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Computational design of proteins with altered ligand specificity is an emerging method for the creation of new biosensing systems. In this work, we investigated the outcome of site-directed mutagenesis on the Escherichia coli ribose binding protein (RBP), which is frequently used as a design scaffold for computational searches. A ribose biosensor was first constructed whereby an environmentally sensitive fluorescent probe was covalently attached to RBP at position S265C. This protein conjugate displayed a 54% decrease in emission intensity upon the addition of saturating ribose concentrations and exhibited an apparent dissociation constant ( Kd) of 3.4 μM. Site-directed mutants within the RBP binding pocket were created and examined for ribose binding ability and overall structural stability. Because as many as 12 mutations are needed to alter ligand specificity in RBP, we measured the effect of single and multiple alanine mutations on stability and signal transduction potential of the ribose biosensor. Single alanine mutations had significant impact on both stability and signaling. Mutations of N190A and F214A each produced melting temperatures >8°C below those observed for the wild-type protein. Residue Q235, located in the hinge region of RBP, appeared to be a hot spot for global protein stability as well. Additional single alanine mutations demonstrated as much as 200-fold increase in apparent Kd but retained overall protein stability. The data collected from this study may be incorporated into design algorithms to help create more stable biosensors and optimize signal transduction properties for a variety of important analytes. [ABSTRACT FROM AUTHOR]

Objectives: Determine whether various techniques used to promote tympanic membrane (TM) perforation closure at the time of ventilation tube removal impact the likelihood of TM perforation healing in children. Methods: A retrospective chart review was conducted of 265 children (341 ears) who underwent tympanostomy tube removal at a tertiary care pediatric hospital between January 2010 and November 2013. Techniques performed at the time of ventilation tube removal included tube removal only, freshening the perforation edges, performing paper patch myringoplasty, or both freshening edges and paper patch myringoplasty. Results: The persistent TM perforation rate was 9.97% (34/341). Technique was not found to significantly influence the likelihood for the TM to heal (P =. 81), with 11/86 (12.7%) having persistent perforations with tube removal only, 6/62 (9.7%) with freshened perforation edges, 7/50 (14.0%) with paper patch myringoplasty and 10/109 (9.2%) with both edges freshened and paper patch myringoplasty. There was no significant interaction between technique and age, tube retention length, number of previous ventilation tubes placed, or type of tube removed. Additionally, no significant difference in TM perforation rate was observed for number of previous tympanostomy tubes, tube duration, previous adenoidectomy, or indication for tube removal. Factors associated with higher rates of persistent perforation included older age, longer‐acting ventilation tubes, and trisomy 21. Conclusions: There is no reduction in persistent TM perforation rate following ventilation tube removal if edges are freshened and/or a paper patch myringoplasty is performed. Increased pediatric age, longer‐acting tympanostomy tubes and history of trisomy 21 may negatively influence the likelihood of closure. [ABSTRACT FROM AUTHOR]