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The effect of a pencil scanning proton beam in two regions of the Bragg curve with different linear energy transfer (LET) relative to X-ray radiation on the induction of micronuclei (MN) in cytochalasin-blocked binucleate lymphocytes (CBBLs) during in vitro irradiation of human peripheral blood at doses ranging from 0 to 2.0 Gy was studied. A nonlinear dose-response relationship was observed in the dose range of 0 to 1.0 Gy. The frequency of MN in CBBLs during proton irradiation was 2 times lower than during X-rays and did not depend on the LET value. In the dose range above 1 Gy, the dose dependences were linear, the value of the relative biological effectiveness depended on LET and was equal to 0.76 before the Bragg peak, and 1.16 at the peak.
Effective gene therapy depends on efficient delivery of genetic material into cells. Polyplexes, complexes of positively charged polymers and DNA, are a common non-viral delivery system. In this study, polyplexes were formed using a plasmid encoding the yellow fluorescent protein, TurboYFP, and polyethylenimine-polyethylene glycol-(TAT peptide) (PEI-PEG-TAT) block copolymers. These were used to transfect human lung adenocarcinoma A549 cells. Results showed that increasing the PEI nitrogen to DNA phosphate ratio and/or increasing the polyplex concentration significantly improved transfection efficiency, from a few percent up to 100 percent. These findings are valuable for producing specific proteins in cells, which may have applications in research and in the treatment of various diseases.
Effective approaches for selecting nuclei in which a gene of interest is in an active state are necessary for studying spatial organization and gene expression regulation. In this work, a novel two-component genetic system was created for the parasegment-specific labeling of nuclei in which the regulatory domain iab-5, which stimulates the homeotic gene Abd-B, is active. The system is based on the integration of a transgene expressing the yeast GAL4 activator under the control of the minimal hsp70 gene promoter into the iab-5 domain, near the early embryonic enhancer. As a result, the iab-5 enhancer induces specific expression of GAL4, which strongly amplifies the expression of the mScarlet fluorescent protein, allowing for the efficient selection of labeled nuclei in which the iab-5 domain is activated. This approach can be used for the selection of target nuclei in which any regulatory element or gene of interest is in an active or repressed state.
Aliphatic aldehydes are involved in many important biological processes, but detection of them at low concentrations requires expensive laboratory equipment and labor-intensive analytical methods. Typically, chromatographic and chromatograph mass spectrometric methods are used for this purpose. Therefore, developing a simple method for detecting aldehydes at low concentrations is a pressing scientific and technical challenge. We have demonstrated that a bacterial bioluminescent system can be effectively used for the semiquantitative determination of biogenic aldehydes in vitro at nanomolar concentrations. Furthermore, this system holds promise for detecting biogenic aldehydes in vivo.
We previously demonstrated that fragments of the central loop of the non-conventional toxin WTX reduce blood pressure in rats and inhibit certain nicotinic acetylcholine receptor subtypes [10]. In the present study, we examined the effects of hexamethonium and methylicaconitine, which are nicotinic acetylcholine receptor inhibitors, as well as of atropine, a muscarinic acetylcholine receptor inhibitor, on the hypotensive effect of the WTX central loop fragment. For this purpose, these compounds were administered intravenously before the peptide. We found that hexamethonium and methylicaconitine enhanced the fragment's hypotensive effect, while atropine weakened this effect. Only methylicaconitine enhanced the tachycardic effect of the peptide. These data indicate the involvement of both nicotinic and muscarinic acetylcholine receptors in the hemodynamic effects of the WTX central loop fragment.
The genotypic variability of the SARS-CoV-2 virus is extremely high, and the emergence of new strains raises concerns about their possible high virulence and ability to bypass responses of the body's immune system induced by previous infection or vaccination. Therefore, one of the main tasks is to study the pathogenesis of various variants of the virus using experimental animal biomodels of SARS-CoV-2 to quickly find methods and approaches to fighting new viruses. The study was performed on humanized mice of the C57BL/6-Tgtn line. Mice were infected intranasally at different doses with three variants of the SARS-CoV-2 virus. We showed that humanized hACE2 mice, when infected with all three variants of the SARS-CoV-2 virus, showed typical pathological changes in lung consistency comparable to those found in COVID-19 in humans. At a dose of 4 log plaque-forming unit (PFU), all variants showed 100% mortality. In a comparative assessment of different variants of the SARS-CoV-2 virus in hACE2 humanized mouse model, it was found that the Delta variant leads to more severe damage compared to Wuhan or Omicron.
