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Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092


Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
Crit Care Clin ; 16(1): 83-100, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10650501


Bedside fiberoptic bronchoscopy is a valuable tool in the diagnosis and treatment of various respiratory conditions in critically ill patients. The fiberoptic bronchoscope allows direct airway inspection, facilitating the diagnosis of benign and malignant airway lesions. In addition, pulmonary secretions or tissue samples can be collected using the bronchoscope and techniques that allow sampling of the lower airways with minimal or no upper airway contamination. Collection of lower airway samples is important in the diagnosis of pulmonary infiltrates in immunocompromised patients, in many patients with ventilator-associated pneumonia, and in selected patients with CAP. The fiberoptic bronchoscope can be used for therapeutic interventions, such as insertion of an endotracheal tube, removal of an aspirated foreign body, clearance of tenacious secretions, promotion of hemostasis in patients with hemoptysis, instillation of drugs, and assistance in the placement of tracheobronchial prostheses (i.e., airway stents). If proper preprocedural training and planning are done and the patient is monitored carefully during the procedure, fiberoptic bronchoscopy can be performed quickly and safely at the bedside in most critically ill patients.

Broncoscopia , Broncoscópios , Broncoscopia/métodos , Estado Terminal , Tecnologia de Fibra Óptica , Corpos Estranhos/diagnóstico , Corpos Estranhos/terapia , Humanos , Inalação , Pneumonia/diagnóstico , Troca Gasosa Pulmonar , Mecânica Respiratória , Traqueia