Aptamers are short single-stranded nucleic acid molecules with a unique ability to bind with high specificity andaffinity to a wide range of biomolecules, including proteins, viruses, and small molecules, due to the formation of tertiary structures, making them a powerful alternative to antibodies in biosensing, diagnostics, and therapy. Their advantages, such as high stability, the possibility of targeted chemical modification, and reproducible solid-phase synthesis without the use of cell-based systems or bioreactors, have driven the active development of methods for their design and optimization. This review systematizes the key stages in the evolution of aptamer development technologies, starting with the classic SELEX technology, and considers its modifications, including CE-SELEX, M-SELEX, Cell-SELEX, HT-SELEX, and others, aimed at improving the efficiency, specificity, and automation of the process. Special attention is paid to the integration of computational methods such as secondary and tertiary structure prediction (RNAfold, MXfold2, RNAComposer), molecular docking (AutoDock Vina, HADDOCK, ZDOCK), molecular dynamics (GROMACS, AMBER, NAMD), and SELEX data analysis (AptaSUITE, FASTAptamer). The implementation of machine learning and deep learning algorithms (AptaDiff, RaptGen, Apta-MCTS, Xelari) is also described, opening up new opportunities for the rational design of de novo aptamers, optimization of their affinity, and minimization of experimental costs. The review discusses the limitations of the current methods, such as limited amount of structural data, the complexity of predicting noncanonical pairs and pseudoknots, and the insufficient generalizability of ML models to new sequence families. This review highlights the transformative potential of aptamers in biomedicine but emphasizes the need for rigorous experimental validation of computational predictions to overcome current limitations.
RecQ helicases are a conserved family of DNA-unwinding enzymes that play a key role in maintaining genomic stability by participating in DNA damage repair, recombination, replication, and telomere homeostasis. Five RecQ helicases are known in humans: BLM (the Bloom syndrome protein), WRN (Werner syndrome helicase), RECQL4, RECQL1, and RECQL5. All members of the RecQ family possess 3' → 5' helicase activity and are capable of unwinding DNA, including its complex secondary structures, in the 3'-to-5' direction by using the energy of ATP hydrolysis. WRN is unique among RecQ helicases in possessing additional 3' → 5' exonuclease activity, which expands its functionality in maintaining genomic stability. Mutations of the RecQ helicase genes lead to Werner, Bloom, and Rothmund–Thomson syndromes and are associated with a predisposition to cancer and premature aging. The enzymes consequently attract significant interest of the scientific community. Many viruses rely on host cell replication and repair mechanisms for their reproduction, and RecQ helicases may therefore influence the development of viral infections. The review discusses the role of RecQ helicases in replication of various viruses, including socially significant ones, such as the human immunodeficiency virus, herpes simplex virus, Epstein–Barr virus, hepatitis C virus, and others.
The main causes of early mortality in patients with schizophrenia are comorbid somatic diseases (primarily cardiovascular diseases, diabetes, and obesity) and suicide, which reduces life expectancy on average by ten years compared to the general population. The COMT gene encodes catechol-O-methyltransferase. This enzyme is responsible for O-methylation, one of the main pathways for catecholamine inactivation. The MAOA gene encodes monoaminoxidаse type A, an enzyme that carries out oxidative deamination of dopamine, norepinephrine, and serotonin. We hypothesized that polymorphic variants of the COMT and MAOA genes are associated with suicidal behavior in patients with schizophrenia. Accordingly, the aim of our study was to investigate the associations between the presence or absence of a history of suicidal behavior in patients with schizophrenia and polymorphic variants of the COMT and MAOA genes. A total of 150 patients of Slavic ethnicity with schizophrenia were examined. The presence or absence of a history of suicidal tendencies was determined based on medical records and a clinical interview. The control group consisted of 134 apparently healthy military personnel. Seven polymorphic variants in the COMT gene and two polymorphic variants in the MAOA gene were selected for pilot genotyping. Polymorphic variants of the COMT and MAOA genes were initially determined using PCR. An association was found between the COMT rs4680 and rs4633 polymorphic variants and a history of suicide attempts in patients with schizophrenia. The results of our study demonstrate the possible contribution of the catechol-O-methyltransferase gene to the mechanisms of suicidal behavior in patients with schizophrenia.