The patterns of radiation adaptive response (RAR) induction and transgenerational genomic instability in mice following exposure to carbon ions (12C) with a linear energy transfer (LET) of ~39 keV/μm and to X‑rays with a LET of ~2 keV/μm at a dose of 10 cGy were studied. Low doses of 12C, as well as X‑rays, induce RAR, the magnitude of which depends on the quality of the challenge radiation. In the first- and second- generation offspring of males irradiated 12C at a dose of 10 cGy, an increased spontaneous level of cytogenetic damage and the absence of RAR in the first generation were detected, in contrast to the offspring of males after irradiation with X‑rays.
The design of prodrugs for nucleoside antiviral agents is the primary approach to achieving target profiles not only in terms of safety but also efficacy. We have proposed a new chemotype of inhibitors of tick-borne encephalitis virus (TBEV) reproduction among N6-substituted adenosine derivatives, characterized by the presence of a hydrophobic aromatic substituent at the N6 position of adenine and benzoic acid residues attached via ester bonds to the ribofuranose scaffold. The compounds inhibited TBEV reproduction at micromolar concentrations only in their benzoylated form, while exhibiting no detectable cytotoxicity regardless of the substitution on the ribofuranose moiety. Our study demonstrates that modifying nucleoside analogs with hydrophobic groups can yield compounds with improved antiviral activity, and the identified compounds may subsequently serve as prototypes for the development of new antiviral drugs against epidemiologically significant pathogenic RNA viruses affecting humans.
of the study is to evaluate the efficacy, safety, and immunogenicity of divozilimab (DIV), anti-CD20 monoclonal antibody, in patients with systemic sclerosis (SS). . Patients with SS according to ACR/EULAR (American College of Rheumatology/European Alliance of Associations for Rheumatology) 2013 criteria with modified Rodnan skin score (mRSS) ≥10 and ≤20 and forced vital capacity (FVC) ≥40% from the due took part in the study. Infusions of DIV 250 mg were administered on weeks 0 and 2, and DIV 500 mg on week 24 with the subsequent use in open mode, starting from week 48. This publication presents data obtained for 48 weeks of trial (before the DIV infusion at week 48). Primary endpoint was the change in the mRSS from baseline to week 48. The dynamic of FVC was estimated as the secondary endpoint. The safety evaluation included the frequency and profile of adverse events and adverse reactions (ARs). Immunogenicity was assessed by detection of binding and neutralizing anti-drug antibodies on weeks 2, 24, and 48. : The patients (151) were randomized into two groups: DIV (n = 76) and placebo (n = 75). The most were female; the median duration of the disease was about 3-4 years. The initial value of the mRSS was 14 and 13 points in DIV and Placebo groups, respectively. The change of mRSS from baseline to week 48 was -5.8 ± 1.1 points in DIV group and -2.7 ± 1.0 points in placebo group (adjusted mean difference (AMD) with 95% confidence interval -3.1 (-4.5; -1.7); p < 0.0001). The lung function was stable in patients treated with DIV. A comparable safety profile of DIV and placebo was demonstrated. The most frequent ARs were infusion reactions and a decrease in the number of lymphocytes. There were no severe and serious ARs in DIV group. All infusion reactions were mild and moderate. 5.3% (4/76) patients in DIV group had binding antibodies without neutralizing activity. : Divozilimab has demonstrated a significant decrease in severity of skin fibrosis, a positive effect on the respiratory function, and a favorable safety profile, which allows it to be considered as a promising therapeutic option for SS.
