Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium

A Self-Organized ECM-Mimetic Model Based on an Amphiphilic Multiblock Silk-Elastin-Like co-Recombinamer with a Concomitant Dual Physical Gelation Process

Although significant progress has been made in the area of injectable hydrogels for biomedical applications and model cell niches, further improvements are still needed, especially in terms of mechanical performance, stability, and biomimicry of the native fibrillar architecture found in the extracellular matrix (ECM). This work focuses on the design and production of a silk-elastin-based injectable multiblock corecombinamer that spontaneously forms a stable physical nanofibrillar hydrogel under physiological conditions. That differs from previously reported silk-elastin-like polymers on a major content and predominance of the elastin-like part, as well as a more complex structure and behavior of such a part of the molecule, which is aimed to obtain well-defined hydrogels. Rheological and DSC experiments showed that this system displays a coordinated and concomitant dual gelation mechanism. In a first stage, a rapid, thermally driven gelation of the corecombinamer solution takes place once the system reaches body temperature due to the thermal responsiveness of the elastin-like (EL) parts and the amphiphilic multiblock design of the corecombinamer. A bridged micellar structure is the dominant microscopic feature of this stage, as demonstrated by AFM and TEM. Completion of the initial stage triggers the second, which is comprised of a stabilization, reinforcement, and microstructuring of the gel. FTIR analysis shows that these events involve the formation of β-sheets around the silk motifs. The emergence of such β-sheet structures leads to the spontaneous self-organization of the gel into the final fibrous structure. Despite the absence of biological cues, here we set the basis of the minimal structure that is able to display such a set of physical properties and undergo microscopic transformation from a solution to a fibrous hydrogel. The results point to the potential of this system as a basis for the development of injectable fibrillar biomaterial platforms toward a fully functional, biomimetic, artificial extracellular matrix, and cell niches.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea a través del Fondo Europeo de Desarrollo Regional (ERDF), por el del MINECO (MAT2013-41723-R, MAT2013- 42473-R, PRI-PIBAR-2011-1403 y MAT2012-38043), la Junta de Castilla y León (VA049A11, VA152A12 y VA155A12) y el Instituto de Salud Carlos III bajo el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Crosstalk between GSK-3, c-Fos, NFκB and TNF-α signaling pathways play an ambitious role in Chitosan Nanoparticles Cancer Therapy

Nanotechnology is a promising era of medicine for developing targeted drug delivery system. Chitosan nanoparticles (CNPs) have attracted increasing attention for their wide applications as anticancer drugs. This article is concerned with the therapeutic index of chitosan nanoparticles against diethyl nitrosamine (DEN) induced hepatocellular carcinoma (HCC). HCC was induced in rats via repeated DEN administration in a dose of 200 mg/kg BW IP, 2 weeks later rats received (2 ml/kg BW) CCl4 orally for 2 months followed by daily treatment with chitosan nanoparticles in an oral dose of 12 mg/kg for 1 month. Then the gene expression of glycogen synthase kinase-3 (GSK-3), (c-FOS), nuclear factor kappa-B (NFκB) and tumor necrosis factor- α (TNF-α) were reported in rats sera and the correlation between GSK-3, C-Fos, NFƘB and TNF-α and liver tumorigenesis was investigated. The results elucidated that DEN significantly increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Marked increments in serum malondialdehyde (MDA) and nitric oxide (NOx) levels along with a slight reduction of glutathione (GSH) level were evidenced in HCC. Liver injury triggered an inflammatory response by enhancing the mRNA gene expression of NFκB and TNF-α. DEN effectively activated apoptotic markers GSK-3 and c-FOS. Oral administration of CNPs alleviated the oxidative, inflammatory and apoptotic hazards induced via DEN. The histopathological examination reinforced these results. The present study highlights the anti-inflammatory and anti-apoptotic potentials of CNPs against DEN-induced HCC. Keywords: Chitosan nanoparticles, HCC, Inflammatory markers, Apoptotic marker

