Biomedical Chemistry: Research and Methods

The Impact of Sequencing Depth on the Number of Transcript Splice Variants Revealed by MinION Nanopore Sequencing

2025-10-14T14:47:52+00:00

Alternative splicing (AS) of a precursor mRNA is a fundamental regulatory process implicated in physiology and pathology. The long-read RNA sequencing with a nanopore sequencer such as ONT MinION allows for direct AS profiling. In this study the impact of sequencing depth on the number of transcribed genes and the overall number of transcripts (splice variants), revealed by MinION-based sequencing, has been investigated. This is of importance in AS profiling for the issue of comparability for biospecimens analyzed in different MinION runs. The sequencing depth was described in terms of the output of high-quality mapped reads produced by a MinION sequencer. The human liver tissue samples and hepatocytederived cell lines HepG2 and Huh7 were employed as model objects. It has been found that the yield of detected genes and transcripts substantially depends on the sequencing depth. While the number of transcribed genes levelled off at about 12 thousand when the reads output exceeded 1.2 million, the number of revealed transcripts steadily increased up to about 20 thousand splice variants at the highest reads output of 2.3 million, achieved in the study. At that reads output, the ratio of the number of revealed transcripts to that of genes was slightly below 1.7. The yield of more than 2.3 million high-quality mapped reads would be required in the MinION-based nanopore sequencing to approach the level of 1.8 transcripts (splice variants) per gene, expected from the known numbers of annotated genes and transcripts for human genome. The sequencing data used were produced for human liver tissue and hepatocyte-derived cells and it is still to be seen whether the findings are general and valid for other types of cells and tissues.

Metabolic Profiling of 5xFAD Mice Hippocampal Interstitial Fluid after Cognitive Stimulation: a Pilot Study

2025-09-22T09:13:47+00:00

Alzheimer`s disease (AD) is increasingly viewed as a condition associated with disruptions in brain energy metabolism, contributing to synaptic and cognitive impairments. While metabolic disturbances in AD are well characterized under resting conditions, much less is known about the hippocampal response to cognitive stimulation that requires high metabolic flexibility. In this pilot study, using in vivo microdialysis and highresolution NMR spectroscopy, we analyzed the metabolic profile of interstitial fluid in the hippocampus of 5xFAD transgenic mice and control C57BL/6 animals on days 7 and 28 following cognitive training in a conditioned passive avoidance paradigm. Among the metabolites examined, significant differences were found for acetone: in 5xFAD animals on day 28, its concentration was markedly lower compared to controls, indicating a limited ability to maintain ketone metabolism in the delayed post-training period. Other metabolites (acetate, lactate, caprate, isobutyrate, and glycine) did not show significant differences between groups; however, the observed changes may indicate shifts in the utilization of alternative energy substrates. These data suggest reduced hippocampal metabolic plasticity in transgenic animals, particularly in the late phase following cognitive stimulation, which could potentially limit long-term cognitive adaptation. Despite the limitations of this pilot study, the results highlight the potential of an approach combining metabolic profiling in vivo for the early detection of metabolic disturbances and the search for potential biomarkers of neurodegenerative processes in AD.

Justified Choice of a Substrate for Alkaline Phosphatase Based on the Enzymatic Kinetics and Electrochemical Characteristics of Screen-Printed Carbon Electrodes in Immunosensors

2025-06-20T07:36:53+00:00

In this study, a substantiated selection of a substrate for further use in electrochemical enzyme immunoassay is carried out based on the parameters describing the enzymatic kinetics and the electrochemical characteristics of the products of electroenzymatic reactions. Data from an analysis of the electrochemical characteristics of screen-printed graphite electrodes (SPE) using various surface pretreatments are presented. Using SPE pretreatment in sulfuric acid, it was possible to achieve a significant decrease in the background current and lower the detection limit of 1-naphthol, which is a product of the enzymatic reaction catalyzed by alkaline phosphatase, to 0.73 μM in the differential pulse voltammetry (DPV) mode and to 0.09 μM in the chronoamperometry (CA) mode without the use of additional catalysts. The selected substrate and pretreatment were used in a quantitative electrochemical enzyme-linked assay for the determination of procalcitonin (PCT). The sensitivity coefficient and detection limit were 109 nA×mL/mg and 0.8 ng/mL, respectively.