The lipid envelope of the influenza A virus contains two major types of protein spikes formed by the transmembrane hemagglutinin trimers (HA, MW 80 kDa) and neuraminidase tetramers (NA, MW 55 kDa), in quantities of 500 and 120, respectively. The third transmembrane protein, M2 (MW 14 kDa), forms tetramers of ion channels in quantities of about 10-20 per virion. Modeling of the molecular structure using the AlphaFold software tool showed a novel model for a heterocomplex of HA0-M2 proteins, in which the M2 tetramer located inside the HA0 trimer like a "matryoshka doll." Similar models of the HA0-M2 heterocomplex were obtained for the A/Aichi/2/68 (H3N2) and A/WSN/33 (H1N1) viruses. The resulting HA0-M2 heterocomplex possessed a high structural complementarity of the macromolecular interfaces (ipTM = 0.65), had no structural clashes of atoms in the molecular interfaces (clash score = 0.0), and exhibited stable and reliable intermolecular topology with a high ranking score of 0.79. The constructed model allows to explain the phenomenon of blocking the function of M2 ion channels by the rigid conformation of the uncleaved HA0 protein and, in contrast, the activation of M2 channels after (i) specific point proteolysis of HA0 into HA1 (55 kDa) and HA2 (25 kDa) subunits and (ii) exposure to acidic pH of 4.0-5.5, leading to the disclosure of the 3D structure of the HA1/2 molecule and the opening of the M2 channel.
Genetic predisposition without doubt is one of the risk factors of cancer initiation. It is known that single nucleotide polymorphisms (SNP) of genes that maintain the genome stability, including SNP of DNA repair, may contribute to the initiation of carcinogenesis. Single-nucleotide polymorphisms of genes that support genome stability, including SNP of DNA repair genes, can contribute to cancer initiation. Polymorphism of the excision repair gene OGG1 causes interest of leading scientific groups from various countries. It is assumed that there is relationship between the rs1052133 polymorphism in the gene and predisposition to cancer initiation. The objective of this study was to establish association between rs1052133 polymorphism of base excision repair gene OGG1 and the risk of cancer initiation in people chronically exposed to ionizing radiation. Residents (888 people) of the Techa riverside settlements, chronically exposed to low or medium dose radiation from the Techa River and the East-Urals Radioactive Trace were included in the study. The study allowed researchers to establish that exposed to chronic radiation people, carriers of the rs1052133*G allele, have increased risk of malignant neoplasm initiation: OR = 1.38; 95% CI [1.05-1.83], p = 0.023. The multifactorial synergistic interactions between the dose to the red bone marrow and the rs1052133 polymorphism of the OGG1 gene was found: Testing Balanced Accuracy (TBA) = 0.56; Cross Validation Consistency (CVC) = 10/10; p = 0.01). The study found that the rs1052133 polymorphism may be considered as genetical marker of risk of cancer initiation in people chronically exposed to radiation with doses ranged from 0.74 to 3507.07 mGy (average 523.10 ± 33.89 mGy). It was found that the presence of the rs1052133*G in combination with radiation exposure can modify the risk of solid cancers initiation, as it is indicated by the synergistic relationship between the SNP and the radiation dose.
Boundary elements in the Drosophila Bithorax complex partition the Abd-B regulatory landscape into autonomous domains that require insulation and boundary bypass. We surveyed Large Boundary Complex (LBC) occupancy across Abd-B boundaries and detected binding at Fab-7 and Fab-8, but not at Fab-6 or at Mcp (the boundary between Abd-B and abd-A). Despite lacking LBC binding, Fab-6 supports boundary bypass, demonstrating that bypass can occur without LBC. Fab-7 and Fab-8 share two conserved motifs ("green" and "blue"); however, mutational analysis shows that neither motif is required for LBC recruitment. These findings reveal selective, context-dependent LBC occupancy at Abd-B boundaries and imply that additional factor(s) and/or higher-order sequence organization underlie bypass activity. Thus, LBC is not universally required for boundary bypass, and its recruitment to Fab-7/8 is independent of the shared "green/blue" motifs.