Anti-Termitic Activity of Three Plant Extracts, Chlorpyrifos, and a Bioagent Compound (Protecto) against Termite Microcerotermes eugnathus Silvestri (Blattodea: Termitidae) in Egypt

A trend towards environmentally friendly chemicals for use in termite management has been occurring globally. This study examined three naturally occurring plant extracts from Lavandula latifolia (Spike lavender), Origanum vulgare (Marjorum), and Syzygium aromaticum (Clove) against the termite Microcerotermes eugnathus. Plant extract results were compared to two commercially used termite pesticides, the bio-insecticide, Bacillus thuringiensis var. kurstaki (Protecto 9.4% WP) and Dursban (Chlorpyrifos 48%). Gas chromatography–mass spectrometry (GC-MS) analysis was used to identify the main compounds in the three plant extracts. The main compounds in Lavandula Latifolia were linalool (21.49%), lavandulol (12.77%), β-terpinyl acetate (10.49%), and camphor (9.30%). Origanum vulgare extract contained thymol (14.64%), m-cymene (10.63%), linalool (6.75%), and terpinen-4-ol (6.92%) as main compounds. Syzygium aromaticum contained eugenol (99.16%) as the most abundant identified compound. The extract of O. vulgare caused the highest termite death rate, with an LC50 of 770.67 mg/L. Exposure to lavender extract showed a high death rate with an LC50 of 1086.39 mg/L. Clove extract did not show significant insecticidal activity with an LC50 > 2000 mg/L. Significant termiticide effects were found, with LC50 values of 84.09 and 269.98 mg/L for soldiers and workers under the application of Dursban and Protecto, respectively. The LC50 values reported for nymphs were <120, <164.5, and 627.87 mg/L after exposure to Dursban, Protecto, and O. vulgare extract, respectively. The results of the study show that some of the extracts have low toxicity compared to the bioagent and Dursban, and may show promise as natural termiticides, particularly as extracts from O. vulgare

GC–MS, quantum mechanics calculation and the antifungal activity of river red gum essential oil when applied to four natural textiles

Abstract The most important uses of old fabrics include clothing, mummification, and bookbinding. However, because they are predominantly constructed of natural materials, they are particularly susceptible to physical and chemical deterioration brought on by fungi. The treatments that are typically used to preserve old textiles focus on the use of synthetic fungicides, which have the potential to be dangerous for both human health and the environment. Essential oils (EOs), which are safe for the environment and have no negative effects on human health, have been widely advocated as an alternative to conventional antifungals. Four natural fabrics—linen, cotton, wool, and silk—were utilized in the current work. The extracted EO from leaves of river red gum (Eucalyptus camaldulensis Dehnh.) were prepared at 125, 250, and 500 µL/L. Aspergillus flavus, Fusarium culmorum and Aspergillus niger were inoculated separately into the treated four fabrics with the EO at concentrations of 125, 250, and 500 µL/L or the main compounds (spathulenol and eucalyptol) at the concentrations of 6, 12, 25, and 50 µL/L and were then compared to the un-treated samples. GC–MS was used to analyze the EO chemical composition, while visual observations and scanning electron microscopic (SEM) were used to study the fungal growth inhibition. Spathulenol (26.56%), eucalyptol (14.91%), and p-cymene (12.40%) were the principal chemical components found in E. camaldulensis EO by GC–MS. Spathulenol molecule displayed the highest electrostatic potential (ESP) compared with the other primary compound, as calculated by quantum mechanics. In the untreated textile samples, SEM analysis revealed substantial proliferation of hyphae from A. flavus, F. culmorum, and A. niger. The fungal growth was completely inhibited at a concentration of 500 µL/L from the EO. Both eucalyptol and spathulenol completely inhibited the formation of the fungal spores at a concentration of 50 µL/L, although eucalyptol was more effective than spathulenol across the board for all four textiles. The results support E. camaldulensis EO functionalized textiles as an effective active antifungal agent