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In silico modelling of cascade enzymatic proteolysis is an exceedingly complex and challenging task. Here, we study partial proteolysis of insulin by pepsin: a process leading to the release of a highly amyloidogenic two chain 'H-fragment'. The H-fragment retains several cleavage sites for pepsin. However, under favorable conditions H-monomers rapidly self-assemble into proteolysis-resistant amyloid fibrils whose composition provides snapshots of early and intermediate stages of the proteolysis. In this work, we report a remarkable agreement of experimentally determined and simulation-predicted cleavage sites on different stages of the proteolysis. Prediction of cleavage sites was based on the comprehensive analysis of the docking interactions from direct simulation of coupled folding and binding of insulin (or its cleaved derivatives) to pepsin. The most frequent interactions were found to be between the pepsin's active site, or its direct vicinity, and the experimentally determined insulin cleavage sites, which suggest that the docking interactions govern the proteolytic process.

PURPOSE: We studied the feasibility of labeling hydrogel scaffolds with a fluorine nanoemulsion for F-19 magnetic resonance imaging (MRI) to enable non-invasive visualization of their precise placement and potential degradation. PROCEDURE: Hyaluronan-based hydrogels (Activated Hyaluronan, HA) at increasing concentrations of fluorine nanoemulsion (V-sense, Vs) were prepared to measure gelation time and oscillatory stress at 1 h and 7 day after the beginning of gelation. All biomechanical measurements were conducted with an ARES 2 rheometer. Diffusion of fluorine from the hydrogel: Three hydrogels in various Vs to HA volumetric ratios (1:50, 1:10 and 1:5) were prepared in duplicate. Hydrogels were incubated at 37°C. To force the diffusion, three hydrogels were agitated at 1000 rpm. 1H and F-19 MRI scans were acquired at 1, 3, 7 days, and 2 months after gel preparation on a Bruker Ascend 750 scanner. To quantify fluorine content, scans were analyzed in Voxel Tracker 2.0. Cell viability in vitro and in vivo: Mouse, luciferase-positive, Glial-restricted Progenitors (GRPs) were embedded in 0:1, 1:50, 1:10, and 1:5 Vs:HA mixtures according to the manufacturer’s protocol (final cell conc. 1×l0(7)/ml). For the in vitro assay, mixtures were placed on white 96-wells plate in triplicate and bioluminescence was measured at 1, 3, 7, 14, 21, and 28 days later. For in vivo experiments, GRPs in Vs/HA mixture were injected subcutaneously in SCID mice and BLI was acquired at 1, 3, 7, and 14 days. RESULTS: Mixing of perfluorocarbon nanoemulsions (V-sense) at increasing ratios of 1:50, 1:10, and 1:5 v/v of fluorine/activated hyaluronan (HA) hydrogel gradually elongated the gelation time from 194 s for non-fluorinated controls to 304 s for 1:5 V-sense:HA hydrogels, while their elastic properties slightly decreased. There was no release of V-sense from hydrogels maintained in stationary conditions over two months. The addition of V-sense positively affected in vitro survival of scaffolded GRPs in a dose-dependent manner. CONCLUSIONS: These results show that hydrogel fluorination does not impair its beneficial properties for scaffolded cells, which may be used to visualize scaffolded GRP transplants with F-19 MRI.

The physiological spaces (lateral ventricles, intrathecal space) or pathological cavities (stroke lesion, syringomyelia) may serve as an attractive gateway for minimally invasive deployment of stem cells. Embedding stem cells in injectable scaffolds is essential when transplanting into the body cavities as they secure favorable microenvironment and keep cells localized, thereby preventing sedimentation. However, the limited migration of transplanted cells from scaffold to the host tissue is still a major obstacle, which prevents this approach from wider implementation for the rapidly growing field of regenerative medicine. Hyaluronan, a naturally occurring polymer, is frequently used as a basis of injectable scaffolds. We hypothesized that supplementation of hyaluronan with activated proteolytic enzymes could be a viable approach for dissolving the connective tissue barrier on the interface between the scaffold and the host, such as pia mater or scar tissue, thus demarcating lesion cavity. In a proof-of-concept study, we have found that collagenase and trypsin immobilized in hyaluronan-based hydrogel retain 60% and 28% of their proteolytic activity compared to their non-immobilized forms, respectively. We have also shown that immobilized enzymes do not have a negative effect on the viability of stem cells (glial progenitors and mesenchymal stem cells) in vitro. In conclusion, proteolytic rafts composed of hyaluronan-based hydrogels and immobilized enzymes may be an attractive strategy to facilitate migration of stem cells from injectable scaffolds into the parenchyma of surrounding tissue.