The history of research on lactate is quite interesting. At first, it was considered an unnecessary product of glycolysis, which humans use only by converting it back into glucose (gluconeogenesis, Cori cycle). Then it turned out that it is a ready-made source of food for cells and, moreover, that some cells (neurons) may prefer it to glucose. Lactate receptors were discovered, and it became a regulator. Finally, in 2019 (after more than 200 years of study), the lactylation of histones and other proteins ( hundreds of targets) was discovered. Lactate became an epigenetic regulator. A single, very small molecule combines many functions. One of the main functions of interest to oncologists is that lactate, together with hypoxia, is a sign of supply dysfunction in the body and causes reactions aimed at normalization (angiogenesis, etc.). As a result, tumors, with the immune system silent, undergo “normalization,” with blood vessels growing in them to reduce hypoxia and the lactate concentration. In addition, in the tumor microenvironment, where the concentration of lactate is dozens of times higher than normal, lactate acts as an immunosuppressant. In recent years, antitumor therapy has been reoriented from targeting tumor cells themselves to targeting immune cells (using “checkpoints”). Lactate has become one of the targets of such therapy. Various aspects of the molecular biology of lactate are covered in this review. Particular attention is paid to the role of lactate in antitumor therapy.
To date, the genomes of several mycoplasmas have been cloned in Saccharomyces cerevisiae cells, including Mycoplasma genitalium, M. pneumoniae, and M. mycoides. In this work, the 1-Mb genome of Mycoplasma gallisepticum S6 was cloned in S. cerevisiae. A transposon vector was constructed to deliver a synthetic S. cerevisiae replicon and a selective marker into M. gallisepticum genome. Transplantation of the M. gallisepticum genome with the integrated yeast replicon to yeast cells was performed using two approaches, polyethylene glycol (PEG)-induced transformation of yeast spheroplasts with intact genomic DNA isolated from M. gallisepticum cells and prepared in aragose plugs and PEG-induced fusion of M. gallisepticum cells with yeast spheroplasts. Both of the approaches yielded S. cerevisiae strains with the cloned M. gallisepticum genome, as was verified by nanopore sequencing.
Acquired resistance to endocrine therapy poses a major limitation in the treatment of estrogen receptor (ER)-positive breast cancer (BC). Somatic mutations of the ESR1 gene, which encodes ERα, are one of the key resistance mechanisms. However, the effect of mutations and especially double mutations on the binding of novel drugs remains insufficiently understood. To evaluate the effect of the mutations at the molecular level, a comprehensive computational approach was employed, including molecular docking, binding free energy calculations (MM-GBSA), and molecular dynamics simulations. A comparative analysis revealed opposite effects of ESR1 mutations on affinities of various selective ER degraders (SERDs). Vepdegestrant and fulvestrant were found to bind far less efficiently with ERα mutants and especially double mutants (e.g., Y537C/D538G) than with the native ERα. In contrast, the SERD ZB716 demonstrated the same or even better binding with most mutants. A potential structural cause of its resilience to mutations was identified via a comprehensive analysis, which included the evaluation of individual amino acid residue mobility through root-mean-square fluctuation (RMSF) calculations and an assessment of intermolecular hydrogen bond stability. ZB716 was shown to maintain a stable hydrogen bond with Glu353, and the bond was preserved in the ERα mutants. A lower stability of this key interaction was observed with fulvestrant. The findings elucidate the molecular mechanism of ESR1 mutation-driven resistance and support the promise of ZB716 as a compound capable of overcoming resistance conferred by a subset of the most common somatic ESR1 mutations in BC patients. The results justify the targeted design of new SERDs aimed at interacting with ERα mutants and outline an avenue of further research in the field of personalized therapy for ER+ BC.
