Expression of a Novel Chimeric Truncated t-PA in CHO Cells Based on in Silico Experiments

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms

Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power

Abstract Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm

Antioxidant and cytotoxic effect of biologically synthesized selenium nanoparticles in comparison to selenium dioxide

The present study was designed to evaluate antioxidant and cytotoxic effect of selenium nanoparticles (Se NPs) biosynthesized by a newly isolated marine bacterial strain Bacillus sp. MSh-1. An organic–aqueous partitioning system was applied for purification of the biogenic Se NPs and the purified Se NPs were then investigated for antioxidant activity using DPPH scavenging activity and reducing power assay. Cytotoxic effect of the biogenic Se NPs and selenium dioxide (SeO2) on MCF-7 cell line was assesed by MTT assay. Tranmission electron micrograph (TEM) of the purified Se NPs showed individual and spherical nanostructure in size range of about 80–220 nm. The obtained results showed that, at the same concentration of 200 �g/mL, Se NPs and SeO2 represented scavenging activity of 23.1 ± 3.4% and 13.2 ± 3.1%, respectively. However, the data obtained from reducing power assay revealed higher electron-donating activity of SeO2 compared to Se NPs. Higher IC50 of the Se NPs (41.5 ± 0.9 �g/mL) compared to SeO2 (6.7 ± 0.8 �g/mL) confirmed lower cytotoxicity of the biogenic Se NPs on MCF-7 cell line

Licence plate recognition using Viola-Jones method

This paper contains description of licence plate's recognition algorithm using Viola-Jones method. The results of the work program implementing the method of Viola-Jones C{++} OpenCV library are performed

COVID-19 related stigma among the general population in Iran

Funding Information: GT is supported by the National Institute for Health Research (NIHR) Applied Research Collaboration South London at King’s College London NHS Foundation Trust, and by the NIHR Asset Global Health Unit award. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. GT is also supported by the Guy’s and St Thomas’ Charity for the On Trac project (EFT151101), and by the UK Medical Research Council (UKRI) in relation to the Emilia (MR/S001255/1) and Indigo Partnership (MR/R023697/1) awards. Publisher Copyright: © 2022, The Author(s).Peer reviewedPublisher PD

Selection of Mother Wavelet Function for Multi-Channel EEG Signals Analysis during a Working Memory Task

We performed a comparative study to select the efficient mother wavelet (MWT) basis functions that optimally represent the signal characteristics of the electrical activity of the human brain during a working memory (WM) task recorded through electro-encephalography (EEG). Nineteen EEG electrodes were placed on the scalp following the 10–20 system. These electrodes were then grouped into five recording regions corresponding to the scalp area of the cerebral cortex. Sixty-second WM task data were recorded from ten control subjects. Forty-five MWT basis functions from orthogonal families were investigated. These functions included Daubechies (db1–db20), Symlets (sym1–sym20), and Coiflets (coif1–coif5). Using ANOVA, we determined the MWT basis functions with the most significant differences in the ability of the five scalp regions to maximize their cross-correlation with the EEG signals. The best results were obtained using “sym9” across the five scalp regions. Therefore, the most compatible MWT with the EEG signals should be selected to achieve wavelet denoising, decomposition, reconstruction, and sub-band feature extraction. This study provides a reference of the selection of efficient MWT basis functions

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field