The aim to evaluate the effectiveness of long-term therapy with Russian rituximab (RTX) biosimilar in Sjögren's disease (SjD) in real-life clinical practice. MATERIALS AND METHODS : The retrospective study included 53 patients with SjD (Russian 2001 criteria and ACR/EULAR (American College of Rheumatology/European Alliance of Associations for Rheumatology) 2016 criteria), observed at the Nasonova Research Institute of Rheumatology from 2017 to 2024 and receiving long-term RTX therapy (Russian biosimilar Acellbia®, BIOCAD). The signs of clinical and laboratory activity of the disease, stomatological and ophthalmological tests, as well as the incidence of new systemic manifestations and lymphomas were assessed dynamically. RESULTS : The median duration of RTX therapy was 27 [19; 55] months, and the median total dose was 4 [3.5; 5.5] g. Before the therapy, 13 (25%) patients had recurrent parotitis, which was relieved in all patients during the therapy. Persistent enlargement of the salivary glands was observed in 10 (20.4%) patients, in 9 of them it was relieved. A significant increase in stimulated saliva flow was found (from 1.5 [0.5; 3] to 2.4 [1.4; 3.5] mL; p = 0.002), an increase in salivation was found in 51% of patients, stabilization in 28.6%, and deterioration in 20.4%. When assessing the ultrasound dynamics of the salivary glands, the size of hypoechoic avascular lesions significantly decreased (from 1.8 [1.3; 2.3] to 1.3 [1.1; 1.5] mm; p<0.001), and according to the ultrasound activity index, stabilization was noted in 67.4% of patients, improvement in 27.9%, and deterioration in 4.7% of patients. When assessing the dynamics of sialography, the size of cavities significantly decreased (from 1.5 [1.5; 2.5] to 1.0 [0; 1.5] mm; p < 0.001), and according to the assessment of sialographic stages, stabilization was noted in 67.5% of patients, improvement in 32.5% of patients, and deterioration was not noted in any patient. When assessing the lacrimal glands function, a significant increase in lacrimation was found according to the stimulated Schirmer's test (from 6 [3.75; 12] to 8 [5; 15] mm; p = 0.005); an increase in lacrimation was noted in 38% of patients, stabilization in 40.6%, and a decrease in 21.4%. When assessing the tear break-up time, a tendency towards its increase was noted, but statistically insignificant (from 5 [3.75; 9.25] to 5.5 [4; 9] sec; p = 0.35). Corneal epitheliopathy during the therapy was relieved in 44% and persisted in 56% of patients; worsening of corneal epitheliopathy during the treatment was observed in a few patients, while no cases of ulcer formation or perforation of the cornea were recorded. During the therapy, a significant decrease in the levels of erythrocyte sedimentation rate, gamma globulins, IgG, IgA, IgM, rheumatoid factor, an increase in the C3 complement level, and the elimination of monoclonal gammopathy were observed, while the dynamics of the C4 complement level and cryoglobulinemia were multidirectional. The median duration of B lymphocyte depletion was 5 [4; 6] months, constant depletion could be maintained only in 59.6% of patients. During the therapy, the SjD systemic activity index (ESSDAI, EULAR Sjögren's Syndrome Disease Activity Index) significantly decreased (from 5 [2; 8] to 1 [0; 3] points; p<0.001), and minimal clinically important improvement of this index was achieved in 66.6% of patients. During the observation, one patient developed a new skin lesion (lupus chilblain); no other new systemic manifestations or lymphomas were registered. CONCLUSIONS : According to our retrospective study conducted in real-life clinical practice, long-term therapy with Russian RTX biosimilar in most cases (60-80%) led to stabilization or improvement of SjD manifestations. RTX can be used to treat not only systemic but also glandular manifestations of the disease. Given the lack of an optimal response to RTX therapy in a number of SjD patients, it is necessary to study the effectiveness of drugs that lead to a deeper depletion of B lymphocytes.
Tag proteins, widespread among prokaryotes, are DNA glycosylases that participate in the repair of alkylated DNA excising 3-methyladenine. Among eukaryotes, Tag homologs are found only in plants, but they have not yet been characterized. Here, we report the first study of a plant Tag homolog, BvTAG1 from sugar beet (Beta vulgaris). Unlike bacterial Tags, BvTAG1 lacks activity towards alkylated DNA, which can be explained by the mutation of the critical catalytic Glu residue to Ser. At the same time, BvTAG1 exhibits a high affinity for undamaged DNA, mediated by a long N-terminal tail characteristic of all plant Tag homologs. This binding further increases in the presence of free 3-methyladenine. Our results suggest that plant Tag homologs may be required for regulating the plant response to genotoxic stress, rather than for DNA repair.
