Frog foams and natural protein surfactants.

Foams and surfactants are relatively rare in biology because of their potential to harm cell membranes and other delicate tissues. However, in recent work we have identified and characterized a number of natural surfactant proteins found in the foam nests of tropical frogs and other unusual sources. These proteins, and their associated foams, are relatively stable and bio-compatible, but with intriguing molecular structures that reveal a new class of surfactant activity. Here we review the structures and functional mechanisms of some of these proteins as revealed by experiments involving a range of biophysical and biochemical techniques, with additional mechanistic support coming from more recent site-directed mutagenesis studies.

Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

Aqueous solubilization of C60 fullerene by natural protein surfactants, latherin and ranaspumin-2.

C60 fullerene is not soluble in water and dispersion usually requires organic solvents, sonication or vigorous mechanical mixing. However, we show here that mixing of pristine C60 in water with natural surfactant proteins latherin and ranaspumin-2 (Rsn-2) at low concentrations yields stable aqueous dispersions with spectroscopic properties similar to those previously obtained by more vigorous methods. Particle sizes are significantly smaller than those achieved by mechanical dispersion alone, and concentrations are compatible with clusters approximating 1:1 protein:C60 stoichiometry. These proteins can also be adsorbed onto more intractable carbon nanotubes. This promises to be a convenient way to interface a range of hydrophobic nanoparticles and related materials with biological macromolecules, with potential to exploit the versatility of recombinant protein engineering in the development of nano-bio interface devices. It also has potential consequences for toxicological aspects of these and similar nanoparticles.

Resonance assignments for latherin, a natural surfactant protein from horse sweat.

Latherin is an intrinsically surfactant protein of ~23 kDa found in the sweat and saliva of horses. Its function is probably to enhance the translocation of sweat water from the skin to the surface of the pelt for evaporative cooling. Its role in saliva may be to enhance the wetting, softening and maceration of the dry, fibrous food for which equines are adapted. Latherin is unusual in its relatively high content of aliphatic amino acids (~25% leucines) that might contribute to its surfactant properties. Latherin is related to the palate, lung, and nasal epithelium carcinoma-associated proteins (PLUNCs) of mammals, at least one of which is now known to exhibit similar surfactant activity to latherin. No structures of any PLUNC protein are currently available. (15)N,(13)C-labelled recombinant latherin was produced in Escherichia coli, and essentially all of the resonances were assigned despite the signal overlap due to the preponderance of leucines. The most notable exceptions include a number of residues located in an apparently dynamic loop region between residues 145 and 154. The assignments have been deposited with BMRB accession number 19067.

The structure of latherin, a surfactant allergen protein from horse sweat and saliva.

Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting microbial biofilms. How these proteins work as surfactants while remaining soluble and cell membrane-compatible is not known. Nor have their structures previously been reported. We have used protein nuclear magnetic resonance spectroscopy to determine the conformation and dynamics of latherin in aqueous solution. The protein is a monomer in solution with a slightly curved cylindrical structure exhibiting a 'super-roll' motif comprising a four-stranded anti-parallel ß-sheet and two opposing α-helices which twist along the long axis of the cylinder. One end of the molecule has prominent, flexible loops that contain a number of apolar amino acid side chains. This, together with previous biophysical observations, leads us to a plausible mechanism for surfactant activity in which the molecule is first localized to the non-polar interface via these loops, and then unfolds and flattens to expose its hydrophobic interior to the air or non-polar surface. Intrinsically surface-active proteins are relatively rare in nature, and this is the first structure of such a protein from mammals to be reported. Both its conformation and proposed method of action are different from other, non-mammalian surfactant proteins investigated so far.

Construction and validation of a low-cost laparoscopic simulator for surgical education.

INTRODUCTION: To construct a trainer that would achieve the equivalent goals of the Fundamentals of Laparoscopic Surgery (FLS) trainer at an economical cost. A validation study comparing our homemade (HM) trainer vs the FLS trainer was performed. A literature search as well as a price comparison with other commercially available laparoscopic trainers is presented. METHODS: The HM laparoscopic trainer was constructed using a prefabricated hard plastic frame with a vinyl plastic sheet affixed as the roof. A row of light-emitting diode lights and a charge-coupled device camera were mounted on the inside roof of the frame. Electrical wires were spliced to supply power to both the light-emitting diode lights and the camera. The charge-coupled device camera was connected to a liquid crystal display screen which was affixed directly across from the user. Subjects were prospectively randomized to perform the 5 tasks put forth by the McGill Inanimate System for Training and Evaluation of Laparoscopic Skills on both the HM trainer and the FLS trainer (pegboard transfer, pattern cut, placement of ligating loop, extracorporeal knot suture, and intracorporeal knot suture). Simple paired t test was performed to compare times between the trainers. SETTING: The construction of the trainer and the validation study were performed at the Central Michigan University College of Medicine Department of Simulation. PARTICIPANTS: Subjects consisted of third- and fourth-year medical students (n = 30). RESULTS: A laparoscopic trainer box was constructed and assembled in 2 hours. The HM trainer cost $309 representing a cost savings of $1371. Results of the validation study demonstrated no statistical difference in times to complete 3 out of the 5 tasks as well as no difference in total time to complete all 5 tasks (p value< 0.05). CONCLUSION: Valid laparoscopic simulators can be constructed at an economical cost.

An emergency department guideline for the diagnosis of pulmonary embolism: an outcome study.

OBJECTIVE: To assess the clinical outcome of patients suspected of pulmonary embolism (PE) following implementation of an emergency department (ED) diagnostic guideline. METHODS: A prospective observational study of all patients suspected of PE who presented to the ED during a four-month study period. The authors' modification of the Charlotte criteria recommended D-dimer testing in those younger than 70 years of age with a low clinical suspicion of PE and no unexplained hypoxemia, unilateral leg swelling, recent surgery, hemoptysis, pregnancy, or prolonged duration of symptoms. The primary outcome was the identification of venous thromboembolism during a three-month follow-up period. The negative predictive value of the overall diagnostic strategy and the test characteristics of D-dimer were calculated. RESULTS: A total of 1,207 consecutive patients were evaluated for suspected PE; 71 (5.8%) were diagnosed with venous thromboembolism. One missed case of PE was identified on follow-up, yielding a negative predictive value of 99.9% (95% confidence interval [CI] = 99.5% to 100%). The missed case was a patient who presented with pleuritic chest pain and shortness of breath; a chest radiograph revealed pneumothorax, and the physician decided not to pursue the positive D-dimer result. The patient returned six weeks later with PE. Subgroup analysis of patients having D-dimer performed (n = 677) yields a sensitivity of 0.93 (95% CI = 0.77 to 0.98) and a specificity of 0.74 (95% CI = 0.70 to 0.77). CONCLUSIONS: Implementation of a PE diagnostic guideline in a community ED setting is safe and has improved the specificity of the enzyme-linked immunosorbent assay D-dimer test when compared with previous studies.