICAS), but these techniques are known to underestimate the true extent of the disease. High-resolution (HR) vessel wall imaging (VWI) has recently gained recognition as a valuable tool in the assessment of ICAS. The aim of this study is to determine the association between cardiovascular risk factors and specific intracranial vessel segment involvement using HR-VWI. MATERIALS AND METHODS: From January 2017 to January 2020, consecutive patients ≥ 18 years-old undergoing HR-VWI of the brain were identified. Patients with history of primary or secondary vasculitis, reversible cerebral vasoconstriction syndrome, or moya-moya were excluded. The presence of vessel wall thickening and enhancement were assessed in the perpendicular plane for each vessel segment by two neuroradiologists. Univariate and multivariate analyses were performed to assess associations between imaging findings and cardiovascular risk factors. Interrater reliability was calculated. RESULTS: Seventy-one patients (39 men; mean age: 55.9 years) were included. Vessel wall enhancement was seen in 39/71 (55%). A total number of 105 vessel segments demonstrated abnormal enhancement and 79/105 (75%) had an eccentric pattern. Eccentric vessel wall enhancement was independently associated with age >65 years-old in the ICA (OR 9.0, CI 2.1 - 38.2, p < 0.01) and proximal MCA (OR 4.0, CI 1.2 - 13.2, p = 0.02), and with hyperlipidemia in the posterior circulation (OR 44.0, CI2.9-661.0, p<0.01). CONCLUSION: There is a significant association between eccentric vessel wall enhancement of the ICA and proximal MCA in patients with age > 65; and of the proximal posterior circulation (basilar - PCA1) with hyperlipidemia.
Assuntos
Angiografia Cerebral , Artérias Cerebrais/diagnóstico por imagem , Arteriosclerose Intracraniana/diagnóstico por imagem , Angiografia por Ressonância Magnética , Adulto , Idoso , Bases de Dados Factuais , Feminino , Fatores de Risco de Doenças Cardíacas , Humanos , Arteriosclerose Intracraniana/etiologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Medição de RiscoRESUMO
OBJECTIVES: Endovascular thrombectomy (EVT) has revolutionized stroke care for large vessel occlusions (LVOs). However, over half treated remain functionally disabled or die. Patients with tandem lesions, or severe stenosis/occlusion of the cervical internal carotid artery (ICA) with intracranial LVO, may have technical EVT challenges and worse outcomes. We sought to compare treatments and outcomes for patients with anterior circulation tandem lesions versus isolated LVOs. MATERIALS AND METHODS: Consecutive tandem lesion and isolated intracranial LVO patients were identified at a single center. Demographics, medical history, presentations, treatments, and outcomes were collected and analyzed. RESULTS: From 381 EVT patients, 62 had tandem lesions related to atherosclerosis (74%) or dissection (26%). Compared to isolated intracranial LVOs, they were younger (63 vs 70, pâ¯=â¯0.003), had less atrial fibrillation (13% vs 40%, p < 0.0001), less adequate reperfusion (TICI 2b-3, 58% vs 82%, p < 0.0001), more intracranial hemorrhage (ICH, 13% vs 5%, pâ¯=â¯0.037), but similar 90-day functional independence (mRS 0-2, 34% vs 43%, pâ¯=â¯0.181). The cervical ICA was treated before intracranial EVT (57%), after (13%), not acutely (22%), or was inaccessible (8%). Acute cervical ICA treatments were stenting (57%) or angioplasty alone (13%). Neither acute stenting nor order of treatment was associated with outcomes (TICI 2b-3, ICH, or 90-day mRS 0-2). Among acutely stented, neither alteplase nor antiplatelets were associated with outcomes or stent patency. CONCLUSIONS: Tandem lesions were associated with less reperfusion, more ICH, but similar 90-day functional independence. No treatment approach was associated with outcomes. These data illustrate the technical challenges of tandem lesion treatment and underscore the importance of developing new approaches.
