Molekulyarnaya Biologiya最新文献

The emergence of new data on the association between the composition of the intestinal microbiota and various human diseases has generated increasing interest in microbiome research. In this context, selection of the DNA extraction method represents a critical stage in the design of the experiment, significantly affecting the reliability and reproducibility of results. This study presents a comparative analysis of 12 DNA extraction methods, including nine commercial kits and three laboratory protocols. We evaluated the taxonomic representation, including Gram-positive (Lactobacillaceae, Coprococcus spp., Streptococcus sp., Clostridium leptum) and Gram-negative bacteria (Enterobacteriaceae, Akkermansia muciniphila, Fusobacterium nucleatum, Bacteroides fragilis). The extraction efficiency was assessed by the DNA yield, expressed in GE/pL of eluate or in GE/-µL of feces, as well as by the frequency of low-abundance taxa loss. Clustering of the methods according to the type of lysis was demonstrated: mechanical lysis provided stable and high DNA yields, particularly for Gram-positive bacteria, while chemical and enzymatic methods showed lower efficiency. We determined that the lysis type and pre-processing of intact fecal samples are the key factors affecting the DNA extraction efficiency and preservation of the native taxonomic profile. The best results were demonstrated by the QIAamp® PowerFecal® Pro DNA Kit (Qiagen) and the combination of AmpliTest UniProb + AmpliTest RIBO-prep kits (Center for Strategic Planning, Federal Medical-Biological Agency, Russia), both of which outperformed other methods in terms of DNA yield. The QIAamp® Fast DNA Stool Mini Kit (Qiagen) showed minimal losses of low-abundance taxa. These findings can be used for the standardization of gut microbiota DNA extraction methodologies and the development of domestic protocols.

Ovarian cancer remains one of the most lethal malignancies with a five-year survival rate around 20% at III-IV stages, which determines the urgent need to develop new therapeutic approaches. Newcastle disease virus (NDV) has demonstrated considerable promise as an oncolytic agent, capable of selectively lysing tumor cells, suppressing the metastatic potential and stimulating anti-tumor immunity. Despite the established therapeutic potential, studies that investigate oncolytic properties of this virus within the context of ovarian cancer remain limited. In this work, we evaluated oncolytic activity of the NDV vaccine strain H in SC-OV-3, TOV-21G and OV-90 ovarian cancer cell lines. Such parameters as ability to support viral replication and cell viability after infection were investigated. As a result, all three lines were permissive to NDV-H infection. Therapeutic efficacy in vivo was assessed using a model of TOV-21G subcutaneous xenografts in BALB/c nude mice. Upon intravenous administration of the virus, a statistically significant reduction in tumor volume was observed compared to the control group. Based on these results, NDV-H strain can be considered as a potential oncolytic agent for the treatment of ovarian cancer.

Colorectal cancer remains one of the leading causes of cancer-related mortality, highlighting the importance of optimizing approaches for its early diagnosis and therapy. One promising area in this field is the investigation of the role of the gut microbiome in the initiation and progression of colorectal cancer. This review examines three principal hypotheses explaining the contribution of microbiota to carcinogenesis: the "Alpha-bug", the "Keystone pathogen", and the "Driver-Passenger" models. We analyze data on the mechanisms of microbiota-tumor cells interactions, including the induction of inflammation, genotoxicity, and disruption of the intestinal barrier function. Findings are also presented indicating that certain microorganisms previously considered markers of the advanced stages may possess pro-oncogenic properties, thereby refining existing carcinogenesis models. Overall, the data suggest that the microbiota and its dysbiotic alterations can be considered potential targets for colorectal cancer diagnosis and therapy.

