Animal-model studies used to predict vaccine efficacy in humans are challenging because direct extrapolation of experimental results to humans is not possible for a number of reasons. Interspecies differences in pathogen-recognition receptors may explain differences between humans and rodents in the response to microbial vaccines. There are also species-specific B-cell and T-cell repertoires, as well as HLA presentation of dominant epitopes to CD4 and CD8 T cells. Because of these limitations, studies of candidate vaccines are conducted in several species of small and large animals, which is expected to yield data more transferable to humans. The aim of this study was a comparative assessment in mice and guinea pigs of the protective effect of the differing in antigenic composition 231ΔnlpD and EVΔlpxM plague pathogen strains upon a single immunization with them separately or in a mixture. Materials and methods. The protectivity of the studied strains was characterized by the immunity index in subcutaneous infection of mice and guinea pigs. Results. As in previous experiments [1], the introduction of monopreparations based on attenuated 231ΔnlpD or EVΔlpxM strains selectively induced protection only in mice or guinea pigs, respectively. Immunization with a mixture of attenuated strains provided an immunity index of more than 108 for both types of laboratory animals. Conclusion. Single-stage subcutaneous immunization of mice and guinea pigs with attenuated Y. pestis strains 231ΔnlpD and EVΔlpxM enhances immunity to plague following subcutaneous challenge with wild-type strain 231.
Objective. To examine changes in lactobacilli composition in the vaginal microbiocenosis associated with bacterial vaginosis (BV) and to assess the frequency of individual species. Material and methods. The study included 40 patients who presented with pathological discharge from the genital tract and had a confirmed diagnosis of BV based on the Amsel criteria. Quantitative assessment and typing of lactobacilli were performed using real-time PCR. Results. All women with BV were found to have lactobacilli present in quantities ranging from 0.01% to 100% of the total bacterial content. Analysis of the distribution of the lactobacilli index (LI), which indicates the proportion of lactobacilli in the total bacterial content, revealed three peaks corresponding to samples with low, intermediate, and high values. During the typing, all four species of lactobacilli characteristic of the vaginal microbiocenosis were identified: L. iners, L. crispatus, L. jensenii, and L. gasseri/L. johnsonii. No samples were found that lacked at least one of these species; L. crispatus and L. iners were the most frequently detected. Despite a general trend toward decreased LI levels in BV, patients were categorized into three subgroups based on their LI values: high, moderately reduced, and low. Conclusion. The positive correlation observed for L. iners and the negative correlation for L. crispatus with the degree of dysbiosis do not, in our opinion, reflect direct cause-and-effect relationships regarding BV, as these correlations are more closely associated with lactobacilli content than with clinical manifestations of the disease. A significant proportion of BV cases with the preservation of lactobacilli dominance suggests the existence of at least two forms of this syndrome. One of them, despite the presence of clinical symptoms, is not associated with a decrease in the number of lactobacilli in the vaginal microbiome.
The aim of the research was to study the effect of volatile organic compounds (VOCs) of various types, secreted by microorganisms (ketones, alcohols, terpenes, dimethyl disulfide (DMDS)), on the planktonic growth and formation of the Gram-negative marine bacterium Vibrio harveyi BB120 biofilms. This pathogen bacterial species causes infectious diseases of various marine organisms (vertebrates and invertebrates), which hinders the development of marine aquaculture. Materials and methods. The cells were grown on 96-well polystyrene plates with light shaking. The planktonic growth was determined by optical density. The level of formed biofilm was determined by staining them with crystal violet with subsequent ethanol extraction. The level of biofilm formation was estimated by the color intensity of the solution. Results. It was demonstrated that the effect of all studied VOCs led to a decrease of the planktonic growth of V. harveyi BB120. (–)-Limonene, β-ionone, and (+)-α-pinene had the strongest effect on bacteria; 2-phenylethanol had a slightly weaker effect. The level of biofilms (as a percentage of the control without VOC) in all cases was higher when exposed to the indicated VOC than the level of planktonic growth of V. harveyi BB120, which indicates a higher resistance of the cells in biofilms to VOC. The patterns of the effect of various VOCs on the biofilm formation in the studied VOC ranges were different. With an increase in the amount of the added 2-pentanone and DMDS, an increase in biofilm formation was observed; the addition of (–)-limonene, 2-phenylethanol, isoamyl alcohol, and (+)-α-pinene to the culture led to a decrease in the level of biofilms. Conclusions. The inhibitory effect of VOCs with different structures (2-pentanone, β-ionone, isoamyl alcohol, 2‑phenylethanol, (–)-limonene, (+)-α-pinene, dimethyl disulfide) on the growth of V. harveyi BB120 was for the first time demonstrated. These data are of interest for the control of infections caused by V. harveyi and other pathogenic representatives of the genus Vibrio.
The aim of the study was to analyze biotechnological approaches to the construction of an internal control sample (ICS) in the manufacture of gene diagnostic preparations and the development of such an ICS for PCR test systems produced by the Microbe Russian Anti-Plague Institute. Materials and methods. To create an artificial nucleotide sequence that no known organism has, the online resource Random DNA Sequence Generator was used. Specific primers for generation of artificial nucleotide sequences were selected using DNA-Works software (v3.2.4.). Oligonucleotides were synthesized using the standard solid-phase amidophosphite method on an ASM-800 automatic DNA synthesizer. Cloning of the amplification products used as the ICS was performed in the pAL2-T vector. Bacteria culturing and bacterial clone selection were carried out on Luria-Bertani (LB) agar medium containing 50 μg/mL of ampicillin as a selective factor. Results. The paper presents data on biotechnological approaches to constructing ICSs. In the first case, a 174-bp region of the green fluorescent protein gene sGFP-206 (gfp) of the jellyfish Aequorea victoria was selected to create an exogenous ICS, since the presence of this sequence in the studied samples was virtually excluded. In the second case, an artificial nucleotide sequence was created that no known organism possesses. When developing a diagnostic set of reagents for detecting V. cholerae strain DNA in the studied clinical samples using PCR with hybridization-fluorescence registration of results, one of the tasks was to construct an endogenous internal control sample. It was proposed to use human DNA genes as a DNA target for the ICS. Studies were conducted to assess the efficiency of amplification of ICS fragments with primer annealing temperatures of 49, 58, and 60°C according to the working protocols of the produced test systems and the absence of a negative impact on the efficiency of the studied gene-fragment amplification reaction. Conclusions. The employed approaches allowed three variants of ISOs to be created for implementation of control with the help of RT-PCR of produced sets of reagents to identify the DNA of pathogens of especially dangerous infectious diseases, such as cholera, plague, anthrax, brucellosis, and tularemia with hybridization-fluorescence registration of results.
Abstract—Objective. New advances in genomic editing field have led to the creation of DNA base editors. Such a tool allows to make precise changes to the genome without double-stranded DNA breaks, which reduces its mutagenicity. As a result, DNA base editors are becoming a popular tool for gene therapy and transgenesis of cell lines and laboratory animals. Certainly, there is a need not only to evaluate the effectiveness of editing, but also to create new forms of DNA base editors through evolution. The purpose of our work is to create system to evaluate the effectiveness of DNA base editors and their evolution to obtain new forms. Materials and methods. Classical methods of genetic engineering were used in the work. The proportion of cells with the target effect was assessed by flow cytometry. Results. It is shown that the created systems simulate transgene expression depending on the state of its start-codons. It was revealed that editing of the first start-codon plays a key role in the initiation of translation or its inhibition. Conclusions. Based on the developed systems, it is possible to evaluate the effectiveness of editing the first start-codon of the HIV-1 gag gene and the evolution of DNA base editors to obtain new forms.
Objective—to investigate the possibility Aim was to study a possibility of encapsulation of hydrophilic B/C and P forms of riboflavin into calcium alginate particles, to study riboflavin release from the particles and its antibacterial properties. Materials and methods. Alginate particles were obtained by cross-linking of sodium alginate with calcium ions and loaded in situ with B/C and P types of riboflavin. The strains of Pseudomonas aeruginosa bacteria were used to evaluate antibacterial activity. Results. Two polymorphic modifications and salt form of riboflavin (types A, B/C, and P) were successfully encapsulated into calcium alginate particles of about 600 μm in size. Using the methods of optical and electron microscopy, it was demonstrated that types A and B/C retain the shape of crystals and crystalline intergrowths, while type P riboflavin is uniformly distributed in the gel matrix. X-ray phase analysis confirmed the preservation of the crystalline form of type A riboflavin, but revealed that the crystalline aggregates of type B/C become amorphous during encapsulation. It was demonstrated that alginate particles containing type B/C riboflavin are characterized by a prolonged release of the active substance into deionized water, and the equilibrium concentration for type P is already reached within 30 min. It was established that the particles loaded with types B/C and P exhibit a pronounced antibacterial activity against Gram-negative bacteria. Conclusions. The encapsulation of types B/C and P riboflavin into alginate particles allows us to preserve the antibacterial activity of the substance and provide its controlled release.
