Biomedical Chemistry: Research and Methods

Lamp Primer Designing Software: The Overview

2024-06-06T09:34:10+00:00

The loop-mediated isothermal amplification (LAMP) of DNA is considered as the most promising method for DNA diagnostics of infectious diseases in a “point-of-care testing” format. Currently, the online services and downloadable programs have been developed for design of LAMP primers, some of them (freely available) are overviewed here. Among them, the PrimerExplorer software appears to be mostly used, followed by the NEB LAMP Primer Design Tool that is steadily gaining popularity. The other non-commercial programs such as GLAPD, MorphoCatcher, LAMPrimer iQ, and LAVA have been utilized for LAMP primer design much more rarely. The advantages and disadvantages of the currently existing free software for the construction of LAMP primers, alongside with directions for its further development, are discussed.

The Phospholipid System with the Targeted Peptide Angiopep-2 for Delivery of Chlorine e6 an in vitro Study

2024-07-31T20:28:05+00:00

The previously obtained phospholipid nanosystem for the delivery of the photosensitizer chlorine e6 was modified with a targeting ligand, the oligopeptide angiopep-2, exhibiting a high ability to transcytose through the blood-brain barrier. This feature of angiopep-2 is especially relevant for the targeted delivery of therapeutic and diagnostic agents to pathological area (tumor) of the brain. According to the analysis of the physico-chemical parameters of the developed composition, the particle size was 31.98±1.98 nm (PdI 0.453±0.03), the ζ-potential corresponded to -27.43±1.14 mV, while the substance was almost completely (98.6±0.43%) incorporated into nanoparticles. An in vitro experiment on the human glioblastoma cell line U-87 MG showed an increase in the total accumulation and internalization of chlorine e6 in the variant with the phospholipid form containing the targeted peptide compared with the free substance by 33% and 40%, respectively. The study of the cytotoxic action without irradiation showed no differences between the samples in the concentration range from 0.125 μg/ml to 2.5 μg/ml (in terms of to chlorine e6); the percentage of living cells was about 100%. The study of the photoinduced activity (with a dose 1,5 J/cm² irradiation) showed that the IC₅₀ value for the obtained composition was 1,33 times lower than that for the free substance and amounted to 2.85±0.1 μg/ml. The results of the experiments suggest the prospects of the developed composition and the clear need for further studies in vitro and in vivo.

Modeling the Blood-brain Barrier Using Rat Brain Cell Cultures

2024-07-04T16:43:21+00:00

Permeability of the blood-brain barrier (BBB) represents a significant problem for most promising drugs used for treating brain diseases due to its high selectivity. The neurovascular unit, which includes neurons, interneurons, astrocytes, the basal membrane, smooth muscle cells, pericytes, endothelial cells, and the extracellular matrix, forms an anatomically and functionally cohesive structure that ensures effective regulation of cerebral blood flow. In vitro modeling of the BBB is a relevant and practically significant task for studying the penetration of therapeutic agents into the brain. This study presents a BBB model consisting of endothelial cells, pericytes, and astrocytes, which partially mimics the in vivo layers of the BBB. Despite some limitations, such as incomplete matching of astrocyte location, the model demonstrates high expression of tight junction proteins and optimal TEER values of the endothelial cell monolayer, making it suitable for studying the permeability of the BBB to various substances, including drugs and nanoparticles.

Comparison of the Results of SPR Analysis of Antigen-antibody Interactions Performed Using Optical Biosensors Biacore X-100 (“Cytiva”, USA) and MI-S200D (“Inter-Bio”, China)

2024-09-09T12:33:06+00:00

Limited availability of Western scientific equipment, explains a growing need to switch to using scientific instruments made in China. This is especially true in the case of optical biosensors operating on the surface plasmon resonance (SPR) effect. However, comparability of experimental data obtained using Western and Chinese biosensors has not been investigated yet. In this work we have comparedresults of SPR analysis of the kinetics and affinity of interaction of IgG2a and IgG1 antibodies with protein A. Two biosensos Biacore X-100 (“Cytiva”, USA) and MI-S200D (“Inter-Bio”, China) have been used. It was shown that the values of the association rate constants obtained on both devices for two antibodies were close within one order of magnitude. For IgG1 antibodies, the dissociation rate constants were almost identical. Both devices provide high-quality data that are well described by a simple 1:1 model (Langmuir binding).

Obtaining and Properties Study of Human Granulocyte-macrophage Colony-stimulating Factor in a Vehicle for Drug Delivery

2024-07-23T11:31:23+00:00

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that stimulates the growth and development of granulocyte and macrophage progenitor cells. It also increases the activity of mature neutrophils, monocytes, and eosinophils. The preparations of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) are used to treat the diseases associated with myelosuppression, such as neutropenia of different etiologies, chronic infections, etc. One problem in the widespread use of GM-CSF is its instability in biological fluids, which leads to a short half-life and reduced biological activity. To solve this problem, a method has been developed to incorporate rhGM-CSF into a drug delivery vehicle based on polyglucin. It has been found that the conjugation of rhGM-CSF and polyglucin enhances the resistance of the protein against degradation by trypsin. The dosage form of rhGM-CSF incorporated into the drug delivery vehicle increased the number of segmented neutrophils in CBA mice with cyclophosphamide-induced myelosuppression to a similar extent as the initial rhGM-CSF preparation (300% and 350%, respectively, compared with the control level, 5 days after cyclophosphamide administration). Additionally, it contributed to a more rapid recovery of the total number of bone marrow karyocytes. Thus, a preparation containing a conjugate of rhGM-CSF and polyglucin exerts a more significant effect on activating bone marrow myelopoiesis than the unmodified protein.

β1-adrenergic Receptor within Nanodiscs of 10-16 nm in Diameter Retains Ligand-binding Properties

2024-09-27T12:07:31+00:00

The detection of autoantibodies against the β1-adrenergic receptor (ADRB1 Ab) in the blood of patients and the monitoring of the levels of these antibodies is an urgent need in clinical practice. The solid-phase enzyme-linked immunosorbent assay (ELISA), using ADRB1 in native conformation as antigen, seems to be the most suitable for this task. We have previously tested various amphipathic polymers for their ability to solubilize ADRB1 in the form of nanodiscs so that ADRB1 retains its antigenic properties. The aim of the present work was to investigate the ligand binding properties of ADRB1 in nanodiscs prepared with amphipathic polymers such as Ultrasolute^TM Amphipol (UA17) and AASTY 11-45 and to determine the size of the nanodiscs by dynamic light scattering. The binding of the ligands isoproterenol (agonist) and cyanopindolol (antagonist) was assessed by their ability to compete with recombinant hAB2367 antibodies specific for the second extracellular loop of ADRB1 in ELISA. It was found that ADRB1 solubilized with UA17 and AASTY 11-45 retained its ligand-binding properties. This fact supports the assumption that ADRB1 retains its native structure in nanodiscs. The size of nanodiscs prepared with UA17 was determined for the first time by dynamic light scattering. In the range of polymer concentrations from 0.0625% to 0.5%, no significant differences were observed in the size of the nanodiscs, which varied between 10 and 16 nm.

Interaction of Mouse and Sheep Polyclonal Antibodies with the Main Forms of Human and Rat Renalase

2024-10-01T13:46:23+00:00

The interaction of sheep and mouse polyclonal antirenalase antibodies obtained by immunization with full-length human (RNLS1-human) and rat (RNLS2-rat) renalases, respectively, has been studied. The target recombinant proteins, RNLS1-human and RNLS2-rat, were expressed in E. coli cells and isolated by Ni-agarose chromatography. Sheep polyclonal antibodies against RNLS1-human interacted more effectively with both RNLS1-human than with RNLS2-rat. Mouse polyclonal antibodies against RNLS2-rat effectively interacted mainly with RNLS2-rat, but not with RNLS1-human. The data obtained indicate the preferential selectivity of the antibody interaction with the proteins against which they were obtained. This should be taken into consideration in the case of selection of commercially available antibody preparations for quantitative immunodetection of target proteins in biological objects.

Hepatoprotective Effect of Xymedon and its Conjugate with L-ascorbic Acid during Poisoning of Mice with a Semilethal Dose of Paracetamol

2024-10-21T11:04:52+00:00

There is currently an urgent need to develop effective therapies aimed at preventing the consequences of drug-induced liver injury (DILI). In this context, our study focused on assessing the hepatoprotective effect of pyrimidine derivatives of xymedon (1,2-dihydro-4,6-dimethyl-1-(2- hydroxyethyl)-pyrimidin-2-one) and its conjugate with L-ascorbic acid in a mouse model of paracetamol (acetaminophen) poisoning. ICR (CD-1) mice were treated with a single oral LD₅₀ dose of paracetamol (425 mg/kg), followed by intraperitoneal injections of various doses of the test compounds at various doses half an hour later. The animals` survival was monitored for 5 days. On the day 6, biochemical and histopathological evaluations of the effects of the test compounds were conducted. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were analyzed. Malondialdehyde (MDA) levels in blood and liver tissue were also determined. Xymedon attenuated the toxic effects of paracetamol on the liver, improving animal survival and enhancing the macro- and micro-structure of the liver, normalizing the biochemical indicator of liver condition ALT and the serum marker of oxidative stress MDA. The conjugate of xymedon with L-ascorbic acid exhibited a more pronounced hepatoprotective effect compared to the native xymedon molecule.

Alignment and Normalization of Mass Spectrometry Data Using the Hydrophobicity Index

2024-08-18T06:56:18+00:00

This paper presents a program for the alignment of data from mass spectrometry experiments by retention time on a chromatographic column. The program uses the experimentally obtained data set as a reference against which the alignment procedure is performed. The primary advantage of this approach consists in its capacity to align data sets that had significant variations in both peptide composition and substance amount, such as individual fractions derived from multivariate separation. To illustrate this, two datasets were employed. The first dataset contains data obtained after multivariate separation, while the second dataset exhibited comparable peptide composition across all samples. The second dataset was used to assess the efficacy of the alignment program in normalizing signal intensity between individual samples. The results were compared with the normalization results obtained by the Progenesis LC-MS program. The normalization multipliers obtained for 22 of the 24 samples exhibited good correlation with those calculated by the Progenesis LC-MS (R² = 0.68). The program is freely available at http://lpcit.ibmc.msk.ru/AlignRT.