Sertraline, a selective serotonin reuptake inhibitor, is widely used as a first-line drug for anxiety and depressive disorders. The clinical efficacy and adverse reactions observed with antidepressants are closely related to the concentration of this drug in the patient's blood, but the vast majority of antidepressants demonstrate significant pharmacokinetic variability, leading to pronounced interindividual differences in the steady-state concentration of the drug in the blood and its efficacy even with the same dosing regimen. In this regard, it has become obvious that genetic markers alone are not enough to obtain the most complete profile of the efficacy and safety of a drug; a combination of genotyping methods with omics biomarkers is necessary. As a result of examination of patients, residents of the Republic of Bashkortostan, that had been diagnosed with mixed anxiety-depressive disorder (F41.2), it was found that polymorphic variants rs16947 (CYP2D6*2), rs389209 (CYP2D6*4), and rs1065852 (CYP2D6*10) of the CYP2D6 gene do not have a significant effect on the activity of CYP2D6. Genetically determined variations in the activity of the CYP2D6 isoenzyme lead to differences in the metabolism of sertraline and its active metabolite N-desmethylsertraline in different patients, which causes variability in their concentrations in the blood plasma. A statistically significant increase in the plasma concentrations of sertraline and N-desmethylsertraline was found in patients carrying the slow allelic variants rs3892097, rs1065852, and rs16947 of the CYP2D6 gene. A statistically significant moderate inverse correlation was found between the dose and the metabolic ratio C6-HO-THBC/CP. The results obtained are preliminary, which makes it necessary to continue this study with an expanded sample size.

Current Alzheimer's disease (AD) therapies offer only symptomatic relief and fail to halt disease progression, underscoring the urgent need for novel therapeutic strategies. We have previously shown that selective positive allosteric modulator of TRPC6, benzopyran derivative (C20), exhibits synaptoprotective properties at nanomolar concentrations, restores synaptic plasticity in 5xFAD mice, and enhances hippocampus-dependent memory. Here, we further evaluate the preclinical efficacy and safety of C20, focusing on its effects on chronic toxicity, mutagenicity, amyloidosis, astrogliosis, synaptic plasticity, and behavior in a transgenic AD model. Chronic and acute toxicity studies were performed on female wild type mice. The Ames test was conducted using Salmonella typhimurium and E. coli strains to evaluate the mutagenic potential of C20. 8 months old 5xFAD were used as model of AD. Electrophysiological recordings were applied to study long term potentiation in hippocampal slices of 5xFAD mice following intraperitoneal injections of C20. Behavioral testing included the open field test. Immunohistochemical analyses were performed to quantify amyloid plaques and astrogliosis in the hippocampus. Chronic and acute toxicity studies revealed no significant adverse effects on mice weight and survival, indicating that C20 is well-tolerated at the tested dose. The Ames test confirmed that C20 is almost non-mutagenic. Behavioral testing demonstrated that C20- treated mice exhibited increased exploration in the open field test. Immunohistochemical analyses detected a significant reduction in amyloid plaques and astrogliosis. Our findings suggest that C20 is a safe and effective therapeutic candidate for AD, with the potential to restore synaptic plasticity, improve cognitive function, and reduce pathological hallmarks of the disease.

Histone lysine methyltransferase G9a is an important regulator of transcription of various genes and cell processes, but its role in determining aging and radioresistance is still poorly understood. The selective G9a inhibitor UNC0646 was tested for effect on lifespan and resistance to γ radiation and paraquat in Drosophila melanogaster. UNC0646 used at concentrations of 0.1-100 μM exerted a geroprotective effect on females, increasing the average lifespan by 1.6-13.9% (p < 0.05). Resistance to γ radiation was found to decrease in D. melanogaster receiving UNC0646 with food for 2 weeks. A positive effect of the inhibitor on lifespan in females might be due to the activation of genes involved in the DNA damage response and repair (D-Gadd45, mei-9, spn-B, and Ku80) and proteostasis (Hsp27, Hsp68, Atg1, and Ire1).

