Innovative Biosystems and Bioengineering

Designing a Multi-Epitope Vaccine Candidate to MERS-CoV: An in silico Approach

2024-01-14T14:20:03+02:00

Background. Middle East Respiratory Syndrome Coronavirus (MERS-CoV), associated with severe respiratory illness, originates from the Middle East region. The virus is transmitted from animals to humans, with the dromedary camel serving as a significant reservoir. The virus's high fatality rate has spurred research into vaccine development and therapeutics.

Objective. This study aimed to employ an in silico approach to design a potential vaccine candidate against MERS-CoV, focusing on the M protein as an antigen.

Methods. The FASTA sequence of M protein was used to predict B cell and major histocompatibility complex class I and class II epitopes. The best epitopes were selected from these predicted epitopes. The vaccine candidate's construct consisted of epitopes, linkers, and a tag. The sequence of the vaccine candidate's construct, consisting of 390 amino acids, was back-translated, optimized, and then inserted into a plasmid for cloning and expression using SnapGene. The 3D structure of the vaccine candidate is docked with TLR-4 receptor. Molecular dynamics simulation was run for this docked complex using GROMACS gmx, version 2021.4.

Results. Through computational modeling and analysis, we developed a novel vaccine candidate with promising structural and functional properties. Our results suggest that the designed vaccine candidate has the potential to induce a robust immune response.

Conclusions. This in silico approach presents a promising MERS-CoV vaccine candidate designed to trigger both humoral and cellular immune responses. This candidate holds the potential to provide broad-spectrum protection against MERS-CoV.

Antimicrobial Activity of Composite Preparations Based on Biosurfactants and Heterocyclic Amino-Containing 1,4-Naphtoquinone Derivativesantimicrobial Activity of Composite Preparations Based on Biosurfactants and Heterocyclic Amino-Containing 1,4-Naphtoquinone Derivatives

2024-09-16T12:42:49+03:00

Background. The development of highly effective and environmentally safe composite drugs with antimicrobial properties is an important challenge in biotechnology and pharmacy.

Objective. To determine the antimicrobial activity of new composite preparations based on rhamnolipids combined with heterocyclic amine-containing derivatives of 1,4-naphthoquinone against the test-bacteria Escherichia coli B-906, Staphylococcus aureus 209-P, Mycobacterium luteum B-917, and fungi Candida tenuis UCM Y-70 and Aspergillus niger UCM F-1119.

Methods. The in vitro antimicrobial activity of heterocyclic amine-containing derivatives of 1,4-naphthoquinone, biosurfactants, and composite preparations based on them was tested against cultures of Escherichia coli B-906, Staphylococcus aureus 209-P, Mycobacterium luteum B-917, and fungi Candida tenuis UCM Y-70 and Aspergillus niger UCM F-1119 using the agar diffusion method on solid nutrient media, as well as the serial dilution method (minimum inhibitory and bactericidal concentrations). The toxicity of the heterocyclic amine-containing derivatives of 1,4-naphthoquinone was predicted using in silico methodology via the ProTox-II software.

Results. It was found that 2-chloro-3-((1-methyl-1H-pyrazol-3-yl)amino)naphthalene-1,4-dione 3.2 and 2-chloro-3-((3-(n-tolyl)- 1H-pyrazol-5-yl) amino) naphthalene-1,4-dione 3.3 exhibit antimicrobial activity. Their composite preparations, due to the combination with rhamnolipids, demonstrated improved solubility and enhanced antimicrobial effects. Based on the results of the calculated assessment, the studied heterocyclic amine-containing derivatives of 1,4-naphthoquinone are predicted to fall into toxicity class IV.

Conclusions. The antimicrobial activity of 2-chloro-3-((1-methyl-1H-pyrazol-3-yl)amino)naphthalene-1,4-dione 3.2 and 2-chloro-3-(3-( 3-(p-tolyl)-1H-pyrazol-5-yl)amino)naphthalene-1,4-dione 3.3 is enhanced. This suggests the potential of the proposed biotechnological approaches in the development of new biocidal preparations.

Effect of Magnetic Field and Magnetic Nanoparticles on Choice of Endothelial Cell Phenotype

2023-12-07T15:44:02+02:00

Background. Endothelial cells as participants in angiogenesis choose their phenotype as tip cells (leading, migratory) or stalk cells (following). It has been experimentally found and theoretically modeled that rapid oscillations in intracellular calcium concentration play a key role in controlling phenotype selection and possible vessel architecture. In addition, the intracellular calcium concentration in endothelial cells is known to be regulated by mechanical wall shear stress induced by blood flow, which controls mechanosensitive calcium ion channel gating. Experimental methods of controlling mechanosensitive ion channel gating in external magnetic fields with application of magnetic nanoparticles are developed that affect magnetic nanoparticles artificially attached to cell membranes.

