Biomedical Chemistry: Research and Methods

The Blood-Salivary Barrier as a Multifaceted System: a Review

E.I. Dyachenko, E.A. Sarf, L.V. Bel’skaya — Thu, 28 May 2026 00:00:00 +0000

The purpose of this review is to provide a comprehensive description of the blood-salivary barrier and systematize the result of recent studies on its structure and function in healthy and diseased states. The blood-salivary barrier (BSB) is considered a multifaceted system, which includes following key components: the salivary glands, oral epithelium, intercellular junction proteins that maintain barrier strength, and also saliva and mucus. The barrier’s blood supply, neural connections, local immune responses, and the oral microbiome create the microenviroment, in which BSB operates. The BSB requires consideration of a number of additional variables, including innervation, blood supply, the presence of circulating metabolites in the vessels supplying the oral epithelium and salivary glands, as well as the volume of secreted saliva and its rheological properties, the composition of the oral microbiome, and the state of the immune system.

Validation of the Sample Preparation Protocol Based on 1DE-gel Concentration for Proteomic Analysis of HepaRG Cell Culture

Thu, 28 May 2026 00:00:00 +0000

HepaRG is a promising cell line for the study of drug metabolism, which requires an integrated approach that includes genomics, transcriptomics, and proteomics. The genome and transcriptome of HepaRG have been studied quite well, while the proteome remains poorly characterized, which may be due, in particular, to the lack of a standardized sample preparation method for proteomic analysis. In this study, two methods of sample preparation for MS analysis of HepaRG cells were compared: protein extraction with RIPA buffer based on sodium dodecyl sulfate followed by 1DE-gel concentration and trypsinolysis in the gel (protocol 1), protein extraction with urea/thiourea buffer followed by trypsinolysis in solution (protocol 2). The efficacy of trypsinolysis was evaluated using an external standard, recombinant cytochrome P450 BM3 from the bacterium Bacillus megaterium. The search for peptides and proteins was carried out using the domestic proteomic machine IdentiProt. Trypsinolysis in gel (Protocol 1) allowed the identification of 82.7 ± 1.5 BM-3 peptides, whereas trypsinolysis in solution identified 76.0 ± 0.0 peptides, which averaged 49.8% and 45.8% of the theoretically possible amount, respectively. Both methods demonstrated a high correlation (r = 0.84) between the intensity values of the reported BM3 peptides. 8487 + 235 peptides and 1242 + 22 HepaRG proteins (protocol 1), 9415 + 276 peptides and 1478 + 34 proteins (protocol 2) were identified. Analysis of the identified proteins showed that both protocols made it possible to identify proteins in the molecular weight range of 6–629 kDa, with the majority of proteins (75%) in both cases being represented by low molecular weight proteins (10–100 kDa). The analysis of the protein composition of HepaRG cells made it possible to identify a greater number of membrane proteins, liver cell proteins and drug metabolism proteins for the sample preparation protocol using urea-based buffer, while the sample preparation protocol based on RIPA buffer solubilization with 1DE-gel concentration and trypsinolysis in the gel made it possible to identify a greater number of proteins associated with biological processes specific to the liver. Thus, the method of sample preparation based on 1DE-gel concentration and trypsinolysis in the gel has shown its effectiveness for characterizing the proteome of human hepatoma HepaRG cells and searching for proteins involved in the metabolism of xenobiotics and drugs.

Fluoroquinolone-Induced NAD⁺ Depletion as a Novel Mechanism of Cytotoxicity

I.V. Kuzminov, D.I. Boyarintsev, K.V. Bryutova, A.V. Melnik, Yu.S. Rodina — Thu, 30 Apr 2026 00:00:00 +0000

A potential mechanism for the cytotoxic and antitumor effects of fluoroquinolones has been discovered. Treatment of HCT-116wt cells with norfloxacin, ciprofloxacin, and ofloxacin decreased intracellular NAD⁺ and cell survival. The IC₅₀ values for the effect of fluoroquinolones were 0.314 mM (for norfloxacin), 0.547 mM (for ciprofloxacin), and 0.612 mM (for ofloxacin). The MTT test for cytotoxicity and mitochondrial functional status assessment revealed similar IC50 values of 0.32 mM (for norfloxacin), 0.35 mM (for ciprofloxacin), and 0.67 mM (for ofloxacin). The suggest unveil a new potential mechanism of fluoroquinolone action on cells beyond their antimicrobial activity.

Protocol for the Preparative Isolation of LDL and HDL Fractions from Human Blood Plasma

Thu, 11 Jun 2026 00:00:00 +0000

The pathogenesis of atherosclerosis, the main cause of cardiovascular disease, including coronary heart disease and stroke, involves plasma lipoproteins, which differ in protein and lipid composition, size, charge and function. At present, chromatographic methods, in particular gel filtration (SEC), are widely used to isolate lipoproteins. In this article, we present a protocol for the preparative isolation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) fractions from human blood plasma using gel chromatography. To determine the boundaries of the elution zones of apolipoproteins A-1 and B-100, HDL and LDL markers, respectively, we used liquid chromatography with tandem mass spectrometry (LC-MS/ MS) with ELISA cross-validation. Lipoprotein fractions obtained with the help of the SEC can be used for various studies of scientific and applied importance.