Head and neck paragangliomas (HNPGLs) are rare neuroendocrine tumors that originate in the parasympathetic paraganglia of the head and neck. The diagnosis of these tumors is challenging, and the therapeutic options are limited. The study of HNPGLs is fraught with challenges at every stage. One of the main problems is the absence of HNPGL cell lines in cell repositories, which is associated with the difficulty of their culturing and low division rate. In this regard, neither functional nor preclinical studies are available for this category of tumors. This significantly slows down the study of the molecular mechanisms of HNPGL pathogenesis and the development of effective therapeutic approaches. Here, we investigated the molecular genetic characteristics of the primary HNPGL culture. Using the single-cell RNA sequencing method, expression patterns were analyzed, and cell types were annotated. The results demonstrated that the HNPGL primary culture cells were optimally divided into three clusters and had different degrees of differentiation, expressing neural tissue cell and stem cell markers. Exome sequencing revealed genetic abnormalities in the HNPGL culture, including mutations in the IGSF3, DHH, EXOSC8, SERPINA1, TYR, and NQO1 genes, aneuploidy, as well as multiple chromosomal duplications and deletions. These results enhance our knowledge of the molecular genetic features of successfully cultured HNPGL tumor cells.

The epidermal growth factor receptor (EGFR) is among the research subjects of most interest and remains genuinely attractive due to its key role in regulating the main conserved signaling pathways responsible for cell growth, survival, and proliferation. Dysregulation of the signaling pathways leads to cell malignant transformation, tumor progression, and metastasis. Therefore, EGFR is considered as one of the main targets for anticancer drug development. Although several generations of novel anti-EGFR drugs have been successfully developed, acquisition of drug resistance and the mutation status of the downstream effector protein KRAS significantly reduce the tumor response to therapy. The review focuses on the current approaches to anti-EGFR therapy. Drugs designed to block the EGFR-mediated signaling are described, including monoclonal antibodies, tyrosine kinase inhibitors, and immunotoxins. Mechanisms of acquired resistance to anti-EGFR therapy are discussed, and combination treatment strategies are proposed to improve the efficacy of the available drugs. Finally, promising antitumor agents, including ribonucleases (RNases) of various origins, are considered.

Although the role of NADPH oxidase 2 (NOX2) in the development of Alzheimer's disease (AD) is widely recognized, its contribution to the initial stages of amyloid-induced pathology remains unclear. Intraventricular administration of β-amyloid (Aβ) causes acute amyloid toxicity, leading to neurodegenerative changes similar to AD. The acute phase, lasting several days, is a critical time window for studying early pathological mechanisms. In this work, we assessed the level of oxidative stress in the brain of BALB/c mice at the early stages of amyloid toxicity and the role of NOX2 in these processes. Analysis of key markers of oxidative stress in various fractions of brain homogenate on day 4 after Aβ administration showed that individual parameters demonstrated only a tendency to change, without reaching statistical significance. However, principal component analysis (PCA) revealed a clear separation between the Aβ-treated and control groups, indicating the need for a comprehensive rather than isolated analysis of biochemical changes at early stages of pathology. It is noteworthy that the centroids of the groups in PCA were located along the same straight line, and the group receiving Aβ together with the NOX2 inhibitor occupied an intermediate position between the control and Aβ groups. This indicates partial suppression of oxidative stress through NOX2. At the same time, the NOX2 inhibitor completely prevented Aβ-induced microgliosis in the hippocampus, confirming that the concentration used was sufficient to suppress NOX2-dependent microglial activation. The in vivo data demonstrate that oxidative stress induced by Aβ administration may not be entirely mediated by NOX2, although this mechanism plays an important role in the initiation of the pathological process in AD.

In PCR with Taq polymerase on the Staphylococcus aureus genomic DNA template, the substrate properties of eight fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTP and Cy5- dCTP), which are dU-dC pairs with similar cyanine substituents, were compared during simultaneous introduction of such pairs in PCR. The different Cy5-dUTP and Cy5-dCTP pairs each had substituents with different linker lengths between the nitrogenous base and the fluorophore and between the quaternary ammonium group and the second heterocycle of the Cy5 fluorophore. The amplification efficiency, as well as the yield of the product, and the density of label incorporation were determined. It was found that, with the simultaneous introduction of Cy5-modified dU and dC into the reaction at equimolar concentrations, the inhibitory effect was not directly proportional to the concentration, in contrast to that with separate (individual) introduction of fluorescently labeled dNTPs. This allows one to use the simultaneous introduction of Cy5-modified dU and dC into PCR to increase sensitivity in methods based on the detection of a fluorescent signal, for example, in DNA-microarray technology.