Fibrosis of burn-related wounds remains an unresolved clinical issue that leads to patient disability. The aim of this study was to assess the efficacy of the transplantation of adipose-derived stromal cells seeded onto a collagen-based matrix in the reconstruction of burn-related scars. Here, we characterized an in vitro interaction between adipose-derived stromal cells and a collagen-based matrix, Integra&reg, DRT. Our results show that transcription of pro-angiogenic, remodeling, and immunomodulatory factors was more significant in adipose-derived stromal cells than in fibroblasts. Transcription of metalloproteinases 2 and 9 is positively correlated with the collagenolytic activity of the adipose-derived stromal cells seeded onto Integra&reg, DRT. The increase in the enzymatic activity corresponds to the decrease in the elasticity of the whole construct. Finally, we validated the treatment of a post-excision wound using adipose-derived stromal cells and an Integra&reg, DRT construct in a 25-year-old woman suffering from burn-related scars. Scarless healing was observed in the area treated by adipose-derived stromal cells and the Integra&reg, DRT construct but not in the reference area where Integra&reg, DRT was applied without cells. This clinical observation may be explained by in vitro findings: Enhanced transcription of the vascular endothelial growth factor as well as remodeling of the collagen-based matrix decreased mechanical stress. Our experimental treatment demonstrated that the adipose-derived stromal cells seeded onto Integra&reg, DRT exhibit valuable properties that may improve post-excision wound healing and facilitate skin regeneration without scars.

Despite the significant progress that has been made in recent years in parathyroid imaging, improvements in surgical techniques and availability of surgical quality control based on intraoperative parathyroid hormone levels (PTH) assay, approximately 1-5% of patients undergoing surgery have state of persistent hyperparathyroidism. The most common causes of persistent hyperparathyroidism are: limited surgical experience, a failure to recognize multiglandular parathyroid disease, ectopic parathyroid adenoma location, insufficient range of resection of diseased parathyroid glands, parathyroid capsule tearing leading to parathyromathosis, as well as parathyroid cancer. In this clinical observation the case of a 52-years old man is described who underwent surgical removal of 2 parathyroid adenomas, and within few days he was found to have persistent hypercalcemia. After completing the diagnostic imaging and biochemical work-up that patient underwent bilateral neck re-exploration with removal of ectopic giant supernumerary parathyroid adenoma (60 mm in diameter and 22.8 g in weight) which was localized in the upper part of the posterior mediastinum, resulting in stable normocalcemia afterwards.

Purpose Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. Methods For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. Results Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro—one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. Conclusions The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.

Risk of recurrence after surgical treatment of a recurrent fistula is up to 50%. It has be known that more aggressive surgical treatment is associated with a high risk of anal sphincter damage and leads to incontinence. Several studies have been designed to elaborate minimally invasive treatment of rectovaginal and anal fistulas. The properties of Adipose-derived Stem Cells (ASC) significantly enhance a natural healing potency. Here, we present our experience with combined surgical and cell therapy in the treatment of fistulas. Materials and Methods: Four patients were enrolled in our study after unsuccessful treatments in the past – patients 1-3 with rectovaginal fistulas including two women after graciloplasty, and patient 4 - a male with complex perianal fistula. Adipose tissue was obtained from subcutaneous tissue. ASCs were isolated, cultured up to 10+/-2 mln cells and injected into the walls of fistulas. Follow-up physical examination and anoscopy were performed at 1, 4, 8, and 12 weeks, 6 and 12 months after implantation. Results: Up to 8 weeks after ASC implantation, symptoms of fistulas’ tracts disappeared. At 8 weeks, in patients 1-3, communication between vaginal and rectal openings was closed and at 12-16 w. intestinal continuity was restored in patient 3 and 4. After a 6-month follow-up, the fistula tract of patient 4 was closed. Up to 12 m. after ASC implantation no recurrences or adverse events were observed. Conclusion: ASCs combined with surgical pre-treated fistula tracts were used in four patients. All of them were healed. This encouraging result needs further trials to evaluate the clinical efficiency and the cost-effectiveness ratio.

