Enhanced Vascularization of Cultured Skin Substitutes Genetically Modified to Overexpress Vascular Endothelial Growth Factor11The authors declared in writing to have no conflict of interest.

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications

Topical Nutrients Promote Engraftment and Inhibit Wound Contraction of Cultured Skin Substitutes in Athymic Mice

Routine treatment of burns with cultured skin substitutes (CSS) has been limited by poor engraftment and by scarring. Hypothetically, topical application of essential nutrients and/or growth factors may support epithelial survival temporarily during graft vascularization, CSS, composed of human epidermal keratinocytes and dermal fibroblasts attached to collagen-glycosaminoglycan substrates, were incubated for 19 d in media optimized for keratinocytes. CSS, human xenografts, murine autografts, or no grafts were applied orthotopically to full-thickness skin wounds (2 × 2 cm) in athymic mice. Wounds were irrigated for 14 d with 1 ml/d modified cell culture medium or with saline containing epidermal growth factor, or were treated with dry dressings. After 6 weeks, treated sites were scored for percentage original wound area (mean ± SEM) and percentage HLA- ABC-positive healed wounds [(number positive/n) × 100], and tested for significance (analysis of variance, p < 0.0001; Tukey test, p < 0.05). The data showed that CSS irrigated with nutrient medium were not statistically different in wound area (67.8 ± 5.1%) from murine autografts (63.3 ± 2.9%) but were statistically larger than human xenograft, no graft, or CSS treated with saline irrigation or dry dressings. HLA- ABC expression was 100% in CSS with nutrient irrigation, 86% in CSS with saline irrigation, 83% In CSS without irrigation, and 75% in xenografts with nutrient irrigation. These findings suggest that availability of essential nutrients supports keratinocyte viability during graft vascularization of CSS

The geography of biodiversity change in marine and terrestrial assemblages

This work was supported by funding to the sChange working group through sDiv, the synthesis center of iDiv, the German Centre for Integrative Biodiversity Research Halle-Jena-Leipzig, funded by the German Research Foundation (FZT 118). S.A.B., H.B., J.M.C., J.H., and M.W. were supported by the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig. S.R.S. was supported by U.S. National Science Foundation grant 1400911. LHA was supported by Fundação para a Ciência e Tecnologia, Portugal (POPH/FSE SFRH/BD/90469/2012), and by the Jane and Aatos Erkko Foundation. M.D. was supported by a Leverhulme Trust Fellowship. A.E.M., F.M., and M.D. were supported by ERC AdG BioTIME 250189 and PoC BioCHANGE 727440. A.G. is supported by the Liber Ero Chair in Biodiversity Conservation.Human activities are fundamentally altering biodiversity. Projections of declines at the global scale are contrasted by highly variable trends at local scales, suggesting that biodiversity change may be spatially structured. Here, we examined spatial variation in species richness and composition change using more than 50,000 biodiversity time series from 239 studies and found clear geographic variation in biodiversity change. Rapid compositional change is prevalent, with marine biomes exceeding and terrestrial biomes trailing the overall trend. Assemblage richness is not changing on average, although locations exhibiting increasing and decreasing trends of up to about 20% per year were found in some marine studies. At local scales, widespread compositional reorganization is most often decoupled from richness change, and biodiversity change is strongest and most variable in the oceans.PostprintPostprintPeer reviewe

Novel Anti-bacterial Activities of β-defensin 1 in Human Platelets: Suppression of Pathogen Growth and Signaling of Neutrophil Extracellular Trap Formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria