Biotechnology and applied biochemistry最新文献

The emergence or reemergence of monkeypox (Mpox) and Ebola virus (EBOV) agents causing zoonotic diseases remains a huge threat to human health. Our study aimed at designing a multi-epitope vaccine (MEV) candidate to target both the Mpox and EBOV agents using immunoinformatics tools. Viral protein sequences were retrieved, and potential nonallergenic, nontoxic, and antigenic epitopes were obtained. Next, cytotoxic and helper T-cell (CTL and HTL, respectively) and B-cell (BCL) epitopes were predicted, and those potential epitopes were fused utilizing proper linkers. The in silico cloning and expression processes were implemented using Escherichia coli K12. The immune responses were prognosticated using the C-ImmSim server. The MEV construct (29.53 kDa) included four BCL, two CTL, and four HTL epitopes and adjuvant. The MEV traits were pertinent in terms of antigenicity, non-allergenicity, nontoxicity, physicochemical characters, and stability. The MEV candidate was also highly expressed in E. coli K12. The strong affinity of MEV-TLR3 was confirmed using molecular docking and molecular dynamics simulation analyses. Immune simulation analyses unraveled durable activation and responses of cellular and humoral arms alongside innate immune responses. The designed MEV candidate demonstrated appropriate traits and was promising in the prediction of immune responses against both Mpox and EBOV agents. Further experimental assessments of the MEV are required to verify its efficacy.

Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram-negative sepsis and COVID-19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin-antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome-wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery.

A new and innovative rolled graphene oxide (roll-GO)/poly-m-methylaniline (PmMA) core–shell nanocomposite has been successfully synthesized using an in situ polymerization technique. This eco-friendly and cost-effective material shows great promise due to its antimicrobial properties. The characterization of the nanocomposite involved X-ray diffraction and Fourier transform infrared spectroscopy to analyze its structure and functional groups, whereas scanning electron microscopy and transmission electron microscopy (TEM) were utilized to examine its morphology. TEM analysis revealed the formation of roll-GO, forming multi-walled tubes with inner and outer diameters of 50 and 70 nm, respectively. Optical analysis demonstrated an enhanced bandgap in the nanocomposite, with bandgap values of 2.38 eV for PmMA, 2.67 eV for roll-GO, and 1.65 eV for roll-GO/PmMA. The antibacterial efficacy of the nanocomposite was tested against Gram-positive bacteria, including Bacillus subtilis and Staphylococcus aureus, as well as Gram-negative bacteria such as Escherichia coli and Salmonella sp. The well diffusion method was used to determine the inhibition zones, revealing that the nanocomposite demonstrated broad-spectrum antibacterial activity against all the pathogens tested. The largest inhibition zones were observed for B. subtilis, followed by S. aureus, E. coli, and Salmonella sp. Notably, the inhibition zones increased when the samples were exposed to light compared to dark conditions, with increases of 33 and 18 mm noted for B. subtilis. This enhanced activity under light exposure is attributed to the photocatalytic properties of the nanocomposite. The antibacterial mechanism is based on both adsorption and degradation processes. Moreover, antibacterial activity was found to increase with increasing concentrations of nanoparticles, ranging from 100 to 500 ppm. This suggests that the nanocomposite has potential as an alternative to antibiotics, especially considering the growing issue of bacterial resistance. The promising results obtained from the inhibition zones make these nanocomposites suitable for various applications. Currently, the research team is working on the development of a prototype utilizing these antimicrobial particles within commercial bottles for sterilization purposes in factories and companies.

Mounting studies have shown that the oncoproteins E6 and E7 encoded by the human papillomavirus (HPV) genome are essential in HPV-induced cervical cancer (CC). Ca²⁺ binding protein 1 (CABP1), a downstream target of HPV18-positive HeLa cells that interferes with E6/E7 expression, was identified through screening the GEO Database (GSE6926). It was confirmed to be down-regulated in CC through TCGA prediction and in vitro detection. Subsequent in vitro experiments revealed that knocking down E6/E7 inhibited cell proliferation, migration, and invasion, whereas knocking down CABP1 promoted these processes. Simultaneously knocking down CABP1 reversed these effects. Additionally, the results were validated in vivo. Previous studies have indicated that CABP1 can regulate Ca²⁺ channels, influencing Ca²⁺ influx and tumor progression. In this study, it was observed that knocking down CABP1 enhanced Ca²⁺ inflow, as demonstrated by flow cytometry and confocal microscopy. Knocking down E6/E7 inhibited these processes, whereas simultaneously knocking down E6/E7 and CABP1 restored the inhibitory effect of knocking down E6/E7 on Ca²⁺ inflow. To further elucidate that E6/E7 promotes CC progression by inhibiting CABP1 expression and activating Ca²⁺ influx, BAPTA/AM treatment was administered during CABP1 knockdown. It was discovered that Ca²⁺ chelation could reverse the effect of CABP1 knockdown on CC cells. In conclusion, our results offer a novel target for the diagnosis and treatment of HPV-induced CC.

In this study, a new amperometric biosensor was developed for glucose determination. For this purpose, polyaniline–polypyrrole–poly(sodium-4-styrenesulfonate) film was prepared by electropolymerization of aniline and pyrrole with poly(sodium-4-styrenesulfonate) on a platinum plate. The best working conditions of the polyaniline–polypyrrole–poly(sodium-4-styrenesulfonate) film were determined. The glucose oxidase enzyme was immobilized by the entrapment method in polyaniline–polypyrrole–poly(sodium-4-styrenesulfonate) film. Glucose determination was made based on the oxidation of hydrogen peroxide, which is formed as a result of the enzymatic reaction on the surface of the prepared biosensor at +0.40 V. The working range for the glucose determination of the biosensor was determined. The effects of pH and temperature on the response of the glucose biosensor were investigated. The reusability and shelf life of the biosensor were determined. The effects of interference in biological environments on the response of the biosensor were investigated. Glucose determination was made in the biological fluid (blood) with the prepared biosensor. This study has a feature that sheds light on biosensor studies to be developed for the detection of substances in the human body, such as glucose, uric acid, and urea. This article will set an example for future scientific research on the development of a sensor for other biological fluids in the human body, such as the sensor developed for blood samples. In addition, this developed sensor provides an innovation that improves the quality of life of patients by allowing them to constantly monitor their glucose levels and intervene when necessary.

Triple-negative breast cancer (TNBC) has short survival rates. This study aimed to prepare a novel formula of sorafenib, carbon nanotubes (CNTs), and folic acid to be tested as a drug delivery system targeting versus TNBC compared with free sorafenib and to evaluate the formula stability, in vitro pharmacodynamic, and in vivo pharmacokinetic properties. The formula preparation was done by the synthesis of polyethylene glycol bis amine linker, CNT PEGylation, folic acid attachment, and sorafenib loading. The prepared formula has been characterized using X-ray diffraction, Flourier-transform infrared, ¹HNMR, UV, high resolution–transmission electron microscope, field emission scanning electron microscopy, and Zeta potential. In vitro studies included drug release determination, MTT assay, flow cytometry to determine the apoptotic stage with percent, cell cycle analysis, and apoptotic marker assays for caspase-3, 8, 9, cytochrome c, and BCL-2. The in vivo study was performed to determine bioavailability and half-life in rats. The in vitro MTT antiproliferative assay revealed that the formula was threefold more cytotoxic toward TNBC cells than free sorafenib, and the flow cytometry showed a significant increase in apoptosis and necrosis. The formula has a greater inhibitory effect on BCL-2 and a lessening effect on cytochrome c and caspases 3, 8, and 9 than free sorafenib. In vivo experiments proved that our novel formula was superior to free sorafenib by increasing bioavailability by eight times and prolonging the half-life by three times. These results confirmed the successful preparation of the desired formula with better pharmacodynamic and pharmacokinetic properties. These promising results may show a novel therapeutic strategy for TNBC patients.