Objective. A key question is raised about the possibility of controlled selection of endothelial cell phenotype in external magnetic fields due to the presence of artificial or biogenic magnetic nanoparticles embedded in the cell membrane.

Methods. The magnetic wall shear stress is calculated due to the influence of the external magnetic field on the magnetic nanoparticles embedded in the cell membrane, which controls the mechanosensitive calcium ion pathways. Numerical modeling of oscillations in intracellular calcium concentration in endothelial cells and determination of their final phenotype was carried out taking into account intercellular communication. The python programming language and scipy, py-pde, matplotlib packages of the python programming language were used for numerical modeling.

Results. The magnetic field flux density and frequency ranges of a uniform rotating magnetic field, as well as the magnitude of the gradient and the frequency of a non-uniform oscillating magnetic field were calculated for controlling the amplitude and frequency of intracellular calcium concentration oscillations in endothelial cells, as well as the selection of their phenotype. It opens the perspective of controlling angiogenesis and vessel architecture.

Conclusions. Phenotype selection by endothelial cells can be controlled in a uniform rotating external magnetic field, as well as in a non-homogeneous oscillating magnetic field.

Fluorescence-Based Study of Oligonucleotide Interactions With Recombinant Proteins: Insulin, Interferon α2-β, Somatotropin, and Their Receptors

2024-05-16T16:25:09+03:00

Background. Oligonucleotides (OLNs) can participate in a wide range of protein-ligand interactions and perform numerous cellular functions by forming structures that enable specific interactions with DNA, RNA, and proteins, what is crucial for many biological processes. Advances in understanding these interactions could lead to the development of new technologies for treating various diseases. However, the mechanism of interaction between proteins and OLNs is complex and still requires detailed study. More research is needed to fully elucidate this process and enhance our understanding of these biomolecular interactions.

Objective. The aim of this study was to synthesize, purify, and investigate the interaction of OLNs with recombinant signaling proteins interferon α2-β and insulin with their receptors and somatropin by assessing binding strength using fluorescence spectroscopy.

Methods. The interactions were analyzed using the Stern–Volmer equation in both general and modified forms, as well as the Hill equation. OLNs were synthesized via the solid-phase phosphoramidite method, purified through solid-phase extraction, and subsequently verified with a spectrophotometer.

Results. Fluorometric titration revealed that OLNs bind to proteins within the medium affinity range, forming non-fluorescent complexes, with the most active interactions observed with shorter OLN. Positive cooperative binding of interferon to G20 and T20, and negative cooperative binding of insulin to C20 and A20, were identified. Additionally, negative cooperative binding of somatropin to C20 was observed.

Conclusions. The study demonstrated the interaction between OLNs and recombinant signaling proteins and receptors through various binding mechanisms, which could potentially affect their conformation and biological activity. These findings have implications for the therapeutic use of OLNs in the context of signaling proteins and receptors.

Selective-Integrative Technology for the Separation of Colostrum Into Components and the Possibilities of Obtaining Protein Substances From Different Sources

2024-03-01T14:48:10+02:00

Background. Obtaining biologically active natural compounds involved in the regulation of metabolism is an important goal in biotechnology. Colostrum is a unique natural source of various biologically active compounds. However, the extremely high natural variability of colostrum composition does not meet the existing requirements for standardization in pharmaceutical preparations.

Objective. To develop a method for separating colostrum into its basic components (lipids, casein, and protein fractions), thereby reducing the variability of whole colostrum composition, obtaining several target products, and demonstrating the possibility of acquiring new protein substances from different sources.

Methods. Colostrum separation was carried out through centrifugation and membrane filtration. Plant proteins (sunflower) and milk proteins were used to obtain protein substances from different sources. The composition of proteins, carbohydrates, and nucleic acids was determined using mass spectrometry, centrifugation, and membrane filtration.

Results. The proposed method for obtaining basic substances from colostrum significantly reduced the variability in composition compared to whole colostrum. The efficiency of protein sedimentation in concentrated protein solutions by centrifugation and ultrafiltration was shown to depend on protein concentration. Additionally, the formation of non-specific protein aggregates in the centrifugal field allowed the extraction of protein substances from various natural sources, which is relevant for functional nutrition.

Conclusions. The proposed selective-integrative technology for obtaining different substances from colostrum significantly reduces the high variability of whole colostrum composition. It increases the efficiency of component separation into lipid, casein fractions, low molecular weight protein fractions, and ultrafiltrate, while also enabling the acquisition of protein substances from diverse sources.