With the widespread adoption of wide- and whole-genome massively parallel sequencing methods, the capabilities of molecular genetic diagnostics of hereditary diseases have expanded significantly. However, the challenge of increasing the detection rate of certain nosologies remains relevant. In recent decades, the use of DNA methylation as a biomarker for pathological conditions, including damage to the epigenetic machinery, has been extensively explored and discussed. Thus, DNA methylation can act as an indicator of damage to the epigenetic machinery. The analysis of the genome methylation pattern can be used to optimize the diagnosis of chromatinopathies, hereditary diseases caused by the emergence of a pathogenic variant in a nucleotide sequence in one of the genes controlling the epigenetic landscape. Highly specific changes in the DNA methylation pattern, or episignature, were identified for over 70 syndromes of this group improving the effectiveness of their differential diagnosis. This review examines the molecular basis for the emergence of the episignatures, the principles of their identification, distinctive features, possibilities and limitations of application, and prospects for the development of methods for analyzing changes in DNA methylation patterns as one of the areas of chromatinopathy diagnostics.
The degradation of intracellular proteins is a fundamental biological process necessary for maintaining cellular homeostasis, controlling the cell cycle, regulating signal transduction, and preventing the accumulation of toxic protein aggregates. Disorders of the proteolytic systems are implicated in the pathogenesis of numerous human diseases, including neurodegenerative diseases, lysosomal storage disorders, metabolic disorders, and certain types of cancer. The development of rudimentary and cost-effective models of these diseases for the purpose of evaluating novel pharmaceutical agents and elucidating the molecular mechanisms underlying disease pathogenesis constitutes a pivotal medical and biological undertaking. The proteolytic apparatus of the yeast species Saccharomyces cerevisiae has become a biochemical model organism of significant importance. This is due to its well-studied nature, low cost, ease of genetic manipulation, and evolutionary conservatism. The mechanisms of proteolytic system dysfunction can be studied in this organism. Furthermore, therapeutic approaches aimed at correcting these dysfunctional mechanisms can be sought.
A set of genes activated by the hormone ecdysone has previously been identified in the salivary glands of wandering Drosophila larvae, along with a group of regulatory elements involved in transcriptional induction by ecdysone. New evidence was obtained to implicate the DNA-binding protein Crol in the transcriptional regulation of ecdysone-dependent genes in larval salivary glands. Crol was shown to bind to the promoters and enhancers of ecdysone-depending genes, demonstrating an increased binding in the salivary glands, where the genes are active, compared to brain tissue, where the genes are silent. Crol was found to prime the recruitment of the ecdysone receptor (EcR) at least on some ecdysone-dependent enhancers. Disruption of Crol binding at these sites in crol knockout larvae reduced the EcR binding level and decreased the transcriptional level of genes possessing the respective regulatory elements. Based on the findings, Crol was assumed to function as one of the many proteins that render regulatory regions susceptible to EcR binding, thereby enabling the specific effect of ecdysone on D. melanogaster tissues.
The immune system consists of a diverse range of cell types, each performing its specific function. T-lymphocytes, B-lymphocytes, and NK (natural killer) cells provide protection against infections and tumors and participate in the regulation of immune responses. Disorders in the development and function of lymphocytes can lead to various immunodeficiencies, autoimmune diseases, and oncological conditions. The functional and morphological diversity of the B- and T-cell immune system in health and disease is maintained by dynamic genetic and epigenetic changes, including those mediated by small regulatory molecules—microRNAs (miRNAs). This review presents current data on the role of miRNAs in the development of B- and T-cells, their activation, and malignant transformation. Aberrant expression of miRNAs plays a key pathogenic role in the development and maintenance of lymphomas, characterizing them as promising diagnostic and prognostic markers.
