Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

Corneal epithelial cell fate is maintained during repair by Notch1 signaling via the regulation of vitamin A metabolism

Integrity and preservation of a transparent cornea are essential for good vision. The corneal epithelium is stratified and nonkeratinized and is maintained and repaired by corneal stem cells. Here we demonstrate that Notch1 signaling is essential for cell fate maintenance of corneal epithelium during repair. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1-deficient corneal progenitor cells differentiate into a hyperplastic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell nonautonomous processes, characterized by secretion of fibroblast growth factor-2 (FGF-2) through Notch1(-/-) epithelium followed by vascularization and remodeling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of cellular retinol binding protein 1 (CRBP1), required to generate a pool of intracellular retinol.     

Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin

Restin and endostatin are C-terminal fragments of the noncollagenous domains of collagen XV and collagen XVIII exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic activity in vivo restricting the growth of solid tumors and metastasis. They are therefore currently being tested in clinical trials as anti-cancer drugs. We present the identification of new endogenous variants of both angiogenesis inhibitors isolated from a human hemofiltrate peptide library. Using an immunological screening approach with time-resolved rare earth metal fluorometry, immunoreactive compounds were purified chromatographically and characterized by mass spectrometry. We discovered four novel proteolytic products of restin as well as four variants of endostatin. Two endostatin products were characterized as short internal fragments (R176-L215 and R176-S219) of the entire molecule containing the recently identified beta1 integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-glycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GalNAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V117-S311-GalNAc-Gal2-NANA) and asialoglycopeptides (V117-S311-GalNAc-Gal2). The four restin variants (R(I)-R(IV)) were identified with identical C- but different N-termini and no posttranslational modification (R(I): P66-A254, R(II): P75-A254, R(III): Y81-A254 and R(IV): A89-A254). Following a differential peptide mass fingerprint approach by reflector mode MALDI-TOFMS, the disulfide patterns of these circulating restins were determined as Cys1-Cys4 and Cys2-Cys3. These endogenous circulating collagen fragments will help to understand the physiological processing of the therapeutic proteins.     

Tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in Leishmania major-infected macrophage-like RAW 264.7 cells

Some polyphenol-containing extracts (Pelargonium sidoides, Phyllanthus amarus) and representatives of simple phenols (shikimic acid 3- and 5-O-gallate), flavan-3-ols (epigallocatechin 3-gallate), proanthocyanidins (a hexamer) and hydrolysable tannins (corilagin, casuariin, geraniin) were studied for gene expressions (iNOS, IL-1, IL-10, IL-12, IL-18, TNF-alpha, IFN-alpha/gamma) by RT-PCR. All extracts and compounds were capable of enhancing the iNOS and cytokine mRNA levels in parasitised cells when compared with those in non-infected conditions.     

Resource tracking in North American Telorchis spp. (Digenea: Plagiorchiformes: Telorchidae)

We examine the evolution of host specificity for species of Telorchis, using the methods developed by researchers studying phytophagous insect-plant systems. Optimization of "generalist" compared with "specialist" onto the phylogeny for Telorchis revealed ambiguous patterns, depending on how the 2 terms were defined. Regardless of that definition, most of the evolutionary diversification of this group has been carried out within eucryptodiran turtles, the ancestral host group. From that plesiomorphic background, there appears to have been 2 episodes of specialization by way of a host switch into caudates (ancestor of T. stunkardi + T. sirenis) and snakes (T. auridistomi), and 1 episode of exuberant expansion producing a true generalist (T. corti). These results, which indicate that most species of Telorchis are tracking widespread plesiomorphic resources, mirror those reported for phytophagous insects and their plants. We believe that establishing a dialogue between the two research groups will be mutually beneficial to both and will strengthen our understanding of the complex factors underlying the evolution of coevolutionary associations.     

Field Demonstration of Acetone Pretreatment and Composting of Particulate-TNT-Contaminated Soil

Solid fragments of explosives in soil are common in explosives testing and training areas. In this study we initially sieved the upper 6 in of contaminated soil through a 3-mm mesh, and found 2,4,6-trinitrotoluene (TNT) fragments. These contributed to an estimated concentration of 1.7 kg per cubic yard soil, or for 2000 ppm TNT in the soil. Most of the fragments ranged 4 mm to 10 mm diameter in size, but explosives particles weighing up to 56 g (about 4 cm diameter) were frequently observed. An acetone pretreatment/composting system was then demonstrated at field scale. The amount of acetone required for a TNT-dissolving slurry process was controlled by the viscosity of the soil/acetone mix rather than the TNT dissolution rate. The amount needed was estimated at about 55 gallons acetone per cubic yard soil. Smaller, 5- to 10-mm-diameter fragments went into solution in less than 15 min at a mixer speed of 36 rpm, with a minimum of 2 g TNT going into solution per 30 min for the larger chunks. The slurries were then mixed with compost starting materials and composted in a vented 1 yd³ container. After 34 days incubation time TNT was below the site-specific regulatory threshold of 44 ppm. TNT metabolites and acetone were also below their regulatory thresholds established for the site.     

Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism

The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.     

Seeking the true time: Exploring otolith chemistry as an age-determination tool

Fish otoliths' chronometric properties make them useful for age and growth rate estimation in fisheries management. For the Eastern Baltic Sea cod stock (Gadus morhua), unclear seasonal growth zones in otoliths have resulted in unreliable age and growth information. Here, a new age estimation method based on seasonal patterns in trace elemental otolith incorporation was tested for the first time and compared with the traditional method of visually counting growth zones, using otoliths from the Baltic and North seas. Various trace elemental ratios, linked to fish metabolic activity (higher in summer) or external environment (migration to colder, deeper habitats with higher salinity in winter), were tested for age estimation based on assessing their seasonal variations in concentration. Mg:Ca and P:Ca, both proxies for growth and metabolic activity, showed greatest seasonality and therefore have the best potential to be used as chemical clocks. Otolith image readability was significantly lower in the Baltic than in the North Sea. The chemical (novel) method had an overall greater precision and percentage agreement among readers (11.2%, 74.0%) than the visual (traditional) method (23.1%, 51.0%). Visual readers generally selected more highly contrasting zones as annuli whereas the chemical readers identified brighter regions within the first two annuli and darker zones thereafter. Visual estimates produced significantly higher, more variable ages than did the chemical ones. Based on the analyses in our study, we suggest that otolith microchemistry is a promising alternative ageing method for fish populations difficult to age, such as the Eastern Baltic cod.