Assuntos
Dissecação da Artéria Carótida Interna/terapia , Estenose das Carótidas/terapia , Procedimentos Endovasculares , Arteriosclerose Intracraniana/terapia , AVC Isquêmico/terapia , Trombectomia , Terapia Trombolítica , Idoso , Idoso de 80 Anos ou mais , Dissecação da Artéria Carótida Interna/diagnóstico por imagem , Dissecação da Artéria Carótida Interna/mortalidade , Dissecação da Artéria Carótida Interna/fisiopatologia , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/mortalidade , Estenose das Carótidas/fisiopatologia , Circulação Cerebrovascular , Bases de Dados Factuais , Procedimentos Endovasculares/efeitos adversos , Procedimentos Endovasculares/instrumentação , Procedimentos Endovasculares/mortalidade , Feminino , Estado Funcional , Humanos , Arteriosclerose Intracraniana/diagnóstico por imagem , Arteriosclerose Intracraniana/mortalidade , Arteriosclerose Intracraniana/fisiopatologia , AVC Isquêmico/diagnóstico por imagem , AVC Isquêmico/mortalidade , AVC Isquêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Estudos Retrospectivos , Fatores de Risco , Stents , Trombectomia/efeitos adversos , Trombectomia/mortalidade , Terapia Trombolítica/efeitos adversos , Terapia Trombolítica/mortalidade , Fatores de Tempo , Resultado do TratamentoRESUMO
The discovery of natural RNA enzymes (ribozymes) prompted the pursuit of artificial DNA enzymes (deoxyribozymes) by in vitro selection methods. A key motivation is the conceptual and practical advantages of DNA relative to proteins and RNA. Early studies focused on RNA-cleaving deoxyribozymes, and more recent experiments have expanded the breadth of catalytic DNA to many other reactions. Including modified nucleotides has the potential to widen the scope of DNA enzymes even further. Practical applications of deoxyribozymes include their use as sensors for metal ions and small molecules. Structural studies of deoxyribozymes are only now beginning; mechanistic experiments will surely follow. Following the first report 21 years ago, the field of deoxyribozymes has promise for both fundamental and applied advances in chemistry, biology, and other disciplines.
Assuntos
DNA Catalítico/metabolismo , DNA Catalítico/químicaRESUMO
OBJECTIVE: Although carotid atherosclerotic-mediated stroke remains a major cause of morbidity and mortality, some have suggested intervention in carotid stenosis should be limited to symptomatic patients given the advances in medical therapy. The present study was conducted to assess the atherosclerotic risk factor profiles, anatomic features, and clinical outcomes of previously asymptomatic patients admitted with stroke of carotid etiology. METHODS: We reviewed the data from 3382 patients admitted to a tertiary referral center with an ischemic stroke during 2005 to 2015. We focused on patients admitted with a radiographically confirmed infarct ipsilateral to a documented carotid artery stenosis ≥50%, with the admitting neurology team adjudicating the stroke etiology as carotid related. Patients were excluded if they had had a previous transient ischemic attack, previous infarct ipsilateral to any carotid lesion, or previous carotid revascularization, intracranial hemorrhage, or malignancy. Patient demographic data, medical treatments before stroke, stroke admission carotid imaging, and stroke treatments and outcomes were assessed. RESULTS: A total of 219 carotid stroke patients (7% of all strokes) were identified, of whom 61% were white and 66% were men, with a mean age of 68 ± 12 years. Hypertension (79%) and smoking (33% current; 29% former) were predominant risk factors. On admission, 50% were receiving antiplatelet therapy (aspirin, n = 92 [41%]; clopidogrel, n = 9 [4%]; dual therapy, n = 11 [5%]) and 55% were receiving lipid-lowering agents (statin, n = 115 [53%]; other, n = 6 [2%]); 77 patients (35%) were receiving both antiplatelet and lipid-lowering therapy. Of the 219 patients, 156 (71%) presented with a moderate or severe stroke (National Institutes of Health stroke scale ≥5 at admission), 54 (25%) received lytic therapy, 96 (43%) presented with an occluded ipsilateral internal carotid artery, and 117 (53%) ultimately underwent carotid revascularization at a median of 4 days. Individuals receiving both antiplatelet and lipid-lowering therapy were significantly less likely to experience a moderate or severe stroke (44% vs 78%; P = .006). CONCLUSIONS: Internal carotid artery occlusion is the culprit lesion in 43% of carotid-related strokes in those without previous symptoms. Previously asymptomatic patients not receiving combined antiplatelet and lipid-lowering medical therapy presenting with carotid-related stroke are significantly more likely to experience a severe, debilitating stroke. However, those receiving appropriate risk-reduction medical therapy are still at risk of carotid-mediated stroke. These results suggest medical therapy alone is unlikely to be sufficient stroke prevention for patients with significant carotid stenosis.
Assuntos
Isquemia Encefálica/etiologia , Artéria Carótida Interna , Estenose das Carótidas/complicações , Acidente Vascular Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , Doenças Assintomáticas , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/terapia , Artéria Carótida Interna/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/terapia , Avaliação da Deficiência , Feminino , Fibrinolíticos/uso terapêutico , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/uso terapêutico , Fatores de Proteção , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/terapia , Fatores de TempoAssuntos
Mucopolissacaridose II/cirurgia , Artéria Vertebral/cirurgia , Insuficiência Vertebrobasilar/cirurgia , Angiografia Cerebral/métodos , Descompressão Cirúrgica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mucopolissacaridose II/diagnóstico , Resultado do Tratamento , Insuficiência Vertebrobasilar/diagnósticoRESUMO
We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N40 or N100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O2-⢠or HOOâ¢) and hydrogen peroxide (H2O2) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H2O2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H2O2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn2+ and Mg2+, although including Mn2+ increases the activity, and Mn2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.
Assuntos
Clivagem do DNA , DNA Catalítico/metabolismo , Peróxido de Hidrogênio/metabolismo , Superóxidos/metabolismo , DNA Catalítico/química , Peróxido de Hidrogênio/química , Espectrometria de Massas , Superóxidos/químicaRESUMO
T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.
Assuntos
DNA Catalítico/química , Oligodesoxirribonucleotídeos/química , FosforilaçãoRESUMO
BACKGROUND AND PURPOSE: Several studies have reported poor outcomes in patients too good to treat with intravenous thrombolysis because of mild or rapidly improving symptoms. We sought to determine baseline clinical and imaging predictors of poor outcome in these patients. METHODS: Among 3950 consecutive stroke admissions (2009-2015) in our local Get With the Guidelines-Stroke database, 632 patients presented ≤4.5 hours and did not receive tissue-type plasminogen activator, with 380 of 632 (60.1%) being too good to treat. Univariate and multivariable analyses explored the clinical and imaging features associated with poor outcome (defined as not being discharged to home) in these 380 cases. RESULTS: Among these 380 cases, only 68% were discharged home; the other 25% to inpatient rehabilitation, 4% to a skilled nursing facility, and 3% expired or were discharged to hospice. Patients with poor outcome were older, were more often Hispanic, had more vascular risk factors, and had higher median National Institutes of Health Stroke Scale. Imaging characteristics associated with poor outcomes included large or multifocal infarction and poor collaterals. In multivariable analysis, only age, initial National Institutes of Health Stroke Scale, and infarct location were independently associated with poor outcome. CONCLUSIONS: Approximately one third of patients deemed too good for intravenous tissue-type plasminogen activator are unable to be discharged directly to home. Given the current safety profile of intravenous tissue-type plasminogen activator, our results suggest that the concept of being too good to treat should be re-examined with an emphasis on the features associated with poor outcome identified in our study. If replicated, these findings could be incorporated into tissue-type plasminogen activator decision-making algorithms.