Perianal fistulas are still a major challenge in everyday clinical practice. Particularly difficult fistulas, like those in Crohn's Disease, often require multiple aggressive surgical interventions, with high risk of sphincter damage and incontinence. Intensive trials have been conducted for over 10 years on the treatment of difficult fistulas with mesenchymal stem cells (MSC). This paper presents a short review of basic mechanisms of mesenchymal stem cells in healing of damaged tissues and results of major clinical trials. MSC in perianal fistulas, regardless of research protocol has a 70% healing rate in recurrent fistulas and in fistulas in Crohn's Disease. Further research is needed to precisely define the indications for MSC-based treatment of tissue damage, and particularly perianal and recto-vaginal fistulas.

The behavior, secondary structure, and orientation of a recently discovered bacteriocin-like peptide BacSp222 in a lipid model system supported at a gold electrode was investigated by chronocoulometry, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and attenuated total reflectance infrared (ATR-IR) spectroscopy. The IR spectra show that the secondary structure of BacSp222 is predominantly α-helical. Analysis of the spectra in the amide I region shows that the α-helical fragment of the peptide is inserted into bilayer at the potential range at which the bilayer is stable and attached to the Au(111) surface, i.e., from -0.5 to 0.3 V vs Ag/AgCl. Insertion of BacSp222 to the membrane significantly changes the conformation of the acyl chains of lipid molecules, from all-trans to partially melted; however, the chains become less tilted. Based on these results, we propose that BacSp222 interacts with the DMPC bilayer through the barrel-stave pore formation. In this model, α-helix of BacSp222 inserts into the membrane with an angle between the α-helix axis and membrane normal equal to ∼18°. The changes in orientation of the α-helical fragment of the peptide indicate that the orientation of BacSp222 with respect to the bilayer surface is potential-dependent. The peptide is inserted into the membrane driven by the electrostatic field generated by negative charge at the metal surface. It is not inserted at negative potentials where the membrane is detached from the metal and no longer exposed to the electrostatic field of the metal.

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.

Insulin is an amyloid-forming polypeptide built of two disulfide-linked chains (A and B), both themselves amyloidogenic. An interesting property of insulin is that agitation strongly influences the course of its aggregation, resulting in characteristic chiral superstructures of amyloid fibrils. Here, we investigate the self-assembly of these superstructures by comparing the quiescent and vortex-assisted aggregation of insulin and its individual A and B chains in the presence or absence of reducing agent tris(2-carboxyethyl)phosphine (TCEP). Our study shows that only the B chain in the presence of TCEP is converted into aggregates with morphology (according to atomic force microscopy) and optical activity (manifested as an extrinsic Cotton effect induced in bound thioflavin T) characteristic of amyloid superstructures that are normally formed by insulin in the absence of TCEP. In contrast to more rigid B-peptide fibrils, elongated aggregates of the A peptide become amorphous upon agitation. Moreover, the aggregation of equimolar mixture of both peptides does not produce highly ordered entities. Our results suggest that the dynamics of the B chain are the driving force for the assembly of superstructures, with the A chain being complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin.

Proteases are recognized for their role in the emergence of highly aggregation-prone protein fragments in vivo. On the other hand, limited proteolysis in vitro is often used to probe different phases of amyloidogenic pathways. Here we show that moderate amounts of pepsin induce “explosive” fibrillation in acidified samples of bovine insulin. Biochemical analysis of the pepsin-induced fibrils reveals previously unreported two-chain peptide with potent amyloidogenic properties as the main building block. The peptide (named ‘H’) comprises of N-terminal fragments of insulin A- and B-chains linked by disulfide bond between Cys7A-Cys7B and conceals up to 8 additional pepsin-cleavage sites which become protected upon fast fibrillation unless concentration of the enzyme is increased leading to complete digestion of insulin. Fibrils built of H-peptides are similar in terms of morphology (as probed by AFM) and infrared features to typical bovine insulin fibrils, but they appear to lack the ability to seed fibrillation of intact insulin. Controlled re-association of these fragments leads to ‘explosive’ fibrillation only under non-reducing conditions implying the key role of the disulfide bonds in the amyloidogenicity of H-peptides.Our study highlights the role of dynamics of the disulfide-bonded N-terminal fragments of A- and B-chains in insulin amyloidogenesis.