Melissa Laura Fernandez

Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid.

Background  Chronic venous leg ulcers are a significant cause of pain, immobility and decreased quality of life for patients with these wounds. In view of this, research efforts are focused on multiple factors in the wound environment to obtain information regarding the healing of ulcers.

Elevated uric acid correlates with wound severity.

Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research. Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise through better understanding the processes involved in the formation of chronic wounds. We have for the first time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may play a critical role in the formation of chronic wounds by prolonging the inflammatory process.

Uric Acid and Xanthine Oxidoreductase in Wound Healing

Chronic wounds are an important health problem because they are difficult to heal and treatment is often complicated, lengthy and expensive. For a majority of sufferers the most common outcomes are long-term immobility, infection and prolonged hospitalisation. There is therefore an urgent need for effective therapeutics that will enhance ulcer healing and patient quality of life, and will reduce healthcare costs. Studies in our laboratory have revealed elevated levels of purine catabolites in wound fluid from patients with venous leg ulcers. In particular, we have discovered that uric acid is elevated in wound fluid, with higher concentrations correlating with increased wound severity. We have also revealed a corresponding depletion in uric acid precursors, including adenosine. Further, we have revealed that xanthine oxidoreductase, the enzyme that catalyses the production of uric acid, is present at elevated levels in wound fluid. Taken together, these findings provide evidence that xanthine oxidoreductase may have a function in the formation or persistence of chronic wounds. Here we describe the potential function of xanthine oxidoreductase and uric acid accumulation in the wound site, and the effect of xanthine oxidoreductase in potentiating the inflammatory response.

Wound Care Innovation for the Tropics-An Industry-Facing Singaporean Initiative Focussed on Wounds and Wound Care in Asian Populations

Wounds are a global and growing problem with ~2%–6% of the population affected by non-healing wounds and their care consuming >5% of healthcare budgets. Despite this huge global burden, wounds and wound healing remain poorly understood and under-researched. Moreover, there are few “wound” research and development (R&D) programmes globally and those that exist are primarily focussed on clinical aspects; none are integrated across the biological, imaging, engineering, health services, Smart Nation and clinical spectrum, and none are focussed on wounds in the tropics or on Asian populations. To address this gap, the Wound Care Innovation for the Tropics (WCIT) Programme was recently established within the Skin Research Institute of Singapore. This initiative, involving 18 partners across the Singapore ecosystem and spanning all 3 integrated healthcare clusters, is supported by SG

Identification of diagnostic and prognostic biomarkers to improve the management of diabetes-related ulcers

22M over 3 years from the Singapore Governments Industry Alignment Pre-Positioning Fund. While this new programme shares some elements similar to the Australian Wound Management Innovation Corporative Research Centre, a centre initially proposed, co-led and established by the authors and Professor Helen Edwards at the Queensland University of Technology, the new initiative is differentiated by its focus on Asian populations and the challenges associated with delivery of care in tropical climates. As most wound research to date has been conducted in cooler, more temperate climates, new research is required to determine the impact and differences in healing outcomes that arise from humidity, infection, differences in pigmentation, genetic factors and microbiomes, as well as cultural and practical aspects related to service delivery. In addition, the WCIT Programme has an intentional focus on fit-for-purpose technology development and an industry-facing mission. Hence, the programme is primarily investing in technologies and approaches that address the key challenges identified by clinicians caring for wounds and by companies developing products for wounds, including: (1) point-of-care diagnostics that enable better stratification of wounds and early confirmation that a wound is on a healing versus a non-healing trajectory, (2) tools that effectively discriminate between a healthily colonised and an infected wound, (3) costeffective dressings and therapies to stimulate healing in non-healing wounds or enable shorter time to closure, and (4) improved design of products and care delivery pathways, with an emphasis on their use in community and home-care settings, and on the patient and consumer experience. The WCIT Programmes industry-facing mission is further supported by:

Biochemical profiling of proteins and metabolites in wound exudate from chronic wound environments

Abstract Introduction Diabetes-related ulcers are a common and severe complication of diabetes which is expected to increase in prevalence in line with projected global growth in rates of diabetes. Caring for these chronic wounds imposes a multi-billion dollar burden on the health care systems. These ulcers can prove lethal if untreated or not recognised and can lead to critical health complications. Methods To investigate underlying causes of wound chronicity, proteomic analyses of swab samples collected weekly from healing and non-healing diabetic foot ulcers was performed. Protein profiling was conducted based on Surface Enhanced Laser Desorption Ionisation Time of Flight (SELDI-TOF) mass spectrometry and statistical softwares were used to short list potential biomarkers. In addition, bottom-up proteomics was performed on healing and non-healing samples by SDS-PAGE and LC-MS/MS analysis using an AB SCIEX Triple TOF ® 5600 System. Trans-proteomic pipeline was used for data analyses and X! Tandem was used to search the database. Label-free quantitative proteomic analyses were performed using a computational tool called Abacus. Results (1) Statistical analyses of healing and non healing samples analysed on SELDI-TOF revealed 5 and 7 potential biomarkers (m/z) for samples with Texas score A1 and C1 respectively. (2) Bottom-up proteomics approach from both healing and non-healing samples identified 15 unique (healing) and 16 unique (non-healing) potential biomarkers. (3) Quantitative proteomic analyses resulted in 24 and 45 up-regulated healing candidates and 67 and 43 up-regulated nonhealing candidates for Texas score A1 and C1 wounds. (4) Relative quantification of 23 proteins related to oxidative stress has been identified through Abacus and 5/23 proteins have been validated using ELISA. Conclusions We are investigating these potential biomarkers using various biochemical, bioinformatics and statistical tools. A thorough investigation and study of the patterns may generate new protein candidates that can be used as potential prognostic and diagnostic biomarkers to improve the management of diabetic ulcers.