A method utilizing hydrogel biological microchips was developed to genotype markers of human Y-chromosome haplogroups. With the method, 14 core haplogroups and their 78 subclades were determined, including (subclade markers are in parentheses) B-M60, C-M130 (F1906, Z18160, F6370, Y4630, Z4063, and M407), D-CTS3946, E-M5388 (P177, M5017, L539, L618, L677, Z827, M123, L29, and L791), G-P257 (F858, P15, Z6552, and L1259), H-M2920, I-U179 (M253, Z2336, Z58, M438, S2648, M436, and L596), J-M304 (PF4641, Z2363, CTS3569, CTS886, M410, PF5125, PF5130, Z7671, Z2221, and Z2417), L-M185 (Z5919, Y6288, and PH3615), N-M231 (L395, CTS10075, CTS10760, and CTS10082), O-M175 (M1422, F36, and M134), Q-M1105 (L472, L74, YP344, M7361, and L275), R-P224 (M420, S224, Z283, Z2906, CTS11962, Z91, Z686, S3357, YP237, Y33, Z93, L657, Z2124, Z2125, Z2123, Y20746, Y934, S23592, Z2122, M343, M73, CTS894, M412, M405, P312, Z2103, and M479), and T-M272. Genotyping included multiplex PCR followed by hybridization on a biochip. Haplogroup frequencies were determined in a sample of 356 males living in European Russia. The direct method can provide an alternative to indirect determination of haplogroups by Y-STR haplotype with the use of predictors. The method was developed with due regard to requirements for forensic genotyping methods and was primarily designed to obtain forensic information on the biogeographical origin of the person in question. The average amplicon length was 72 bp. The study clarified the population structure of Y haplogroups in the Slavic population of European Russia.
Cell therapy is increasingly used to treat a variety of medical conditions, including cancer, immune system disorders, and neurodegeneration. Stem cells secrete growth factors, signaling molecules, and extracellular vesicles, that can be used to treat neurological diseases and promote neuronal regeneration. Transgenic 5xFAD mice, which are a model for Alzheimer's disease (AD), were used in this study. The mice were 7 months old and received retro-orbital injections of glial progenitor cells (GPCs) once a week for 4 months. At 11 months, their behavior was analyzed using a multichannel actigraphy system. Brain tissues from the cortex, hippocampus, and midbrain were collected for postmortem analysis of mitochondrial respiratory chain enzyme activity. The results showed that the GPCs injection significantly improved the response of the hippocampal p2 mitochondrial fraction in 5xFAD mice to succinate, reaching a level observed in control animals. A similar trend was also observed for the cytochrome c oxidase complex. The oxygen consumption rate of mitochondria did not differ from that of clinically healthy mice after ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride administration. A similar decrease in the efficiency of the electron transport chain was detected in the midbrain of 5xFAD mice, but no recovery was observed after GPCs treatment. Behavioral differences between non-transgenic and transgenic groups were observed in a multiparameter analysis using the actigraphy system. The behavior of transgenic mice in the treated and untreated groups was similar, while the behavior of non-transgenic mice varied. Additional analysis of locomotor activity and transient events in particular revealed that the activity of the GPCs-treated 5xFAD mice was differed fundamentally compared to other groups. Specifically, GPCs-treated mice exhibited greater number of transitions between intermediate activity states. In contrast, untreated mice showed transitions between extreme activity states, such as from low to high activity or vice versa. These findings suggest that changes in behavior and activity of the AD mice may be associated not only with hippocampal dysfunction, but also with disruptions in midbrain structures.
