PRODUCTION,PURIFICATION, AND CHEMICAL STABILITY OF RECOMBINANT HUMAN INTERFERON-γ IN LOW OXYGEN TENSION CONDITION: A FORMULATION APPROACH

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.     

Biomechanical and Structural Features of CS2 Fimbriae of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.     

The Role of Copper Oxide Nanomaterials on Ruta graveolens Physiological Response,and IRT1 and CAT Gene Expression

Background

Copper oxide nanomaterials’ (NMs) are important for the critical roles of Cu as a micronutrient that its improper concentration could cause toxicity or deficiency in plant. The Nano form of CuO could amplify the effects due to special characteristic of nano materials.

Method

Treatments of 0.1, 0.5, and 2.5 μM and NM of copper with three replications were applied to plants under hydroponic conditions. Physiological parameters and expression of IRT1 and CAT genes were investigated.

Results

Copper absorption decreased according to MMs-CuO ? NMs-CuO ?? CuSO4 pattern. The positive effects of MMs-CuO on plant copper content were higher than those of nanomaterials and CuSO4. MMs-CuO effect was more significant on plant biomass increase compared to the control. Rue plant needed lower amounts of copper for better plant growth. The treatments increased protein and carotenoids content in leaves compared with control. Changes in total chlorophyll content under three copper forms were very low and were only increased in leaves at 0.1 μM CuSO4. The NMs-CuO and MMs-CuO similarly reduced leaves’ Cu, MDA and ROS contents, and SOD activities. CAT enzyme activity had a similar pattern in three copper forms. CAT enzyme activity was only induced under the lowest level of three forms, while at other levels of Cu, it was reduced. NMs-CuO had a more negative effect on IRT1 relative gene expression in root compared with other iron forms. The IRT1 relative gene expression in shoots was positively affected under 2.5 μM CuSO4, 0.5 μM MMs-CuO, and 0.1 and 2.5 μM NMs CuO treatments.

Conclusion

The effect of micro- and nano-CuO on physiology and gene expression mechanisms in rue plants is shown to be does-dependent.

     

Heterologous expression,purification, and refolding of SRY protein: role of l-arginine as analyzed by simulation and practical study

Molecular Biology Reports - Escherichia coli is a widely-used cell factory for recombinant protein production, nevertheless, high amount of produced protein is seen in aggregated form. The purpose...     

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA.     

An updated overview of the application of CAR-T cell therapy in neurological diseases

Genetically modified immune cells, especially CAR-T cells, have captured the attention of scientists over the past 10 years. In the fight against cancer, these cells have a special place. Treatment for hematological cancers, autoimmune disorders, and cancers must include CAR-T cell therapy. Determining the therapeutic targets, side effects, and use of CAR-T cells in neurological disorders, including cancer and neurodegenerative diseases, is the goal of this study. Due to advancements in genetic engineering, CAR-T cells have become crucial in treating some neurological disorders. CAR-T cells have demonstrated a positive role in treating neurological cancers like Glioblastoma and Neuroblastoma due to their ability to cross the blood–brain barrier and use diverse targets. However, CAR-T cell therapy for MS diseases is being researched and could be a potential treatment option. This study aimed to access the most recent studies and scientific articles in the field of CAR-T cells in neurological diseases and/or disorders.     

Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos

ABSTRACT: BACKGROUND: Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications. RESULTS: We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes. CONCLUSIONS: PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.     

First-principles study of Carbz-PAHTDDT dye sensitizer and two Carbz-derived dyes for dye sensitized solar cells

Dimers of cytosine and its N¹-methylated counterpart were investigated in gas-phase and in various solvents including chloroform, dimethylsulfoxide, and water. The studies were performed at DFT/M06-2X/6-31+G(d,p) level of theory. Relative stabilities of tautomers of cytosine solvated explicitly by a small number of solvent molecules were evaluated. Further solvation effect calculations for homodimers were carried out with conductor-like polarizable continuum model (CPCM). H-NMR shifts and IR frequencies for optimized structures were calculated and compared with available experimental data.