Comparison of the Efficiency of Dendritic Cell Maturation Upon Stimulation with Bacterial Lipopolysaccharide or Tumor Necrosis Factor Alfa

2025-12-01T01:41:24+00:00

Dendritic cells (DCs) are the professional antigen-presenting cells capable of presenting antigens to T-lymphocytes, thereby initiating the primary immune response. The unique immunological properties of this cell population make their use as a cellular vaccine relevant for the treatment of oncological and chronic infectious diseases. A critical stage in obtaining DCs with immunostimulatory properties is their maturation. To compare the effectiveness of alternative methods for stimulating maturation, DCs were obtained in vitro from peripheral blood monocytes through their induced differentiation in the presence of the cytokines GM-CSF and IL-4. As biochemical stimuli for inducing maturation, bacterial lipopolysaccharide (LPS) or tumor necrosis factor alpha combined with prostaglandin E2 (TNFα+PGE2) were used. No significant differences were found in the ability of these factors to stimulate the expression of HLA-DR and costimulatory molecules (CD80, CD83, CD86). The use of alternative maturations did not lead to differences in the ability of DCs to stimulate the proliferation of allogeneic lymphocytes. At the same time, there were morphological signs indicating the ability of LPS to stimulate differentiation into macrophages.

Some Methods for Managing the Degradation of Biomedical Implantable Materials Based on Collagen and Hyaluronic Acid

2025-09-16T16:02:37+00:00

The review considers modern approaches to modifying the in vitro degradation kinetics of implantable matrices based on collagen and hyaluronic acid. It includes data presented in 24 of approximately one hundred articles on similar topics found. Special attention is paid to the effect of polymer additives in collagen- and hyaluronic acid-based materials on their degradation rates in hydrolytic and enzymatic environments. The mechanisms of interaction of components and the impact of their structure on degradation processes are described. Approaches to the development of implantable medical devices based on biopolymers with controlled degradation and biocompatibility properties are proposed.

Heterologous Expression of Recombinant L-asparaginase Genes

2025-01-01T12:07:16+00:00

L-asparaginase (EC 3.5.1.1.) is the enzyme with the highest level of global production and is used in the treatment of cancer and in the food industry. Different expression systems are used for the production of many target proteins, ranging from cell-free to hyperproductive plant, insect, bacterial and mammalian cells. This review attempts to bring together the available literature data on heterologous gene expression and technology for the production of recombinant L-asparaginases.

Design of Highly Specific Structural Fragments for Filtering Compounds with Undesirable Activities

2025-11-05T09:48:47+00:00

The design of chemical compounds with desired properties is a key approach in medicinal chemistry for drug development. Modifying structural formulas it is possible to reduce undesirable biological activities exhibited by the substance under study. Various in silico methods are widely used to predict the types of activity in designed molecules, thereby reducing both the financial and time costs associated with experimental screening of potentially unsafe substances. However, the performance of many computer-based assessment algorithms depends on well-balanced training datasets containing a sufficient number of both positive and negative examples (information on the structural formulas of compounds that do and do not exhibit the desired type of activity). When standard predictive approaches are not applicable, an alternative strategy involves assessing the presence or absence of structural fragments associated with specific biological effects. To support this approach, we have developed a method that estimates the contribution of individual atoms to a given biological activity, taking into account their local structural environment. The applicability of this method to practical drug discovery tasks was demonstrated by analyzing molecular targets linked to a broad range of adverse drug reactions. Moreover, comparison of the constructed fragments with known structural motifs responsible for molecular target binding confirms the robustness of our method and highlights its potential for identifying functionally relevant structural regions in compounds that interact with molecular targets lacking resolved three-dimensional structures.