Organ transplantation is a treatment method for various organ failures and other severe pathologies, applied in critical cases to save a patient’s life. However, there is a severe shortage of donor organs worldwide, resulting in hundreds of thousands of patients being unable to receive the organs they need in time. One possible solution to this problem is xenotransplantation—transplanting organs from animals to humans. Experiments in xenotransplantation began in the mid-1960s, with primates considered as the first potential donors. However, for a number of reasons, they proved unsuitable as a source of organs, while pigs turned out to be the best donors. The development of modern genetic engineering and genome editing methods has led to a new perspective on these animals as a source of human organs. Various genetic modifications have significantly reduced the immune response of the recipient to the graft and improved survival. To date, several successful transplants of organs and tissues from wild-type and genetically modified pigs to humans have been carried out worldwide, and a few companies are developing specialized lines of animals for xenotransplantation. In this review, we provide a detailed overview of the history of xenotransplantation worldwide, as well as all the genetic modifications introduced into the genome of pigs the organs of which have been used successfully for human transplantation, the role of these genetic modifications, and the mechanisms by which they are introduced.
Trehalose disaccharide has a stable neuroprotective effect used in inhibiting experimental neurodegeneration. However, the mechanism of its action on brain neurons remains largely unclear. In hepatocytes, the main target of trehalose is the activation of mTOR-independent autophagy, which is achieved by inhibiting the glucose transporter GLUT8, leading to energy deficiency. An increase in AMP levels activates AMP-dependent kinase AMPK by phosphorylation at Thr172 and further activates autophagy regulator kinase ULK1. In neurons, the GLUT8 transporter inhibitors and other disaccharides also activate autophagy, but less effectively than trehalose. The neuroprotective effect of trehalose includes a chaperone-like effect, inhibition of the accumulation of aberrant proteins, reduction of oxidative stress, increased antioxidant protection, and suppression of neuroinflammation. Similar to the effect on hepatocytes, trehalose triggers the activation of autophagy by the short signaling pathway pAMPK-pULK1. AMPK inhibition prevents the activation of autophagy in neurons and weakens the neurotherapeutic effect of trehalose. AMPK activation is accompanied by the pleiotropic effect of suppression of biosynthetic processes and cellular metabolism related to activation of mTOR-dependent autophagy; however, no such effect has been detected for trehalose. In vivo data on the relationship among GLUT8 expression, AMPK activity, and autophagy levels in the brain are analyzed. The therapeutic advantages of the molecular effects of trehalose in comparison with the activation of mTOR-dependent autophagy and the possibilities of their combined therapeutic use are discussed.
Modern concepts of the mechanisms of protein aggregation with an emphasis on immunoglobulin-like domains (Ig domains) as a structural platform predisposed to the formation of aggregates with amyloid properties are presented in this review. Particular attention is paid to the muscle proteins titin and myosin-binding protein C, which form not fibrillar, but amorphous amyloid aggregates with a cross-β structure without an increase in the total content of the secondary β-structure, capable of binding thioflavin T (ThT) and Congo red. The absence of a nucleation phase during the formation of amyloid aggregates of these proteins, as well as their partial disaggregation, allows us to declare a new, previously undescribed pathway of amyloid protein aggregation. We refer to it as self-templating amorphous β-assembly (conformational conversion by a prion-like mechanism), characteristic of multidomain proteins of the sarcomeric cytoskeleton.
Hemorrhagic shock (HS) is a life-threatening condition that leads to multiple organ failure due to centralization of blood flow and impaired blood clotting. In this study, we investigated the acute and delayed effects of HS on the brain, kidneys, and liver of rats to identify molecular targets for therapy of the consequences of shock. Blood acid-base balance and electrocardiography (ECG) parameters were studied in rats in the acute phase of HS. Gene expression of antioxidant enzymes (Gpx1, Sod1, Cat, Nfe2l2) and inflammatory markers (Ptprc, Cxcl1, Cd86, Itgal, Il1b, Il6, Tnf, Tlr2, Cox2, Cst7, Ccl3, Il10) in brain, kidney, and liver tissues was analyzed, as well as the amount of protein markers for kidney damage (NGAL, KIM-1) in urine 24 h after HS. In addition, markers for the activation of astrocytes (Gfap) and microglia (Aif1) as well as neuronal markers (Eno2, Tubb3) in brain tissue were analyzed. Biochemical markers for liver and kidney damage and total antioxidant activity were determined in blood serum. Acute HS caused decompensated lactic acidosis, arterial hypotension and characteristic changes in the ECG. Although no pronounced inflammatory response was detected in brain, kidney and liver tissue in the late phase after acute blood loss, the brain and liver tissue were more susceptible to the adverse effects of acute blood loss than the kidneys according to a number of indicators. This points to the need to develop targeted strategies to protect organs in the post-resuscitation period by targeting specific molecular targets in specific tissues.