The problem of predicting a culprit of the future influenza pandemic is analyzed in connection with the emerged epizootic of cows caused by the avian influenza H5N1 strain. The emerged epizootic in cows is considered as acquisition of a new host by the H5N1 virus. The reproductive cycle of the H5N1 virus is adapted to a high temperature. In cows, the body temperature varies and is closer to the body temperature in birds than in human. The H5N1 virus nucleoprotein is distinguished by a high content of arginine and lysine, which causes the optimum of viral replication and transcription at elevated host body temperature. In this aspect, the acquisition of cows (but not human) as a host by the H5N1 virus is more optimal. Another peculiarity of the H5N1 virus, which excludes the possibilities of its transmission into the human population, is associated with the peculiarities of its internal proteins (particularly, with a biomarker of influenza virus pandemicity). The invariance matrices of all primary structures of internal proteins of the H5N1 virus differ significantly from those in the strains of influenza pandemics in 1918, 1957, 1968, and 2009, which also does not support the prediction about the H5N1 virus as a herald of a future influenza pandemic. Conclusions. Based on the history of the emergence of last four influenza pandemics and comparison of biological properties and molecular characteristics of hemagglutinins of influenza viruses of the H1N1 and H3N2 subtypes, the H1N1 virus subtype is assumed to be the most likely causative agent of a future influenza pandemic.
The aim of the work. Design and investigation the properties of a prototype for a single intranasal vaccine based on a recombinant Sendai virus Moscow strain expressing as an immunogen the S protein of the JN.1 variant, and an assessment of its cross-activity against a range of other SARS-CoV-2 variants. Materials and methods. A recombinant Sendai virus with transgene S(JN.1) insertion between the P and M genes was obtained by genetic engineering and RT-PCR. Expression of the transgene S(JN.1) was analyzed by immunoblotting. The immunogenicity of the vaccine construct was evaluated by ELISA and virus-neutralization assays in BALB/c mice. The protection was assessed in K18-hACE2 mice by the level of reduced replication (quantitative RT-PCR) and the decrease in the infection titer (on Vero E6 cells) of SARS-CoV-2 in the tissues of the nasal cavity and lungs of vaccinated animals. Results. Based on the Moscow strain of the Sendai virus, the vaccine construct Sen-S-JN.1(M) has been developed, which expresses the S protein of the circulating variant JN.1 of SARS-CoV-2. It has been shown that S protein is effectively exposed on Sen-S-JN.1(M) virions and induces the formation of a mucosal and systemic humoral immune response against SARS-CoV-2 variant JN.1 during a single intranasal vaccination of BALB/c mice. Analysis of the cross-neutralizing activity revealed a significant decrease in antibody titers against variants Delta, BA.1, XBB.1.5, and EG.5.1 while maintaining a high level of neutralization against XBB.1.16 and BA.5.2 variants. The K18-hACE2 mice vaccinated with Sen-S-JN.1(M) were well protected from SARS-CoV-2 (JN.1) infection due to a significant (more than 106 fold) reduction in viral replicative activity. None of the lung and turbinate samples from the vaccinated mice contained detectable levels of infectious SARS-CoV-2, which indicates that the infection had stopped. Conclusions. The recombinant virus Sen-S-JN.1(M) is a promising vaccine construct and can provide cross-protection against circulating and evolutionarily similar variants of the SARS-CoV-2 Omicron lineage.
