An Overview on Application of Natural Substances Incorporated with Electrospun Nanofibrous Scaffolds to Development of Innovative Wound Dressings.

Conventional dressings are cost-effective and highly absorbent, but not effectual enough to promote hemostasis, adherence and in holding a moist wound bed. Thanks to the developments in the field of nanotechnology and bioengineering, one of the promising current trends is to move progress of innovative wound dressings, merging the application of traditional healing agents and modern products/ practices, such as hydrocolloids, hydrogels, films and nanofibers. This review surveys on potentials of electrospun nanofibrous mats for wound dressing applications. Furthermore, loading of bioactive molecules and therapeutic agents into the nanofibrous mats especially natural compounds with the aim of fabrication novel bioactive electrospun nanofibrous mats for skin substitutes and wound dressings are discussed. Systematic literature search was conducted to review all recent progress toward the potential of natural substances incorporated with electrospun nanofibrous scaffolds for wound dressing applications. The electrospun nanofibers webs can provide the essential parameters require for wound dressing to heal wounds including absorptivity, oxygen permeability, and non-adherence to the healing tissue, barrier to bacteria, bioactivity and occlusivity. The modern wound dressings materials made of electrospun nanofibers contain various traditional healing agents such as plant derived compounds could be beneficial to the healing of wounds. Natural substances have been used in skin wound care for many years because of their therapeutic properties, including antimicrobial, antioxidant, anti-inflammatory and mitogenic activities. A screening of natural substances with plant or animal sources having high wound healer activities and cooperating with electrospun nanofiber are an important step toward producing innovative bioactive wound dressings.

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering.

Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es) is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs) is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli) expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A) of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology.

BACKGROUND: Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes. OBJECTIVES: This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3). MATERIALS AND METHODS: The hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression. RESULTS: A maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P < 0.05). The expression of the hES gene was confirmed by western blot. CONCLUSIONS: The obtained results indicate that RSM is an effective method for the optimization of culture conditions for hES gene expression in E. coli.

Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System.

PURPOSE: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. METHODS: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. RESULTS: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. CONCLUSION: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space.

Expression and Secretion of Endostar Protein by Escherichia Coli: Optimization of Culture Conditions Using the Response Surface Methodology.

Endostar as a specific drug in treatment of the nonsmall cell lung cancer is produced using Escherichia coli expression system. Plackett-Burman design (PBD) and response surface methodology (RSM) are statistical tools for experimental design and optimization of biotechnological processes. This investigation aimed to predict and develop the optimal culture condition and its components for expression and secretion of endostar into the culture medium of E. coli. The synthetic endostar coding sequence was fused with PhoA signal peptide. The nine factors involved in the production of recombinant protein-postinduction temperature, cell density, rotation speed, postinduction time, concentration of glycerol, IPTG, peptone, glycine, and triton X-100-were evaluated using PBD. Four significant factors were selected based on PBD results for optimizing culture condition using RSM. Endostar was purified using cation exchange chromatography and size exclusion chromatography. The maximum level of endostar was obtained under the following condition: 13.57-h postinduction time, 0.76 % glycine, 0.7 % triton X-100, and 4.87 % glycerol. The predicted levels of endostar was significantly correlated with experimental levels (R 2 = 0.982, P = 0.00). The obtained results indicated that PBD and RSM are effective tools for optimization of culture condition and its components for endostar production in E. coli. The most important factors in the enhancement of the protein production are glycerol, glycine, and postinduction time.

Diagnostic values of GHSR DNA methylation pattern in breast cancer.

DNA methylation patterns have been recognised as cancer-specific markers with high potential for clinical applications. We aimed at identifying methylation variations that differentiate between breast cancers and other breast tissue entities to establish a signature for diagnosis. Candidate genomic loci were analysed in 117 fresh-frozen breast specimens, which included cancer, benign and normal breast tissues from patients as well as material from healthy individuals. A cancer-specific DNA methylation signature was identified by microarray analysis in a test set of samples (n = 52, p < 2.1 × 10(-4)) and its performance was assessed through bisulphite pyrosequencing in an independent validation set (n = 65, p < 1.9 × 10(-7)). The signature is associated with SFRP2 and GHSR genes, and exhibited significant hypermethylation in cancers. Normal-appearing breast tissues from cancer patients were also methylated at these loci but to a markedly lower extent. This occurrence of methylated DNA in normal breast tissue of cancer patients is indicative of an epigenetic field defect. Concerning diagnosis, receiver operating characteristic curves and the corresponding area under the curve (AUC) analysis demonstrated a very high sensitivity and specificity of 89.3 and 100 %, respectively, for the GHSR methylation pattern (AUC >0.99). To date, this represents the DNA methylation marker of the highest sensitivity and specificity for breast cancer diagnosis. Functionally, ectopic expression of GHSR in a cell line model reduced breast cancer cell invasion without affecting cell viability upon stimulation of cells with ghrelin. Our data suggest a link between epigenetic down-regulation of GHSR and breast cancer cell invasion.

Relationship between vitreous and serum vascular endothelial growth factor levels, control of diabetes and microalbuminuria in proliferative diabetic retinopathy.

BACKGROUND: Diabetic retinopathy is a serious microvascular disorder of the retina. Vascular endothelial growth factor (VEGF) expression, induced by high glucose levels and hypoxia, is a main feature in retinopathy. The aim of this study was to evaluate the relationship between vitreous and serum VEGF levels and control of diabetes and microalbuminuria in patients with proliferative diabetic retinopathy. METHODS: Sixty-five patients were enrolled in this case-control study, comprising 30 patients with proliferative diabetic retinopathy (cases) and 35 patients with nonproliferative diabetic retinopathy (controls). The vitreous VEGF level was compared with the serum VEGF level in both groups. Glycosylated hemoglobin (HbA(1c)), microalbuminuria, serum creatinine, and stage of nephropathy and retinopathy were also measured in patients with proliferative diabetic retinopathy, and the relationship between these parameters and serum and vitreous VEGF levels was investigated. RESULTS: Mean vitreous and serum VEGF levels were significantly higher in cases compared with controls (P = 0.001, P = 0.011, respectively). There was also a significant correlation between vitreous and serum VEGF levels (P = 0.012, r = 0.453). VEGF levels in patients with well controlled blood glucose (P = 0.039), on drug treatment (P = 0.045) and at an early stage of nephropathy (P = 0.042) were significantly lower. There was a significant correlation between VEGF and albumin to creatinine ratio (P = 0.017, r = 0.432). CONCLUSION: Serum and vitreous VEGF levels was significantly lower in patients on oral therapy, in those with well controlled glycemia, and in those with early-stage retinopathy. Administration of anti-VEGF had a good effect in reducing the progression of proliferative diabetic retinopathy.