Collagenolytic Enzymes Research Articles

A tissue-silicone integrated simulator for right ventricular pulsatile circulation with severe functional tricuspid regurgitation

There is a great demand for development of a functional tricuspid regurgitation (FTR) model for accelerating development and preclinical study of tricuspid interventional repair devices. This study aimed to develop a severe FTR model by creating a tissue-silicone integrated right ventricular pulsatile circulatory simulator. The simulator incorporates the porcine tricuspid annulus, valve leaflets, chordae tendineae, papillary muscles, and right ventricular wall as one continuous piece of tissue, thereby preserving essential anatomical relationships of the tricuspid valve (TV) complex. We dilated the TV annulus with collagenolytic enzymes under applying stepwise dilation, and successfully achieved a severe FTR model with a regurgitant volume of 45 ± 9 mL/beat and a flow jet area of 15.8 ± 2.3 cm2 (n = 6). Compared to a normal model, the severe FTR model exhibited a larger annular circumference (133.1 ± 8.2 mm vs. 115.7 ± 5.5 mm; p = 0.009) and lower coaptation height (6.6 ± 1.0 mm vs. 17.7 ± 1.3 mm; p = 0.003). Following the De-Vega annular augmentation procedure to the severe FTR model, a significant reduction in regurgitant volume and flow jet area were observed. This severe FTR model may open new avenues for the development and evaluation of transcatheter TV devices.

(119) Impact of Ethnicity on Clinical Outcomes of Intralesional CCH in Peyronie's Disease Patients Across Kaiser Permanente Southern California Database

Abstract Introduction Intralesional CCH injection is an effective treatment for Peyronie’s disease (PD) that utilizes collagenolytic enzymes to break down collagen plaque, thus reducing the penile deformity. The IMPRESS I and II clinical trials demonstrated efficacy of CCH in a predominant Caucasian population of men (95.8%). Little is known about treatment efficacy in the non-Caucasian population. Objective Using Kaiser Permanente’s ethnically diverse database, we sought to study the impact of race on CCH treatment outcomes. Methods This study examined the efficacy of intralesional CCH injection in a large healthcare system over 15 years. We defined treatment efficacy as percent change in penile curvature abnormality from baseline after a maximum of 4 treatment cycles. Impact of comorbidities on CCH efficacy was also examined. Patients with ventral curvature or initial curvature <30 ̊ or >90 ̊ were excluded from analysis. Standard statistical analysis was performed. Results 112 patients underwent CCH therapy and were included in this analysis. 46.4% of men completed all 4 cycles. Among men who received CCH, 66.1% were Caucasian, 22.3% Hispanic, and 9.8% African American. For co-morbidities, 87.5.% of men were not diabetic, while 22.3% of men had erectile dysfunction. The mean baseline pre-injection penile curvature was 51.63 degrees (range was 32-87 degrees). Caucasian men had the largest treatment response to CCH (45.6% decrease in penile curvature) compared to African American men (43.7%) and Hispanic men (40.5%), p = 0.9554. Non-diabetic men had a larger treatment response to CCH (44.9% decrease in curvature versus 39.3% in diabetics, p = 0.4726). Younger Men (age 18-44) also experienced a greater treatment response compared to other age groups (57.9% reduction in curvature, p = 0.171). There was a significant difference in number of cycles completed across different ethnicities (p = 0.0220). Hispanic men were significantly less likely to complete all 4 cycles of CCH compared to Caucasian and African American men (p=0.0004). Only 28.2% of Hispanic men completed all 4 treatment cycles. 48% of Hispanic patients report the need of language interpretation services during health appointments. Conclusions This is the largest post-market analysis of CCH treatment outcomes in a racially diverse PD population. Caucasian men had a greater treatment response after CCH injection possibly due to higher rate of completing all 4 treatment cycles. Nondiabetic men and younger men (aged 18 to 44) also had a greater treatment response after CCH injection. Hispanic men were significantly less likely to complete 4 cycles of injection therapy; possible explanations include language barriers. Future study is needed to determine if patient resources prepared in Spanish can improve treatment outcomes in Hispanic men with PD, and reduce these health care disparities observed in our study. Disclosure No.

Targeting FGFR3 is a Useful Therapeutic Strategy for Rheumatoid Arthritis Treatment.