The study proposes an experimental approach to determining the rates of individual stages of a reaction catalyzed by bacterial luciferase, based on the tryptophan fluorescence of the protein and the stopped-flow technique. The relationship between the fluorescence intensity of tryptophan residues in luciferase and the presence of substrates and reaction products in the active site of the enzyme is substantiated. The non-steady-state kinetics of bioluminescence in the reaction of two bacterial luciferases with aliphatic aldehydes of different chain lengths, as well as the kinetics of enzyme fluorescence intensity during the reaction, were analyzed. The obtained results confirmed the relationship between the rate of the two kinetic stages of enzyme luminescence and the processes of flavin substrate binding and enzyme activity recovery after the catalytic act.
Polypeptide modular nanotransporters (MNTs) were engineered as a targeted delivery platform for prostate cancer cells. The constructs integrate a ligand module to facilitate specific cellular binding and internalization, and a nuclear localization signal (NLS) to enable subsequent nuclear translocation. Two distinct ligand modules were utilized: (a) a nanobody targeting prostate-specific membrane antigen (anti-PSMA) and (b) a gastrin-releasing peptide (GRP) fragment targeting the GRP receptor (GRPR). It was shown that all modules within MNT-antiPSMA and MNT-GRP retained their functional properties. The MNT-antiPSMA construct demonstrated the capacity to accumulate specifically in the nuclei of prostate cancer cells with both low and high PSMA expression. Conversely, MNT-GRP exhibited selective nuclear entry exclusively in cells characterized by high GRPR expression.
Trans-splicing, the process where exons from two different pre-mRNA molecules are joined, is an efficient mechanism for generating diverse isoforms encoded by a single locus. This process has been most thoroughly studied in the mod(mdg4) locus of Drosophila. This locus comprises one gene encoding the constant N-terminal part of the Mod(mdg4) protein and five genes that encode over thirty 3'-exons clustered together, determining the diversity of C-terminal domains. In this study, transgenic lines were obtained where a strong polyadenylation signal from the SV40 virus was inserted into two 3'-exon clusters with the aim of halting transcription and, consequently, reducing the frequency of trans-splicing between the constant exons of the mod(mdg4) gene and the 3'-exons of the modified clusters. Unexpectedly, it was found that SV40 polyadenylation signals had no effect on either the transcription level of the clusters or the efficiency of trans-splicing. The results indicate the ability of RNA polymerase II, transcribing the 3'-exons of the mod(mdg4) locus, to overcome even strong polyadenylation signals from SV40.
-Background Interleukin (IL)-6 plays an important role in the pathogenesis of comorbid rheumatoid arthritis (RA) depression. IL-6 inhibitors used to treat patients with RA may also have an antidepressant effect. - of this study is to evaluate the effectiveness of 24-week IL-6 inhibitor therapy with olokizumab (OKZ) in combination with or without psychopharmacotherapy (PPT) in patients with moderate to high RA activity. - A total of 125 patients with RA were included, 102 (81.6%) of them were women. The average age of the patients was 48.5 ± 12.6 years; the majority of the patients (86.4%) had high RA activity and had shown ineffectiveness with stable 12-week therapy using conventional synthetic disease modifying antirheumatic drugs (csDMARDs). Additionally, 34 (27.2%) patients had shown inefficiency with one or more biological DMARDs. According to the International Classification of Diseases, 10th revision (ICD-10), a psychiatrist diagnosed varying severity of depression (chronic or recurrent) in all patients during a semi-structured interview. At week 0, all patients were randomized using sequential numbers in a 2:2:1 ratio into one of three groups: in group 1, patients received csDMARDs + OKZ 64 mg subcutaneously once every 4 weeks (q4w) (n = 49); in group 2, patients received csDMARDs + OKZ 64 mg subcutaneously q4w along with PPT (n = 51); in group 