Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.

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.

Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF.

BACKGROUND: While idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease, the aetiology of IPF is poorly understood. Familial cases of pulmonary fibrosis suggest a genetic basis for some forms of the disease. Recent reports have linked genetic mutations in surfactant protein C (SFTPC) with familial forms of pulmonary fibrosis, including one large family in which a number of family members were diagnosed with usual interstitial pneumonitis (UIP), the pathological correlate to IPF. Because of this finding in familial cases of pulmonary fibrosis, we searched for SFTPC mutations in a cohort of sporadic cases of UIP and non-specific interstitial pneumonitis (NSIP). METHODS: The gene for SFTPC was sequenced in 89 patients diagnosed with UIP, 46 patients with NSIP, and 104 normal controls. RESULTS: Ten single nucleotide polymorphisms in the SFTPC sequence were found in IPF patients and not in controls. Only one of these created an exonic change resulting in a change in amino acid sequence. In this case, a T to C substitution resulted in a change in amino acid 73 of the precursor protein from isoleucine to threonine. Of the remaining polymorphisms, one was in the 5' UTR, two were exonic without predicted amino acid sequence changes, and six were intronic. One intronic mutation suggested a potential enhancement of a splicing site. CONCLUSIONS: Mutations in SFTPC are identified infrequently in this patient population. These findings indicate that SFTPC mutations do not contribute to the pathogenesis of IPF in the majority of sporadic cases.

Fiberoptic bronchoscopy for diagnosis and treatment.

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.