Patrick J. M. Murphy

Polyvinylpyrrolidone–sodium hyaluronate gel (Gelclair®): a bioadherent oral gel for the treatment of oral mucositis and other painful oral lesions

Background: Painful oral lesions in patients with cancer, HIV or aphthous ulcers can cause significant pain and discomfort. Those with oral mucositis (OM) resulting from high-dose chemotherapy or irradiation are the most at risk for painful lesions that lead to significant morbidity and mortality, decreased quality of life and increased economic costs. Numerous approaches have been taken to prevent and treat OM in the cancer patient but this condition remains a significant obstacle to cancer therapy. Small studies have been done in healing oral lesions arising from laser surgery and in wound healing but research is needed to support the use of polyvinylpyrrolidone–sodium hyaluronate gel (Gelclair®) in these clinical setting. Objective: The purpose of this paper is to review the benefit of polyvinylpyrrolidone–sodium hyaluronate gel, a topical oral mucosal barrier gel to prevent or diminish the pain of OM and other oral lesions. Methods: Primary resources in peer-reviewed journals, chapters in recognized textbooks and information from international and national oncology and dental medicine scientific meetings. Conclusions: Oncology patients receiving high-dose immunosuppressive therapy are the most at risk with painful OM. Current prevention and treatment options for OM have limited effectiveness. Although opioids are available for pain management, novel agents, to be used alone or in combination with other proven measures to decrease the severity and duration of OM, are emerging. Clinical studies in the use of polyvinylpyrrolidone–sodium hyaluronate to reduce the pain or disruption to the oral mucosa has merit. Few clinical trials or main publications have supported the use of this product. Robust randomized trials are needed to further support the clinical utility of this agent.

The Protein Chaperone HSP90 Can Facilitate the Divergence of Gene Duplicates

The heat-shock protein 90 (HSP90) acts as a chaperone by ensuring proper maturation and folding of its client proteins. The HSP90 capacitor hypothesis holds that interactions with HSP90 allow proteins to accumulate mutations while maintaining function. Following this logic, HSP90 clients would be predicted to show relaxed selection compared with nonclients. In this study, we identify a new HSP90 client in the plant steroid hormone pathway: the transcription factor BES1. Its closest paralog, BZR1, is not an HSP90 client. This difference in HSP90 client status in two highly similar proteins enabled a direct test of the capacitor hypothesis. We find that BES1 shows relaxed selection compared to BZR1, hallmarks of neo- and subfunctionalization, and dynamic HSP90 client status across independent evolutionary paths. These results suggested that HSP90’s influence on gene evolution may be detectable if we compare gene duplicates because duplicates share most other properties influencing evolutionary rate that might otherwise conceal the chaperone’s effect. We test this hypothesis using systematically identified HSP90 clients in yeast and observe a significant trend of HSP90 clients evolving faster than their nonclient paralogs. This trend was not detected when yeast clients and nonclients were compared without considering paralog status. Our data provide evidence that HSP90 influences selection on genes encoding its clients and facilitates divergence between gene duplicates.

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscopes cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.