3, patients received csDMARDs + PPT (n = 25). The study duration was 24 weeks. The severity of depression was assessed using the PHQ-9 (Patient Health Questionnaire 9) and MADRS (Montgomery-Asberg Depression Rating Scale) scales, and anxiety was assessed using the HAM-A (Hamilton Anxiety Rating Scale) scale. Projective experimental psychological techniques were also used. - After 12 and 24 weeks of therapy, a significant decrease in the severity of depression and anxiety was observed in all patients' groups. However, the differences between the final and initial values of the scales filled in by a psychiatrist were statistically significantly greater (p < 0.001) in the groups of patients receiving PPT: in group 2 (ΔMADRS24-0 = -20.2 ± 6.57; ΔHAM-A24-0 = -13.2 ± 5.68) and group 3 (ΔMADRS24-0 = -17.8 ± 4.73; ΔHAM-A24-0 = -13.4 ± 4.41), compared with the group 1 (ΔMADRS24-0 = -5.42 ± 7.14; ΔHAM-A24-0 = -4.58 ± 6.80). There were no significant differences between the groups according to the PHQ-9 depression questionnaire (in group 1, ΔPHQ-924-0 = -4.89 ± 4.87; in group 2, ΔPHQ-924-0 = -6.73 ± 4.97; in group 3, ΔPHQ-924-0 = -7.26 ± 5.58, respectively), despite a greater decrease in the severity of depression observed in the groups with PPT. According to a semi-structured interview with a psychiatrist and in accordance with the criteria of ICD-10 the proportion of patients without depression 24 weeks after the start of therapy was significantly higher in the groups receiving PPT: 84.3% in group 2, 100% in group 3, and 16.3% in group 1. - n patients with moderate/high RA activity and comorbid depression, OKZ without PPT can lead to a decrease in the severity of depression or, less often, to a complete regression of depressive symptoms, mainly in patients with minor depression. OKZ therapy without PPT also reduces the severity of anxiety, but does not eliminate it completely. The combination of OKZ and PPT is optimal for achieving complete regression of depression and anxiety in this category of RA patients.
A novel trend in anticancer therapy is based on the combination of cytotoxic and metabolic drugs. Bach1 is a transcription factor activating glycolysis and increasing proliferation and metastatic potential in cancer cells. The cytotoxic effect was evaluated for a combination of rotenone, an inhibitor of mitochondrial respiration, and two different inhibitors of Bach1 transcription factor in the prostate cancer cell lines (PC3 and Du145) and colorectal cancer (HT-29 and HCT-116) in a kinetic mode. An enhancement of the cytotoxic action of the combination therapy was observed only for the prostate cancer cell lines PC3 and Du145. No enhancement of cytotoxicity of zinc porphyrin in the presence of rotenone was observed for the НСТ-116 line. The HT-29 line was not sensitive to either inhibitor or their combination with rotenone at 24 h incubation. According to the PCR results, HT-29 was the only line showing an extremely high activation of heme oxygenase 1 (HMOX1) in the presence of the Bach1 inhibitor, pointing to the highest level of the antioxidant defense in this cell line. The sensitivity of the prostate cancer cell lines to the combination therapy points to the significant differences in the metabolism between the prostate and colorectal cell lines.
Boron Neutron Capture Therapy (BNCT) is one of the innovative methods for treating oncological diseases. Its selectivity is based on the targeted delivery of the boron-10 isotope to tumor cells, followed by neutron irradiation, the 10B(n, α)7Li reaction occurs with a local release of 2.79 MeV of energy. Budding boron delivery agents are nanoscale systems. This study evaluated in vitro cytotoxicity, accumulation, and retention of elemental boron nanoparticles, synthesized by laser ablation and laser fragmentation, in U87 and BT474 tumor cells and BJ-5ta fibroblasts. It was shown that both types of nanoparticles exhibit low cytotoxicity at therapeutically relevant concentrations. Boron accumulation was maximal after 24 h of incubation and was significantly higher in tumor cells, especially in the BT474 cell line, compared to fibroblasts. The obtained data indicate the promise of these nanoparticles as boron delivery agents for BNCT.