Highlights d Wound microbiota was profiled longitudinally in patients with diabetic foot ulcers d Staphylococcus aureus strains were associated with poor outcomes d S. aureus and other wound isolates promoted differential wound healing responses d Debridement depleted anaerobic bacteria in wounds with favorable outcomes

A diabetic vasculature is detrimental to cardiovascular health through the actions of advanced glycation end products (AGEs) on endothelial cells and platelets. Platelets activated by AGEs agonize endothelial responses promoting cardiovascular disease (CVD) development. While it has been established that AGEs can alter platelet functions, little is known about the specific platelet pathways that AGEs modify. Therefore, we evaluated the effects of AGEs on specific salient platelet pathways related to CVDs and whether the effects that AGEs elicit are dependent on glycation extent. To accomplish our objective, platelets were incubated with reversibly or irreversibly glycated albumin. A time course for adhesion and aggregation agonist receptor expression was assessed. Optical platelet aggregometry was used to confirm the functional activity of platelets after AGE exposure. In general, platelets subjected to glycated albumin had a significantly enhanced adhesion and aggregation potential. Furthermore, we observed an enhancement in dense body secretion and intracellular calcium concentration. This was especially prevalent for platelets exposed to irreversibly glycated albumin. Additionally, functional aggregation correlated well with receptor expression, suggesting that AGE-induced altered receptor sensitivity translated to altered platelet functions. Our findings indicate that under diabetic vascular conditions platelets become more susceptible to activation and aggregation due to an overall enhanced receptor expression, which may act to promote CVD development.

22Chronic, non-healing wounds are a major complication of diabetes associated with high morbidity 23 and health care expenditures estimated at $9-13 billion annually in the US. Though microbial 24 infection and critical colonization is hypothesized to impair healing and contribute to severe 25 outcomes such as amputation, antimicrobial therapy is inefficacious and the role of microbes in 26 tissue repair, regeneration, and healing remains unclear. Here, in a longitudinal prospective 27 cohort study of 100 subjects with non-infected neuropathic diabetic foot ulcer (DFU), we 28 performed metagenomic shotgun sequencing to elucidate microbial temporal dynamics at strain-29 level resolution, to investigate pathogenicity and virulence of the DFU microbiome with respect to 30 outcomes, and to determine the influence of therapeutic intervention on the DFU microbiota. Slow 31 healing DFUs were associated with signatures of biofilm formation, host invasion, and virulence. 32Though antibiotic resistance was widespread at the genetic level, debridement, rather than 33 antibiotic treatment, significantly shifted the DFU microbiome in patients with more favorable 34 outcomes. Primary clinical isolates of S. aureus, C. striatum, and A. faecalis induced differential 35 biological responses in keratinocytes and in a murine model of diabetic wound healing, with the 36 S. aureus strain associated with non-healing wounds eliciting the most severe phenotype. 37Together these findings implicate strain-level diversification of the wound pathogen S. aureus in 38 chronic wound outcomes, while revealing potential contributions from skin commensals and other 39 previously underappreciated constituents of the wound microbiota. 40 41 42 chronic wounds 43 44 MAIN TEXT: 45 INTRODUCTION 46 Chronic, non-healing wounds are common and costly complications of diabetes. Up to one in four 47 persons with diabetes will develop a diabetic foot ulcer (DFU)(Boyko et al., 2015; Martins-Mendes 48 et al., 2014), and approximately 25% of hospital stays for patients with diabetes are due to infected 49 or ischemic DFU(Ramsey et al., 1999). Complications from DFUs account for two-thirds of all 50 non-traumatic lower extremity amputations performed in the United States(Hoffstad et al., 2015; 51 Martins-Mendes et al., 2014) and 5-year mortality rates surpass those of prostate and breast 52 cancer, among others(Armstrong et al., 2007; Moulik et al., 2003). Improved therapeutic 53 approaches are desperately needed, as morbidity, mortality, and health care expenditures only 54 continue to increase as the prevalence of diabetes escalates worldwide. 55 56 Microbial colonization, biofilm formation, and infection are hypothesized to impair healing of DFUs 57 and contribute to severe complications such as osteomyelitis and amputation. Wound infection is 58 believed to underlie up to 90% of amputations(Boulton et al., 2005); yet quantitative cultures of 59 uninfected DFUs were not predictive of outcomes(Gardner et al., 2014). Systemic and topical 60 antimicrobials are of...

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