Angiogenesis and innervation during the integration of engineered skin substitutes

Numerous advances have been made in the field of skin tissue engineering in recent years to overcome skin injuries, burns and pathologies. Most of skin substitutes are exogenous matrices requiring the contribution of both host fibroblasts and endothelial cells and, therefore, a long time to become functional after implantation. In view of this, here we investigate the potential of a specific pre-vascularized dermis (PVD) obtained by seeding freshly isolated fibroblasts onto gelatin microbeads and adding, at a certain culture time, endothelial cells (HUVECs) [1]. More in detail, we implanted our engineered skin on the back of nu/nu mice in a full thickness skin defect model which, respect to the subcutaneous pocket we previously experienced, is more functional for both general and wound healing applications. At different timepoints (3, 7, 14, 21 and 42 days) we retrieved our skin biohybrids and analysed them by histology and immunofluorescence. Animal studies were performed following the guidelines of EU (2010/63/EU). Our main objective is to study the behaviour and the degree of integration of our skin substitute in the host organism. First of all, an appreciable integration of a pre-vascularized substitute with host tissue results in a fast anastomosis between the two vascular networks. However, since integration is a complex process there are other aspects which have to be considered. For example, a major limitation of skin substitutes in clinical application is to mimic the physiological sensitivity of the host skin. Regarding vascularization, we looked for the expression of the lectins Griffonia Semplicifolia and Ulex Europaeus Agglutinin I (UEA I) to mark murine and human vessels, respectively. We noticed, starting from day 14 Abstract 21 onwards, the onset of numerous anastomosis between the two vascular networks (human and murine). Afterwards, from the innervation standpoint, we looked at the expression of Neurofilament-M, PGP9.5, NGF and BDNF along with the corresponding receptors TrkA and TrkB. Interestingly, we noticed a partial reinnervation of our skin substitutes already 42 days after implantation through the appreciable expression of PGP9.5, a promising result in comparison with other skin substitutes where the reinnervation is observed not earlier than after 8 weeks of implantation [2]. Therefore, since neurotrophins guide fibroblasts differentiation into myofibroblasts, also increasing skin tensile strength, we analysed the variation of the Young Modulus of our samples via indentation test. Moreover, to better frame the expression of our markers we performed a quantitative analysis through PCR. In conclusion, our skin substitute, when implanted in a full thickness skin defect model, shows an earlier vascularization and innervation time compared to other substitutes described in the literature leading us to investigate the connection between vascularization and innervation during tissue development and maturation

Easy and Rapid Purification of Highly Active Nisin

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC50 of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure

When the Leader Gets Loose: In Vivo Biosynthesis of a Leaderless Prenisin Is Stimulated by a trans-Acting Leader Peptide

The nisin leader is believed to be crucial for nisin biosynthesis. Here, by using a construct completely lacking the leader peptide, we show that an up to fivefold-dehydrated leaderless prenisin can be obtained, as judged by MALDI-TOF MS, and that some of these species are biologically active, thus suggesting that at least three lanthionine rings are present. Notably, by expressing the leader peptide in trans together with the leaderless prenisin, we were able to increase the dehydration/cyclization efficiency of both NisB and NisC, but still with limited efficiency until the fifth dehydratable residue (Thr13) was processed, thereby enabling three rings to form. This, for the first time, demonstrates that 1) the leader is not absolutely necessary for the dehydration reaction of class I lantibiotics to occur in vivo; 2) the leader acts in trans in vivo; 3) the leader increases the efficiency of modification. Based on previous work and our current study, a model for the interactions of NisB and NisC with prenisin is proposed, in which the leader induces a more active conformation and/or productive complex formation of the biosynthetic machinery, and, when covalently bound, is involved in increasing the efficiency of dehydration to the C-terminal end of the prenisin substrate molecule.

Orexin-A Prevents Lipopolysaccharide-Induced Neuroinflammation at the Level of the Intestinal Barrier

In states of intestinal dysbiosis, a perturbation of the normal microbiome composition, the intestinal epithelial barrier (IEB) permeability is increased as a result of the disruption of the epithelial tight junction protein network, in which occludin is mostly affected. The loss of IEB integrity promotes endotoxemia, that is, bacterial lipopolysaccharide (LPS) translocation from the intestinal lumen to the circulatory system. This condition induces an enhancement of pro-inflammatory cytokines, which leads to neuroinflammation through the gut-brain axis. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions and produced mainly in the brain lateral hypothalamic area, is expressed also in several peripheral tissues. Orexin-producing neurons have been found in the myenteric plexus to project to orexin receptor 1 (OX-1R)-expressing enterocytes of the intestinal villi. In the present study we investigated the protective role of OX-A against LPS-induced increase of IEB permeability and microglia activation in both an in vivo and in vitro model of the gut-brain axis. By exploiting biochemical, immunocytochemical, immunohistochemical, and functional approaches, we demonstrate that OX-A preserves the IEB and occludin expression, thus preventing endotoxemia and subsequent neuroinflammation

Uncovering the Prevalence and Diversity of Integrating Conjugative Elements in Actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria