Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.     

Physicochemical and Structural Properties of Recovered Elastin from Jumbo Squid (Dosidicus gigas) By-Products

Physicochemical and structural properties of soluble jumbo squid (Dosidicus gigas) elastin recovered from skin by-products were evaluated. The molecular weight of isolated elastin was ~40 kDa with an isoelectric point (pI) between 9 and 10. Aspartic, glutamic, arginine, proline, glycine, and lysine amino acids were the most abundant in squid elastin, whereas the hydroxyproline absence, ~0.7% cysteine content, and the calculated 0.35 isoleucine/leucine ratio were used as purity index. Total and reactive sulfhydryl contents were similar (247.0 ± 5.1 vs 242.0 ± 7.5 μmol mg^?1 of protein, P ≥ 0.05) in purified squid elastin but surprisingly higher than previously reported in other elastins. On the other hand, the secondary structures of squid elastin analyzed by Fourier transform infrared spectroscopy (FTIR) were ~45% β-sheets, ~15% α-helices, ~10% β-turns, and ~30% undefined structures. In addition, squid elastin experienced glass transition at 82.01 ± 0.01ºC, denaturation temperature at 110.45 ± 0.64ºC, and aggregation at 197.5 ± 0.23ºC. In conclusion, the prevalence of charged amino acids and pI of squid elastin can facilitate its solubilization in hydrophilic systems, whereas the secondary structure profile and thermostability are desirable features in proteins used for biopolymer designs such as food biofilms or barrier systems.     

A Study of Combining Elastin in the Chitosan Electrospinning to Increase the Mechanical Strength and Bioactivity

While electrospun chitosan membranes modified to retain nanofibrous morphology have shown promise for use in guided bone regeneration applications in in vitro and in vivo studies, their mechanical tear strengths are lower than commercial collagen membranes. Elastin, a natural component of the extracellular matrix, is a protein with extensive elastic property. This work examined the incorporation of elastin into electrospun chitosan membranes to improve their mechanical tear strengths and to further mimic the native extracellular composition for guided bone regeneration (GBR) applications. In this work, hydrolyzed elastin (ES12, Elastin Products Company, USA) was added to a chitosan spinning solution from 0 to 4 wt% of chitosan. The chitosan–elastin (CE) membranes were examined for fiber morphology using SEM, hydrophobicity using water contact angle measurements, the mechanical tear strength under simulated surgical tacking, and compositions using Fourier-transform infrared spectroscopy (FTIR) and post-spinning protein extraction. In vitro experiments were conducted to evaluate the degradation in a lysozyme solution based on the mass loss and growth of fibroblastic cells. Chitosan membranes with elastin showed significantly thicker fiber diameters, lower water contact angles, up to 33% faster degradation rates, and up to seven times higher mechanical strengths than the chitosan membrane. The FTIR spectra showed stronger amide peaks at 1535 cm⁻¹ and 1655 cm⁻¹ in membranes with higher concentrated elastin, indicating the incorporation of elastin into electrospun fibers. The bicinchoninic acid (BCA) assay demonstrated an increase in protein concentration in proportion to the amount of elastin added to the CE membranes. In addition, all the CE membranes showed in vitro biocompatibility with the fibroblasts.     

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.     

Aortopulmonary fistula in a Warmblood mare associated with an aortic aneurysm and supravalvular aortic stenosis

This case report describes the clinical presentation, the necropsy findings, and genetic results of a 13-year-old Warmblood mare presented with colic and a bilaterally loud, holosystolic murmur. Echocardiographic examination revealed the presence of a thoracic aortic aneurysm, an aortic pseudoaneurysm, a periaortic hematoma (circumferential cuffing by perivascular hemorrhage), and aortopulmonary fistulation. A supravalvular aortic stenosis (SVAS) was visible during echocardiography. Necropsy confirmed that the thoracic aortic aneurysm had ruptured and connected to the pseudoaneurysm, which fistulated into the pulmonary artery. Histologically, the aneurysm wall revealed chronic lesions such as fibrosis, mucin depositions, mineralizations, and elastin fragmentation. The mid abdominal aorta showed lesions suggestive of a systemic elastin arteriopathy. Molecular analysis, however, could not attribute this disease to a variant in the elastin gene, the most common causative gene for SVAS. To the authors' knowledge, this case report describes a case of aortopulmonary fistulation in a Warmblood horse associated with the presence of SVAS and an aortic aneurysm.     

牛乳源性金黄色葡萄球菌nEBPS蛋白表达鉴定及多克隆抗体制备

利用PCR方法扩增乳源致病性金黄色葡萄球菌nEBPS全基因,将其定向克隆至原核表达载体pET30a（＋）中,鉴定正确后,转化BL21表达菌,经IPTG诱导获得了以可溶性表达的重组蛋白。通过NiNTAPurificationSystem纯化重组蛋白,纯化蛋白质量浓度为2．16g／L。纯化蛋白经免疫印迹检测显示重组蛋白能够被牛源金黄色葡萄球菌阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,间接EI,1SA测定抗体效价为1：25600,凝集试验测定抗体效价为1：128。