Cardiotoxicity is the leading side effect of anthracycline-based chemotherapy. Therefore, it has gained importance to reveal chemotherapy-supporting strategies and reliable agents with their mechanisms of action. Tannic acid (TA), a naturally occurring plant polyphenol, has diverse physiological effects, including anti-inflammatory, anticarcinogenic, antioxidant, and radical scavenging properties. Therefore, this study was designed to investigate whether TA exerts a protective effect on mechanisms contributing to anthracycline-induced cardiotoxicity in rat heart tissues exposed to doxorubicin (DOX). Rats were randomly divided into control and experimental groups and treated with (18 mg/kg) DOX, TA (50 mg/kg), and DOX + TA during the 2 weeks. Cardiac gene markers and mitochondrial DNA (mtDNA) content were evaluated in the heart tissues of all animals. In addition to major metabolites, mRNA expression changes and biological activity responses of components of antioxidant metabolism were examined in the heart tissues of all animals. The expression of cardiac gene markers increased by DOX exposure was significantly reduced by TA treatment, whereas mtDNA content, which was decreased by DOX exposure, was significantly increased. TA also improved antioxidant metabolism members' gene expression and enzymatic activity, including glutathione peroxidase, glutathione s-transferase, superoxide dismutase, catalase, and thioredoxin reductase. This study provides a detailed overview of the current understanding of DOX-induced cardiotoxicity and preventive or curative measures involving TA.

Glycated proteins are generated by binding of glucose to the proteins in blood stream through a nonenzymatic reaction. Hemoglobin A1c (HbA1c) is a glycated protein with glucose at the N-terminal of β-chain. HbA1c is extensively used as an indicator for assessing the blood glucose concentration in diabetes patients. There are different conventional clinical methods for the detection of HbA1c. However, enzymatic detection method has newly obtained great attention for its high precision and cost-effectiveness. Today, fructosyl peptide oxidase (FPOX) plays a key role in the enzymatic measurement of HbA1c, and different companies have marketed HbA1c assay systems based on FPOX. Recent investigations show that FPOX could be used in assaying HbA1 without requiring HbA1c primary digestion. It could also be applied as a biosensor for HbA1c detection. In this review, we have discussed the recent improvements of FPOX properties, different methods of FPOX purification, solubility, and immobilization, and also the use of FPOX in HbA1c biosensors.

Heparinases, including heparinases I–III (HepI, HepII, and HepIII, respectively), are important tools for producing low-molecular-weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)₅ (R) and a flexible linker (GGGGS)₅ (F) to fuse maltose-binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL-c2X. Compared with their parental fusion protein, MBP-fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)₅ or (GGGGS)₅ all displayed enhanced thermostability (half-lives at 30°C: 242%–464%). MBP-fused HepIs and HepIIs exhibited higher specific activity (127%–324%), whereas MBP-fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP-fused HepIIs showed a significantly decreased affinity toward heparin with increased K_m values (397%–480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP-fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP-fused heparinases with suitable linker designs and their potential for the bioproduction of low-molecular-weight heparin.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial membrane that leads to the destruction of cartilage and bone. Currently, pharmacological targeting of ion channels is being increasingly recognized as an attractive and feasible strategy for the treatment of RA. The present work employs a network analysis approach to predict the most promising ion channel target for potential RA-treating drugs. A protein–protein interaction map was generated for 343 genes associated with inflammation in RA and ion channel genes using Search Tool for the Retrieval of Interacting Genes and visualized using Cytoscape. Based on the betweenness centrality and traffic values as key topological parameters, 17 hub nodes were identified, including FOS (9800.85), tumor necrosis factor (3654.60), TGFB1 (3305.75), and VEGFA (3052.88). The backbone network constructed with these 17 hub genes was intensely analyzed to identify the most promising ion channel target using network analyzer. Calcium permeating ion channels, especially store-operated calcium entry channels, and their associated regulatory proteins were found to highly interact with RA inflammatory hub genes. This significant ion channel target for RA identified by theoretical and statistical studies was further validated by a pilot case–control gene expression study. Experimental verification of the above findings in 75 RA cases and 25 controls showed increased ORAI1 expression. Thus, with a combination of network analysis approach and gene expression studies, we have explored potential targets for RA treatment.