Biomedical Chemistry: Research and Methods

Temperature Dependence of Collateral Activity of Thermostable Recombinant CRISPR Nucleases Cas12b Obtained by One-Step Purification with Metal-Chelate Chromatography after Heterologous Expression

Wed, 01 Oct 2025 00:00:00 +0000

Thermostable CRISPR/Cas nucleases are considered as promising enzymes for development of next-generation DNA diagnostics by coupling loop-mediated isothermal amplification of nucleic acids (LAMP) with selective CRISPR/Cas detection of specific amplicons. In this paper, we present the results of testing the collateral activity of CRISPR nuclease AapCas12b and three variants of CRISPR nuclease BrCas12b (wild type and two mutants) obtained using simplified purification in the typical temperature range of LAMP - from 56°C to 72°C. It was shown that the use of one-step metal-chelate chromatography by excluding a stage of enzymatic removal of N-terminal sequences translated together with the target protein allows for obtaining recombinant CRISPR nucleases BrCas12b with a sufficiently high level of collateral activity. Temperature dependences of collateral activity differed among the studied BrCas12b variants. The obtained results can be useful in selecting thermostable CRISPR nucleases Cas12b for development of test systems based on a combination of LAMP and CRISPR/Cas detection.

The Effect of the Matricellular Protein Tenascin-c on the Functional Activity of Fibroblasts in an Experimental in Vitro Injury Model

Wed, 01 Oct 2025 00:00:00 +0000

Wound healing is a complex, multistep process involving sequential phases of inflammation, proliferation, and remodeling. Tenascin-C (TNC) is a matricellular protein actively involved in tissue regeneration. It is upregulated in response to tissue injury and plays an important role in the regulation of cell adhesion, migration, proliferation, and extracellular matrix protein synthesis. At the same time, during the early stages of wound healing, interleukin-1α (IL1α) exerts a significant effect as an alarmin that initiates inflammatory activation of fibroblasts. The objective of this study was to determine the optimal concentration of TNC stimulating both the migratory and synthetic activity of human dermal fibroblasts in vitro, including under conditions of preliminary inflammatory activation with IL1α. To this end, a comparative analysis of cell migration and proliferation was conducted, along with measurement of type I collagen synthesis using DF-1 human fibroblast cultures pre-incubated with IL1α (50 ng/mL) for 24 h, followed by the addition of recombinant TNC at concentrations of 0.05 μg/ml, 0.2 μg/ml, and 1 μg/mL. TNC exhibited a dose-dependent effect on fibroblasts: at a concentration of 0.2 μg/ml it stimulates cell migration and proliferation, accompanied by a statistically significant increase in type I collagen synthesis compared with the control. However, this level was lower than that observed at 1 μg/mL TNC, where a marked increase in collagen production was detected. Under conditions of IL1α pre-stimulation, the effects of TNC were amplified, particularly at concentrations of 0.2 μg/ml and 1 μg/ml, indicating the potential of TNC as a regulator of cellular activity within an inflammatory microenvironment. Higher concentrations did not further increase the effect. These findings may relevant in the context of to the development of biomaterials and therapeutic agents aimed at accelerating cutaneous wound healing by modulating cellular activity, which is especially relevant for the treatment of chronic or non-healing wounds.

Association of NADPH-Oxidase 2 (NOX2) with the Development of Atopic Asthma

Wed, 01 Oct 2025 00:00:00 +0000

Phagocytic cells destroy bacteria, allergens, and apoptotic cells via antimicrobial reactions coupled with phagocytosis, including generation of reactive oxygen species (ROS) and delivery of hydrolytic enzymes from lysosomes to the phagosome. ROS produced by NADPH oxidase 2 (NOX2), are involved in the mechanism of LC3-associated phagocytosis; they participate in the development of innate and adaptive immune responses, leading to airway remodeling and hyperreactivity. The aim of this study was to investigate an association between NOX2 and the severity of atopic bronchial asthma (ABA). The level of ROS was determined in monocytes of healthy donors and patients with atopic bronchial asthma using dihydrorhodamine 123 (DHR-123). The expression level of NOX2 gene in monocytes of healthy donors and patients with atopic bronchial asthma was analyzed by RT-PCR. In the presence of PMA (phorbol 12-myristate 13-acetate), there was a significant increase in rhodamine-123 (Rho-123) fluorescence in all studied groups. At the same time, a significant increase in the median of Rho-123 fluorescence was found in the group with severe ABA as compared to healthy donors and patients with mild AAA, as well as to the group of patients with moderate ABA. A significant increase in NOX2 gene expression was found in the groups with moderate and severe ABA compared to the other studied groups. Based on the data obtained, it can be assumed that with an increase in the activity of NOX2, the key enzyme of LC3-associated phagocytosis, there is an aggravation of the course of ABA.

Changes in Biochemical Parameters in Acute Pyelonephritis in Different Trimesters of Pregnancy Before and After Treatment

I.M. Kholimenko, A.A. Konoplja, O.N. Bushmina, E.N. Konoplja, A.Ju. Kravcov — Wed, 01 Oct 2025 00:00:00 +0000

Gestational pyelonephritis is characterized by an annual increase in morbidity and complications, which negatively affect the course of pregnancy and childbirth in 3-17% of women in Russia. The discrepancy within the normally balanced oxidant-antioxidant system underlies the pathogenesis of many diseases of pregnant women, with a significant role atributed to oxidative stress and systemic inflammatory response. Biochemical laboratory parameters of systemic inflammation, oxidant-antioxidant system and endothelial dysfunction in peripheral blood plasma and urine were studied.The study included 115 women (mean age 25.1±4.3 years), divided into groups: comparison - 20 healthy women, 20 nonpregnant patients with a verified diagnosis of acute serous pyelonephritis; main groups of 15 women each: three groups with acute pyelonephritis in the 1st, 2nd and 3rd trimesters of pregnancy before and after basic therapy and two groups with acute pyelonephritis in the 2nd and 3rd trimesters of gestation with the inclusion of Viferon in the basic treatment. At all periods of gestation in conditions of acute pyelonephritis, to a greater extent in the 2nd and 3rd trimesters, the development of systemic inflammation, oxidative stress and endothelial dysfunction was established, as evidenced by an increase in the content of stable metabolites of nitric oxide, acyl hydroperoxides, malonic dialdehyde, markers of systemic inflammatory response (C-reactive protein and neopterin) in the plasma of peripheral blood and urine, a decrease in antioxidant defense factors and the level of endothelin-1. Incomplete relief of metabolic disorders by basic therapy was revealed, especially in the late stages of gestation. The inclusion of Viferon in traditional therapy of acute pyelonephritis in the 2nd and 3rd trimesters was more effective of pregnancy.

Metal-Organic Framework Structures in Modern Research: Medicine, Diagnostics, Ecology

Yu.V. Tumanov, P.P. Gladyshev, A.A. Sergeev, A.V. Zaykovskaya — Wed, 01 Oct 2025 00:00:00 +0000

The review presents modern technological developments of means to indicate viruses and toxins using new nanomaterials based on frame structures. The synthesis and functionalization of metal-organic compounds of a frame structure (MOCs) and covalent organic frameworks (COF) are considered as well as the latest achievements in biomedical fields, including the delivery of drugs, nucleic acids, proteins and dyes for cancer therapy, bioimaging, antimicrobial drugs, biosensors and biocatalysis. New trends and promising areas in the development of biomedical materials based on MOC/COF are discussed. Data on the application of new biotechnological products based on simeconductor nanocrystals (quantum dots) and their composites as part of MOCs in solving the problems of modern disease diagnostics that play a strategic role in the development of nanotechnology, biotechnology and nanomedicine are presented. Issues related to the recognition of biomolecules using hybrid MOC/COF structures are discussed. The use of QD nanocomposites with other carbon-based, grapheme-based or MOC-based nanomaterials resulted in the development of new systems for bioimaging, drug delivery, optogenetics and theranostics. Undoubtedly, the rapidly accumulating data on the behavior of QD/MOC in analytical systems in vitro will increase knowledge for the advancement of QD nanotechnology in research in vivo and clinical application.