Assuntos
Fibrinolíticos/administração & dosagem , Avaliação de Resultados em Cuidados de Saúde , Sistema de Registros , Índice de Gravidade de Doença , Acidente Vascular Cerebral/terapia , Ativador de Plasminogênio Tecidual/administração & dosagem , Administração Intravenosa , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alta do Paciente , Prognóstico , Acidente Vascular Cerebral/diagnósticoRESUMO
BACKGROUND AND PURPOSE: Patients with atrial fibrillation and previous ischemic stroke (IS)/transient ischemic attack (TIA) are at high risk of recurrent cerebrovascular events despite anticoagulation. In this prespecified subgroup analysis, we compared warfarin with edoxaban in patients with versus without previous IS/TIA. METHODS: ENGAGE AF-TIMI 48 (Effective Anticoagulation With Factor Xa Next Generation in Atrial Fibrillation-Thrombolysis in Myocardial Infarction 48) was a double-blind trial of 21 105 patients with atrial fibrillation randomized to warfarin (international normalized ratio, 2.0-3.0; median time-in-therapeutic range, 68.4%) versus once-daily edoxaban (higher-dose edoxaban regimen [HDER], 60/30 mg; lower-dose edoxaban regimen, 30/15 mg) with 2.8-year median follow-up. Primary end points included all stroke/systemic embolic events (efficacy) and major bleeding (safety). Because only HDER is approved, we focused on the comparison of HDER versus warfarin. RESULTS: Of 5973 (28.3%) patients with previous IS/TIA, 67% had CHADS2 (congestive heart failure, hypertension, age, diabetes, prior stroke/transient ischemic attack) >3 and 36% were ≥75 years. Compared with 15 132 without previous IS/TIA, patients with previous IS/TIA were at higher risk of both thromboembolism and bleeding (stroke/systemic embolic events 2.83% versus 1.42% per year; P<0.001; major bleeding 3.03% versus 2.64% per year; P<0.001; intracranial hemorrhage, 0.70% versus 0.40% per year; P<0.001). Among patients with previous IS/TIA, annualized intracranial hemorrhage rates were lower with HDER than with warfarin (0.62% versus 1.09%; absolute risk difference, 47 [8-85] per 10 000 patient-years; hazard ratio, 0.57; 95% confidence interval, 0.36-0.92; P=0.02). No treatment subgroup interactions were found for primary efficacy (P=0.86) or for intracranial hemorrhage (P=0.28). CONCLUSIONS: Patients with atrial fibrillation with previous IS/TIA are at high risk of recurrent thromboembolism and bleeding. HDER is at least as effective and is safer than warfarin, regardless of the presence or the absence of previous IS or TIA. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00781391.
Assuntos
Anticoagulantes/uso terapêutico , Fibrilação Atrial/prevenção & controle , Isquemia Encefálica/prevenção & controle , Piridinas/uso terapêutico , Acidente Vascular Cerebral/prevenção & controle , Tiazóis/uso terapêutico , Varfarina/uso terapêutico , Idoso , Fibrilação Atrial/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Prevenção Secundária , Acidente Vascular Cerebral/tratamento farmacológico , Resultado do TratamentoRESUMO
DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.
Assuntos
Amidas/química , DNA/química , Catálise , HidróliseRESUMO
Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening have provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead surprisingly led to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 10(5)-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, we were able to identify deoxyribozymes for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion of chemically modified DNA nucleotides in the catalyst, which we have recently found to enable this cleavage reaction. In numerous other efforts, we have investigated DNA-catalyzed peptide side chain modification reactions. Key successes include nucleopeptide formation (attachment of oligonucleotides to peptide side chains) and phosphatase and kinase activities (removal and attachment of phosphoryl groups to side chains). Through all of these efforts, we have learned the importance of careful selection design, including the frequent need to develop specific "capture" reactions that enable the selection process to provide only those DNA sequences that have the desired catalytic functions. We have established strategies for identifying deoxyribozymes that accept discrete peptide and protein substrates, and we have obtained data to inform the key choice of random region length at the outset of selection experiments. Finally, we have demonstrated the viability of modular deoxyribozymes that include a small-molecule-binding aptamer domain, although the value of such modularity is found to be minimal, with implications for many selection endeavors. Advances such as those summarized in this Account reveal that DNA has considerable catalytic abilities for biochemically relevant reactions, specifically including covalent protein modifications. Moreover, DNA has substantially different, and in many ways better, characteristics than do small molecules or proteins for a catalyst that is obtained "from scratch" without demanding any existing information on catalyst structure or mechanism. Therefore, prospects are very strong for continued development and eventual practical applications of deoxyribozymes for peptide and protein modification.