Toxin-antitoxin systems are modules consisting of genes that encode a stable toxin and a labile antitoxin. These systems are found in the genomes of most bacteria and archaea species. At present, eight types of toxin-antitoxin systems are distinguished. They perform a variety of functions in bacterial cells, the most important of which is adaptation to environmental stress. This review focuses on various types of toxin-antitoxin systems, their functions, and the mechanisms regulating interactions between the main components. These systems are described in detail for Bifidobacterium bifidum, Bifidobacterium longum, Lacticaseibacillus rhamnosus, and Mycobacterium tuberculosis. Furthermore, promising applications of toxin-antitoxin systems in biotechnology and infectious disease therapy are described.
Nowadays, against the backdrop of a global increase in cancer incidence, the search for prognostic markers to assess individual risks is particularly relevant. Early identification of altered DNA methylation patterns may serve as a reliable indicator of malignant transformation and be used for diagnostic purposes. The objective of this study was to assess the methylation level of CpG dinucleotides in the promoter regions of DNA repair genes (AKT1, DDB2, GADD45A, XPC and XRCC3) in the blood of chronically exposed individuals who subsequently developed cancers. The study was conducted in individuals who were affected by chronic low dose-rate exposure in 1950-1960s in the Southern Urals. The main group included exposed individuals in the latent period of cancer development-104 people, the comparison group consisted of exposed individuals without cancers-188 people. The methylation level was assessed in bisulfite-converted DNA samples using methylation-specific high-resolution melting. It was established that in exposed individuals from the main group, there was a statistically significant decrease in the methylation level in the AKT1 and GADD45A gene promoter, relative to the comparison group (4.37 versus 7.23%, p < 0.001 and 10.73 versus 19.58%, p < 0.001, respectively). As for the XRCC3 gene, there was an increase in the methylation level of CpG dinucleotides in the promoter in individuals from the main group relative to the comparison group (5.38 versus 6.18%, p < 0.001). When the prognostic potential of methylation parameters of the AKT1, GADD45A, XRCC3 gene promoters was assessed for the purposes of early diagnosis of cancer development risk, it was found that when these loci were analyzed together, the AUC was 0.89 (95% CI: 0.85-0.93) at p < 0.001.
Brain malignancies, such as glioblastoma, remain one of the most aggressive and difficult-to-treat cancers because of their heterogeneity, invasive growth, and resistance to conventional therapy. Significant recent progress in the treatment of these diseases has been associated with the development of gene and cell technologies, which open up new possibilities for targeted therapy, immunotherapy, and personalized approaches. The review systematically summarizes the data on current treatment strategies, including gene therapy, cell therapy, and combined approaches. Particular attention is paid to preclinical and clinical studies, as well as the prospects for overcoming major limitations, such as the blood–brain barrier, the immunosuppressive tumor microenvironment, and toxicity. Analysis of the literature demonstrates that integration of gene and cell technologies could provide a basis for developing effective treatments aimed at improving the survival of patients with central nervous system tumors.
Lung cancer continues to be the leading reason for deaths related to cancer worldwide, with non-small cell lung cancer (NSCLC) accounting for almost 85