Rheumatoid arthritis (RA) is a systemic inflammatory disease in which TNF-α plays an important role. Fibroblast growth factor receptor 3 (FGFR3) is reportedly involved in RA by regulating the expression of inflammatory cytokines. This study examined the expression profile of FGFR3 in human synovial biopsy tissues and evaluated its gene-silencing effects on behaviors of synovial cells. Immunohistochemical staining was used to measure FGFR3 expression in human RA joint tissues. Cell proliferation, migration, and apoptosis assays were used to monitor behavioral changes in cultured synovial SW-982 cells with siRNA-mediated FGFR3 gene silencing. Immunofluorescent staining and western blotting were used to detect molecular changes in the FGFR3 gene-silenced cells. FGFR3 up-regulation was noted in both cytoplasms and nuclei of synovial cells in human RA joints. FGFR3 siRNA delivery experiments corroborated that FGFR3 knockdown decreased proliferation and migration, and triggered apoptosis of synovial cells. The FGFR3 gene knockdown enhanced constitutive expression of epithelial marker E-cadherin and conversely suppressed expression of epithelial-mesenchymal transition (EMT) markers, including Snail, fibronectin, and vimentin. In addition, FGFR3 silencing significantly reduced the constitutive expressions of TNF-α, transcription factor NF-κΒ, and downstream COX-2 protein and collagenolytic enzyme MMP-9. MAPK inhibition markedly suppressed constitutive levels of NF-κΒ, COX-2, and MMP-9. Genetic interference of FGFR3 could modulate the expression of inflammatory mediators and EMT markers in the synovial cells. Targeting the FGFR3/MAPK signal axis may be considered a useful therapeutic strategy to ameliorate the development of RA.

BIOCHEMICAL SIGNS OF THE COURSE OF REPARATIVE OSTEOGENESIS IN PATIENTS WITH FRACTURES OF THE LOWER EXTREMITY

Background: Some biochemical indicators in patients with disorders of reparative osteogenesis were described in a sufficient number of studies, but the role of factors affecting the metabolism of bone tissue and the ways of their correction are not sufficiently studied .. For effective prediction of the course of reparative osteogenesis in fractures, it is important to identify early markers of impaired consolidation and, accordingly, its pathogenetic correction. Purpose: to determine biochemical indicators of the course of reparative osteogenesis in patients with multiple and monolocal fractures of the bones of the lower limb. Materials and Methods: 44 patients aged 23 to 47 years were examined. To assess the ratio and intensity of the processes of biosynthesis and breakdown of collagen, as the major structural protein of connective tissue, the following indicators were determined in the blood serum of patients: fractions of hydroxyproline, oxyproline, collagenase, cathepsin B, elastase, proteolysis-antielastase inhibitors. In addition, indicators of mineral metabolism were also determined in the blood serum of patients: the content of calcium, phosphorus, alkaline phosphatase activity. Results: A comparative analysis and comparison of the results of the biochemical examination of patients with closed tibial fractures and multiple fractures revealed that already at the beginning of the research, deviations from the norm in the indicators of both mineral and collagen metabolism were observed. Both groups of the patients are characterized by the presence of disturbances in the ratio between calcium and phosphorus, and in patients with multiple fractures - an increase in the level of alkaline phosphatase. The detected changes in the mineral metabolism in patients with multiple fractures had a more pronounced and intense character due to the multiple injuries and presence of a large number of destroyed cells in the first days after the injury. Conclusions: Indicators of the unfavorable course of reparative osteogenesis in patients with bone fractures are a decrease in the content of calcium/phosphorus in blood serum, an increase in the level of free oxyproline and collagenolytic enzymes (collagenase, elastase, cathepsin B). Besides, there is a decrease in the level of proteolysis inhibitors (antielastase, 2-macroglobulin) and a decrease in suspension stability and antioxidant activity, increase in blood fibrinolytic activity. Our findings justify the need for prophylactic drugs that improve reparative osteogenesis soon after the injury (within a month) in patients with multiple fractures in order to activate the processes of reparative regeneration.

A47 THE GUT MICROBIOTA INFLUENCES COLONIC HEALING AFTER SURGERY IN PATIENTS UNDERGOING BOWEL RESECTION FOR COLORECTAL CANCER

Abstract Background The standard treatment of colorectal cancer (CRC) consists of a surgical resection of the colonic segment with the tumor, followed by a reconnection of the remaining bowel ends, or "anastomosis". The anastomosis may fail to heal in up to 20% of patients, which leads to anastomotic leak, a major complication that increases postoperative morbidity and mortality. This complication is unpredictable and its causes remain poorly understood. Purpose The objective of this study is to investigate the possible role of the gut microbiome in anastomotic healing after surgery in patients with CRC. Method We collected preoperative fecal samples and intraoperative mucosal samples from CRC patients undergoing surgery with anastomosis. The gut microbiota of patients with AL and of others that presented optimal healing after surgery was analyzed and compared using the Anchor 16S pipeline. To assess the role of the patients' microbiota in healing, fecal microbiota transplantation (FMT) was performed in mice using preoperative fecal samples from CRC patients with and without AL. Mice were then subjected to colonic surgery using a colonic anastomosis model. Six days after surgery, anastomotic healing was assessed macroscopically and microscopically. The gut barrier function was also assessed. The gut microbiota composition was compared between the groups colonized with samples from patients with and without AL to detect potential differences. Result(s) Mice colonized by FMT with the microbiota of donors with AL displayed poor anastomotic healing macroscopically, and a weaker wound microscopically. These same mice displayed a weaker gut barrier, as objectified by higher bacterial translocation to the spleen. The anastomoses of mice receiving the microbiota of AL donors displayed lower concentrations of collagen and fibronectin and higher inflammatory cytokines and collagenolytic enzymes, indicating poor extracellular matrix formation and collagen degradation locally.The beta-diversity of the gut microbiota was significantly different between mice receiving the microbiota of donors with and without AL, and several bacterial species were differentially abundant between the two groups. Conclusion(s) The preoperative gut microbiota in CRC patients who experience anastomotic leak after surgery induces poor anastomotic healing in mice and a weaker gut barrier after colonic surgery. Several bacterial species were found to be associated with the healing process. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; NSERC, FRQS, New Frontiers in Research, Montreal Cancer Institute. Disclosure of Interest None Declared

Choline and geranic acid (CAGE)ionic liquids inhibit both elastase activity and growth of oral bacteria.

Choline and geranic acid (CAGE) ionic liquids have recently been shown to have applications in the delivery of macromolecules and poorly soluble drugs across epithelial barriers and in bacterial growth inhibition. Ionic liquids are known to denature proteins by the disruption of forces that guide natural protein folding, and the inflammatory enzyme elastase was recently shown to be inhibited by a variety of ionic liquids other than CAGE. Inhibition of collagenolytic enzymes, including elastase, has been shown to improve outcomes in cases of periodontitis via amelioration of periodontal inflammation and alveolar bone resorption. In this study, we investigated whether CAGE prepared with varying stoichiometries was able to inhibit elastase at varying concentrations and whether these CAGE formulations could inhibit the growth of key pathogenic bacterial species associated with oral health conditions. We found that CAGE was capable of inhibiting both porcine elastase and human neutrophil elastase at concentrations as low as 5 mM, and that CAGE formulations were effective at inhibiting the growth of all tested pathogenic oral bacteria. The inhibition of elastase by CAGE may be a mechanism by which CAGE can improve outcomes in periodontitis independent from CAGE's known antibacterial properties.

Capabilities of Single Cell ICP-MS for the Analysis of Cell Suspensions from Solid Tissues

Single cell elemental (SC) analysis of isogenic cell cultures can be done using inductively coupled plasma (ICP-MS) detection. However, 2D cell cultures are just models to simplify the complexity of real tissue samples. Here, we show for the first time the capabilities of the technique (SC-ICP-MS) to analyze single cell suspensions of isolated cells from tissues. An optimized cocktail of proteolytic and collagenolytic enzymes was applied in a single preparation step with cellular yields up to 28% using 0.5 g of fresh rat spleen and liver, respectively. The retrieved cells revealed adequate morphology and stability to be examined by SC-ICP-MS. Quantitative elemental analysis of P, S, Cu, and Fe from disaggregated cells from rat spleen and liver tissues revealed levels of Fe of 7-16 fg/cell in the spleen and 8-12 fg/cell in the liver, while Cu was about 3-5 fg/cell in the spleen and 1.5-2.5 fg/cell in the liver. Evaluation of the transmembrane protein transferrin receptor 1 (TfR1) expression levels in disaggregated cells was also conducted by using a Nd-labelled antibody against this cell surface biomarker. Quantitative results showed significantly lower expression in the disaggregated cells than in the cell model HepG2, in agreement with the overexpression of this biomarker in tumor cells. In this proof of concept study, the tissue disaggregation protocol has shown to maintain the elemental intracellular content of cells as well as the presence of relevant antigens. This opens a completely new area of research for SC-ICP-MS in tissue samples as a complementary strategy with validation capabilities.

Degradation and Stabilization of Resin-Dentine Interfaces in Polymeric Dental Adhesives: An Updated Review

Instability of the dentine-resin interface is owed to the partial/incomplete penetration of the resin adhesives in the collagen fibrils. However, interfacial hydrolysis of the resin-matrix hybrid layer complex activates the collagenolytic and esterase enzymes that cause the degradation of the hybrid layer. Adequate hybridization is often prevented due to the water trapped between the interfibrillar spaces of the collagen network. Cyclic fatigue rupture and denaturation of the exposed collagen fibrils have been observed on repeated application of masticatory forces. To prevent interfacial microstructure, various approaches have been explored. Techniques that stabilize the resin–dentine bond have utilized endogenous proteases inhibitors, cross linking agents’ incorporation in the exposed collagen fibrils, an adhesive system free of water, and methods to increase the monomer penetration into the adhesives interface. Therefore, it is important to discover and analyze the causes of interfacial degradation and discover methods to stabilize the hybrid layer to execute new technique and materials. To achieve a predictable and durable adhesive resin, restoration is a solution to the many clinical problems arising due to microleakage, loss of integrity of the restoration, secondary caries, and postoperative sensitivity. To enhance the longevity of the resin-dentine bond strength, several experimental strategies have been carried out to improve the resistance to enzymatic degradation by inhibiting intrinsic collagenolytic activity. In addition, biomimetic remineralization research has advanced considerably to contemporary approaches of both intrafibrillar and extrafibrillar remineralization of dental hard tissues. Thus, in the presence of biomimetic analog complete remineralization of collagen, fibers are identified.

Anti-inflammatory activity of quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside isolated from Delonix elata against Freund’s complete adjuvant induced inflammation in rats

Delonix elata L. has been used for the treatment of pain and joint stiffness in the traditional medicines of Chitradurga District, Karnataka, India. In the present study, the anti-inflammatory activity of quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside (QRPG) isolated from D. elata stem bark extract was carried out against Freund’s complete adjuvant (FCA) induced inflammation in rats. The anti-inflammatory activity was evaluated by various methods such as radiographic analysis of hind paws, measurement of paw volume, joint diameter assessment, erythrocyte sedimentation rate, serum nitrites concentration, myeloperoxidase activity, the activity of lysosome enzymes (acid phosphatase, β-glucuronidases and collagenolytic enzymes) and histological observations. Indomethacin was used as the standard drug. QRPG showed significant attenuation in paw oedema of FCA-induced rats. Biochemical analysis in QRPG treated animals revealed a significant reduction in the levels of erythrocyte sedimentation rate, serum nitrites concentration, myeloperoxidase activity, lysosome enzyme activities as compared to control animals. Results obtained in biochemical assays were supported by the histological observations. The present investigation demonstrated promising anti-inflammatory activity of quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside and it could be the principal compound in D. elata stem bark extract. Thus, this study provides the scientific basis for the ethnomedicinal uses of D. elata for joint problems.

MMP Inhibitors and Dentin Bonding: Systematic Review and Meta-Analysis.

Objectives Resin-dentin bond strength decreases over time. This reduction is related to the loss of hybrid layer integrity. Collagenolytic enzymes, especially matrix metalloproteinases (MMPs), are responsible for the degradation of the collagen matrix of the hybrid layer. Various MMP inhibitors with the ability to prevent enzymatic degradation have been identified. This study aimed to systematically review the literature for studies which evaluated the effect of MMP inhibitors on the immediate and aged dentin bond strengths. Study SelectionScreening and analysis were carried out by two reviewers. Two databases were searched, and from a total of 740 articles, 43 were accepted for full review. 21 articles with 0.2%–2% chlorhexidine (CHX) treatments were included for meta-analysis. A risk of bias assessment was performed on all studies chosen for meta-analysis. A variety of MMP inhibitors have been studied, CHX being the most widely used. Conclusions A clear trend for a lower loss of dentin bond strength was observed with different MMP inhibitors. In meta-analysis, no significant difference was seen between the CHX and control in the immediate bond strengths. Bond strengths in the CHX group were significantly higher than the control group after aging (P < 0.001). The percentage of fractures occurring at the adhesive interface increased after aging. Five out of 21 studies included in the meta-analysis had high and the rest medium risk of bias. More long-term studies with lower risks of bias should be carried out to increase the reliability of results. Clinical RelevanceThe use of MMP inhibition with chlorhexidine can be recommended to increase the longevity of resin-dentin bond strength.

EXTRACTION OF COLLAGENOLYTIC ENZYME FROM FISH VISCERA BY PHASE PARTITIONING (PEG/CITRATE) AND ITS POTENTIAL FOR INDUSTRIAL APPLICATION

Internal viscera fish are potential sources of protein biomolecules of biopharmaceutical interest. However, this residue is frequently discarded inappropriately. The possibility to obtain by products of higher added value is a reality. Inside this view attention must be given to processes for the recovery and extraction of target molecules. However, the high cost of processing these residues is one of the obstacles to their reuse; techniques that facilitate their handling and make the process cheaper are desirable, such as extraction in a two-phase aqueous system. Thus, the aim of this study was to extract collagenolytic enzymes from common snook (Centropomus undecimalis) using a two-phase aqueous system (polyethylene glycol/citrate), according to the 24 factorial design, using as variables: molar mass of PEG (MPEG), PEG concentration (CPEG), citrate concentration (CCIT), pH, still, considering purification factor (FP), partition coefficient (K), and yield (Y). The collagenolytic activity of the crude extract was 102.41 U mg-1, after partitioning, was purified 3.91 times (MPEG: 8000; CPEG: 20.0%; CCIT: 20.0% and pH 6.0). Inhibition (U mg-1) was observed in benzamidine (22.51), TLCK (21.05), TPCK (21.29), and PMSF (23.05), signaling to be a serine-protease. The results showed the advantage of this semipurification technique as concerns to the low cost of extraction and purification, adding value to the fishing source material and allocating the residues from its processing to the industry.

A Novel Chemically-Modified Curcumin 2.24: Short-Term Systemic Therapy for Natural Periodontitis in Dogs

Periodontitis, a destructive periodontal inflammatory disease, negatively impacts oral-health related quality of life. It's characterized by the generation of inflammatory mediators and the excess-production of collagenolytic tissue-destructive enzymes (especially matrix metalloproteinases, MMPs). Many biomarkers can be used to define/diagnose disease progression. However, there is still a critical lack of specific, fast, and reliable biomarkers that correlate well with early response to treatment, which can be used to predict/monitor disease. Here, we report that an early marker, MMP-9, was found to be sensitive in response to a 1-month systemic therapy of CMC2.24, a novel chemically-modified curcumin, in beagle dogs with naturally-occurring periodontitis. In brief, eight adult female dogs with generalized periodontitis were distributed into placebo and treatment groups (n = 4/group). After a 1-h full-mouth scaling and root planing at time 0, placebo or CMC2.24 (10 mg/kg) capsules were orally-administered once/day for 1-month. Clinical periodontal parameters were measured at time 0 and 1-month; in addition, peripheral blood samples from these dogs were collected and analyzed for the pro-, activated-, and total-forms of MMP-9 by gelatin zymography. Interestingly, we found that the 1-month systemic therapy of CMC2.24 did appear to significantly reduce both pro- and activated-MMP-9 in peripheral blood at this early stage compared to placebo, prior to apparent clinical improvements seen at a later stage in a previous study (3-months). Thus, MMP-9 may serve as an early/sensitive biomarker that can precede/predict future clinical changes in disease severity and response to treatment which we observed in the long-term study in this dog model of natural periodontitis.

Utilisation of collagenolytic enzymes from sierra fish (Scomberomorus sierra) and jumbo squid (Dosidicus gigas) viscera to generate bioactive collagen hydrolysates from jumbo squid muscle.

Crude extracts of collagenases from jumbo squid (Dosidicus gigas) hepatopancreas and sierra fish (Scomberomorus sierra) viscera were used to hydrolyse squid muscle collagen into peptides with inhibitory capacity over angiotensin I-converting enzyme (ACE) and ABTS free radicals [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)], as a measure of their antihypertensive potential and antioxidant activity, respectively. Proteins from 20 to 200kDa were found in both enzyme extracts; however, in comparison to the jumbo squid extract (JSE), the extraction yield and specific activity of the enzymatic sierra fish extract (SFE) were ≈ 40% greater, suggesting the presence of enzymes with different collagenolytic activity. Moreover, the utilised collagen was obtained with a yield of 0.98 ± 0.09g/100g muscle from jumbo squid arms, which after an incubation with JSE and SFE generated peptides with different biological activity. However, the collagen hydrolysates from the enzymatic SFE contained a higher proportion of low-molecular-weight peptides than that obtained from JSE (15.2 and 7.9% of < 3kDa peptides, respectively). Finally, the antioxidant potential and ACE-inhibitory activity were increased after hydrolysis, being the SFE the one that showed a greater increase of both biological activities (82.28% of ACE inhibition and 64% of ABTS inhibition).

Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis

The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60°C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH5.0 and 50°C, and pH5.0 and 60°C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH6.0 and 6.5 for 24h at 4°C, and sensitive to pepstatin A, β-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Recombinant Nidogen-1 Significantly Improves Survival of Hypoxic Human Islets

Daniel Brandhorst,1,2 Heide Brandhorst,1,2 Samuel Acreman,1,2 Yukari Kimura,1,2 Shannon Layland,3 Katja Schenke-Layland,3 Paul R.V. Johnson1,2&#x0D; 1 Research Group for Islet Transplantation, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 2 Oxford Consortium for Islet Transplantation, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 3 Natural and Medical Sciences Institute, Eberhard Karls University, Tübingen, Germany&#x0D; Aim&#x0D; Islet isolation essentially requires dissociation of the islet basement membrane by collagenolytic enzymes. Basement membrane loss is associated with reduced islet function and viability. Previous studies demonstrated that individual extracellular matrix proteins (ECMPs) can increase islet survival pre- and post-transplant. In the present study, we tested our hypothesis, that the combination of different ECMPs, particularly those forming suprastructures, are more efficient than individual ECMPs to protect human islets from hypoxia-induced damage. In contrast to previous studies, we dissolved ECMPs in the media rather than to coat culture surfaces.&#x0D; Methods&#x0D; Islets, isolated from pancreases of 11 human DBD donors (50±2 years, 29.3±1.2 BMI, 5.7±0.3 hours CIT), were cultured for 3–4 days in 2% oxygen and suspended in CMRL 1066 (2% FCS) supplemented with either 40 µg/mL of dissolved collagen-IV, 10 µg/mL laminin-521 or 12.5 µg/mL nidogen-1 used individually or as combination. Sham-treated islets (STIs) cultured without ECMPs served as controls. Post-culture characterisation included IEQ yield or islet number (IN), viability (FDA-PI), early plus late apoptosis (annexin V-PI), glucose stimulation index (SI: 2 vs 20 mM) and reactive oxygen species production. Parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Statistical analysis was performed by Friedman test followed by Dunn’s multiple comparison.&#x0D; Results&#x0D; Compared with STI (41±7%), post-culture recovery was higher when hypoxic islets were treated with collagen-IV (64±7%, p&lt;0.001), laminin-521 (57±6%, p&lt;0.01) or nidogen-1 (65±6%, p&lt;0.001) used individually or combined (61±7% p&lt;0.001). This correlated with islet fragmentation (IN/IEQ ratio) that was lower when collagen-IV (116±13%, p&lt;0.001), laminin-521 (114±12%, p&lt;0.01), nidogen-1 (121±12%, p&lt;0.01) or combined ECMPs (119±13%, p&lt;0.001) were compared with STIs (155±16%). Reactive oxygen species production in STIs was substantially reduced by 71±6% (NS), 73±6% (p&lt;0.05), 90±2% (p&lt;0.001), and 87±4% (p&lt;0.001) in presence of collagen-IV, laminin-521, nidogen-1 or combined ECMPs, respectively. This resulted in improved viability (83±7% [p&lt;0.01], 79±9% [p&lt;0.01], 84±7% [p&lt;0.001], 83±8% [p&lt;0.001]) compared with STIs (63±7%). While individual ECMPs stabilised or reduced pre-culture apoptosis (94±17% [p&lt;0.05], 117±16% [p&lt;0.05], 68±13% [p&lt;0.001]), combined ECMPs (171±18%, NS) were equal to STIs (196±28%). STIs did not adequately secrete insulin after glucose challenge (SI 0.97±0.13) in contrast to the physiological insulin response after treatment with collagen-IV (1.76±1.18 [p&lt;0.01]), laminin-521 (1.53±0.25 [NS]), nidogen-1 (2.27±0.67 [p&lt;0.01]) or combined ECMPs (1.95±0.25 [p&lt;0.05]).&#x0D; Conclusion&#x0D; Among the three individual ECMPs tested, nidogen-1 appears to be most effective to protect human islets from hypoxia-induced damage. As its protective efficiency partially exceeds that of combined ECMPs, we have to reject our hypothesis. Further studies are required to clarify whether collagen-IV, laminin-521 and nidogen-1 spontaneously assemble to suprastructures in vitro.&#x0D;

Whole Porcine Pancreatic Extracellular Matrix Protects Isolated Human Islets from Ischemia-induced Damage

Daniel Brandhorst,1,2 Limor Baruch,3 Heide Brandhorst,1,2 Stasia Krishtul,3 Marcelle Machluf,3 Paul R.V. Johnson1,2&#x0D; 1Research Group for Islet Transplantation, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 2Oxford Consortium for Islet Transplantation, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 3Laboratory for Cancer Drug Delivery and Cell Based Technologies, Faculty of Biotechnology and Food Engineering, Technion, Israel Institute of Technology, Haifa, Israel&#x0D; Aim&#x0D; Islet isolation essentially requires dissociation of islet basement membranes by collagenolytic enzymes. This is associated with reduced islet function and increased cell death. Previous ex-vivo and in-vivo studies demonstrated that individual extracellular matrix proteins (ECMPs) can increase islet survival. As the natural ECM is a tissue-defined complex network we propose a novel concept for creating a specific islet matrix by using the whole pancreatic ECM (WPM). In contrast to previous studies, ECMPs were dissolved in media rather than coating of culture vessel surfaces.&#x0D; Methods&#x0D; Islets, isolated from pancreases of 6 human DBD donors (52±3 years, 28.5±1.5 BMI, 6.4±0.7 hours CIT), were cultured for 4–5 days in hypoxic atmosphere (2% oxygen). Islets were suspended in CMRL 1066 supplemented with 2% FCS and WPM-gel (200 µg/mL) extracted and purified from porcine pancreases. The WPM-gel was compared with a pre-tested combination of human ECMPs composed of 80 µg/mL collagen-IV, 10 µg/mL laminin-521, and 10 µg/mL nidogen-1. Sham-treated islets (STIs) cultured without ECMPs or WPM-gel served as controls. Post-culture characterisation included IEQ yield or islet number (IN), viability (FDA-PI), early plus late apoptosis (annexin V-PI), glucose stimulation index (SI: 2 vs 20 vs 2 mM) and reactive oxygen species production. All parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Statistical analysis was performed by Friedman test and Dunn’s multiple comparison.&#x0D; Results&#x0D; Post-culture recovery was highest when hypoxic human islets were cultured in WPM-gel and compared with STIs (65±10% vs 38±10%, p&lt;0.01). Although fragmentation (IN/IEQ ratio) increased after all treatments, this increase was lowest in the presence of WPM-gel (0.62±0.05 vs 0.80±0.14 vs 0.93±0.27, NS). Pre-culture viability was nearly completely preserved when human ECMPs (99±10% vs 79±10%, p&lt;0.01) or WPM-gel (92±8%, p&lt;0.05) were administered. Reactive oxygen species production in STIs increased nearly three-fold (127±15 AU/IEQ) but was halved in the presence of ECMPs (61±14 AU/IEQ, p&lt;0.01) or WPM-gel (65±18 AU/IEQ, p&lt;0.05). While initial early apoptosis remained stable when human islets were treated with human ECMPs (90±13% vs 136±14%, p&lt;0.01) or WPM-gel (84±10%, p&lt;0.01), apo-necrosis increased substantially in the presence of human ECMPs (172±33%, NS) or WPM-gel (154±195 vs 214±24%, p&lt;0.05). Glucose-stimulated islets did not respond adequately after sham-treatment (SI 0.85±0.14). In contrast, supplementation with human ECMPs (1.29±0.09, p&lt;0.05) or WPM-gel (1.34±0.09, p&lt;0.01) preserved the physiological insulin response during hypoxia. Overall survival, considering the recovery of viable cells only, was increased by human ECMPs (56±8% vs 34±8%, p&lt;0.01) or WPM-gel (58±8%, p&lt;0.01).&#x0D; Conclusion&#x0D; This initial study presents a new approach to protect human islets from hypoxia-induced damage by supplementing culture media with selected ECMPs or with the whole pancreatic ECM. We assume, that the outcome of our approach will be further improved when the ECM is extracted from human pancreases. These promising findings can be used to develop advanced culture media and innovative encapsulation techniques to protect transplanted islets.

First report on Chlorella vulgaris collagenase production and purification by aqueous two-phase system

Collagen is triple helical structured protein, which is undegradable by most proteases and reacts only with collagenases. These enzymes are applied and important in several commercial segments and the interest for new collagenases has only been increasing. Microalgae are not a very prominent source of enzymes but are extremely commercially attractive forms of life due to photosynthetic growth. Yet, the literature is absent on reports about algae collagenases and this work is the first report on Chlorella vulgaris collagenolytic enzyme production, purification and characterization. C. vulgaris was cultivated in autotrophic and mixotrophic conditions and the biomass used to obtain the cell extract. The autotrophic extract displayed a higher collagenolytic activity and was used for ATPS experimental design. Through 24-full factorial design, high collagenolytic activity was observed at pH 8.0, PEG molar mass of 1500 g/mol, PEG concentration of 12.5% (w/w) and phosphate buffer concentration of 15% (w/w). In these conditions, a purification factor of 10.79 was obtained for a partition coefficient of 23.74 with the collagenase retained in top PEG rich phase. Purified collagenase optimum activity was observed under pH 9 and 37 °C, displayed less gelatinolytic and caseinolytic activity, with higher collagenolytic activity with azocoll than crude extract, attesting this enzyme is a true collagenase. The collagenolytic activity was inhibited by EDTA, an indicative of a metallocollagenase.

Serum activity of MMP-2 and MMP-9 and stromielisin-1 concentration as predictors in the pathogenesis of bronchopulmonary dysplasia in preterm neonates

Aim: Bronchopulmonary dysplasia (BPD) is one of the most severe respiratory diseases, mainly related to premature neonates. Previous studies indicated the role of matrix metalloproteinases (MMPs) in the development of BPD. The aim of the study was to determine the relationship between MMP-2, MMP-3, MMP-9 with their tissue inhibitors (TIMP-1 TIMP-2) and BPD occurrence in premature neonates. Material/Methods: Eighty-one patients, divided into four study groups, numbered from 1 to 4, depending on gestational age (25–28; 29–32; 33–36; 37–40 weeks), were enrolled. Venous blood was collected between 5 and 7 days after birth. The activity of MMP-2 and MMP-9 were determined with usage of gelatin zymography, whereas MMP-3, TIMP-1 and TIMP-2 was determined using the immunoassay ELISA. Results: BPD was diagnosed in 50% of patients from group 1 and 11% from group 2. The increase of MMP-2 activity in Group 2, and a decrease in MMP-2/TIMP-2 ratio was noticed in Group 1 compared to Group 2 and 4. A significantly lower incidence of BPD in patients with higher (above the median) values for MMP-2/TIMP-2 (OR = 0.02, CI = 0.00 – 0.55; p &lt;0.05) was noticed in Group 1. The decreased occurrence of BPD in patients with higher MMP-3 concentration, higher MMP-9 activity and the higher value of MMP-9/TIMP-1 did not reach statistical significance. Conclusions: It has been shown that elevated activity of collagenolytic enzyme in serum, especially MMP-2, may have the effect of decreasing the risk of bronchopulmonary dysplasia in premature neonates.