Construction of pVAX-1-based linear covalently closed vector with improved transgene expression.

INTRODUCTION: This study presents a Mammalian Linear Expression System (MLES), a linear covalently closed (LCC) vector based on pVAX-1. The purpose of this system was to improve gene expression in mammalian cells and to test the efficacy of MLES in transient transfection and transgene expression using in vitro and in vivo models. Additionally, we aimed to evaluate potential inflammatory responses in vivo. MATERIALS AND METHODS: MLES was developed by modifying pVAX-1, and the construct was confirmed by gel electrophoresis. Lipofectamine®2000 was used to assess the transfection efficiency and expression of MLES in various cell lines. In vivo studies were conducted in mice injected with MLES/EGFP, and the resulting transfection efficiency, gene expression, and inflammatory responses were analyzed. RESULTS: MLES exhibited higher transfection efficiency and expression levels compared to pVAX-1 when tested on HEK-293, CHO-K1, and NIH-3T3 cells. When tested in vivo, MLES/EGFP showed elevated expression in the heart, kidney, liver, and spleen compared with pVAX-1/EGFP. Minimal changes are observed in the lungs. Additionally, MLES induced a reduced inflammatory response in mice compared with pVAX-1/EGFP. CONCLUSIONS: MLES offer improved transfection efficiency and reduced inflammation, representing a significant advancement in gene therapy and recombinant protein production. Further research on MLES-mediated gene expression and immune modulation will enhance gene therapy strategies.

Synthesis and Structural Characterization of Selenium Nanoparticles-Bacillus sp. MKUST-01 Exopolysaccharide (SeNPs-EPS) Conjugate for Biomedical Applications.

Exopolysaccharides (EPS) are exogenous microbial metabolites generated predominantly during the development of bacteria. They have several biological potentials, including antibacterial, antioxidant, and anticancer actions. Polysaccharide-coated nanoparticles have high biological activity and are used in treatments and diagnostics. In this research, selenium nanoparticles (SeNPs) are synthesized and conjugated with bacterial (Bacillus sp. MKUST-01) exopolysaccharide (EPS). Initially, the creation of SeNPs conjugates was verified through UV-Vis spectral examination, which exhibited a prominent peak at 264 nm. Additionally, X-ray diffraction (XRD) analysis further substantiated the existence of crystalline Se, as evidenced by a robust reflection at 29.78°. Another reflection observed at 23.76° indicated the presence of carbon originating from the EPS. Fourier transform infrared spectroscopy (FT-IR) analysis of the EPS capped with SeNPs displayed characteristic peaks at 3425 cm-1, 2926 cm-1, 1639 cm-1, and 1411 cm-1, corresponding to the presence of O-H, C-H, C=O, and COO-groups. The SeNPs themselves were found to possess elongated rod-shaped structures with lengths ranging from 250 to 550 nm and a diameter of less than 70 nm, as confirmed using scanning electron microscopy and particle size analysis. In contrast to the SeNPs, the SeNPs-EPS conjugates showed no hemolytic activity. The overall antioxidant activity of SeNPs-EPS conjugates outperformed 20% higher than SeNPs and EPS. Additionally, experimental observations involving gnotobiotic Artemia nauplii experiments were also recorded, such as the supplementation of EPS and SeNPs-EPS conjugates corresponding to enhanced growth and increased survival rates compared to Artemia nauplii fed with SeNPs and a microalgal diet.