Introduction. Bacteria need CRISPR-Cas systems to form an adaptive immune response necessary for protection against infectious agents. In addition, in recent years, CRISPR-Cas systems have been actively studied in the context of the development of new methods for editing the genome, which have great potential in the development of new strategies in the fight against infectious and other diseases. The mechanism of formation of bacterial adaptive immunity using CRISPR-Cas systems is in the ability of bacteria to integrate fragments of foreign DNA (for example, bacteriophage DNA), which are called spacers, into their genome, which further allows bacteria to recognize and protect themselves from these infectious agents. However, not much is known about the molecular mechanisms of this process, which limits a lot the ability of scientists to understand and apply CRISPR-Cas systems. In particular, the question of the role of non-CRISPR-Cas components of bacterial cells in the formation of CRISPR adaptation remains open. Such components, for example, are intracellular nucleases, which play a role in the CRISPR adaptation process we aimed to study within our scientific project. It has been previously proven that nucleases are involved in the preparation of a foreign DNA fragment for insertion into the bacterial DNA (CRISPR cassette), which is necessary to ensure CRISPR adaptation –it has to have the certain structure and the correct length. It has been shown that aberrations in the formation of the 5' ends of prespacers do not affect their incorporation into the CRISPR cassette and the efficiency of adaptation. Material and methods. Two mutant strains, Bl21-AI Δxni (Flap endonuclease ExoIX) and Bl21-AI ΔpolA exo (PolI), were constructed. The resulting strains were then transformed with the pCDF Cas1/Cas2 plasmid. Oligoelectroporation experiments were performed with naive adaptation induced by the addition of arabinose and IPTG. High-throughput sequencing was performed using the MiniSeq™ Sequencing System (Illumina, San Diego, CA, USA). Results. Here we determined that DNA polymerase I likely removes extra nucleotide at the 5' ends of spacer precursors, thereby allowing spacer precursors with extended 5' ends to be inserted into a CRISPR cassette as spacers. Conclusion. The knowledge gained significantly expands the understanding of the process of CRISPR adaptation. It is of fundamental and practical significance and opens up new opportunities in the understanding and application of CRISPR-Cas systems, for instance, aiding the development of new methods to combat infectious and other diseases.
Aim. The current study aims to investigate changes in the expression of proprotein convertases (PCs) genes during the development of lung tumors. PCs are a family of highly specific subtilisin-like serine endopeptidases of mammals that process precursors of various proteins and peptides. Nine genes encoding PCs have been identified in the human genome that are essential for normal body functioning. Additionally, PCs can activate proteins involved in carcinogenesis, as well as the expression levels of their genes were shown to correlate with tumor aggressiveness and patient survival. We previously evaluated the expression of all nine PC genes using quantitative PCR on paired samples of lung tumor and adjacent normal tissue. For the first time, we identified four distinct patterns of PCs gene expression change in tumor tissue, which we have called scenarios. For three of them, covering more than two thirds of the samples, a dominant change in the expression of one PC gene in the tumor tissue was shown. These results may indicate the existence of a limited number of possible options for changes in PCs gene expression during the malignant transformation of lung cells. However, these results need to be confirmed using expanded cohort of tumor samples. Materials and methods. To confirm our previous findings, we analyzed the expression of PCs genes using modern methods of mathematical statistics and data from three previously published studies, which evaluated gene expression through high-throughput RNA sequencing in paired tumor and normal tissue samples from 194 patients with non-small cell lung cancer. Results. Our meta-analysis confirmed that the changes in PCs gene expression in lung tumor tissue compared to adjacent normal tissue follow a limited set of possible scenarios, each having a unique profile of PCs gene expression. Conclusions. The reasons for implementing each scenario may be linked to the origin of tumors, their mutation status, characteristics of the tumor microenvironment, and other factors. Correspondingly, these scenarios may correlate, for example, with tumor aggressiveness and resistance to therapy, and therefore may potentially be used to choose the treatment approach and/or to predict the course of the disease. However, this issue requires further research.
Heterochromatic position effect variegation (PEV) of the gene comprises euchromatic gene inhibition upon its transition in the area of heterochromatin either at the same chromosome, for example, in case of inversions (cis-acting PEV), or at the contact of the euchromatic gene with the heterochromatin associated with the allele of the same gene in the homologous chromosome in the three-dimensional nuclear volume (trans-acting PEV). The reverse PEV consists in heterochromatic gene inhibition in case it is placed in euchromatic environment. Most PEV data were obtained using genetic system of Drosophila melanogaster that has four chromosomes combined in the chromocenter. Heterochromatic cis-acting PEV often takes place in case of invertions such as In(2)A4; In(1)wm4. Molecular mechanisms of cis-acting PEV include expression level changes of several genes due to the changes in the quantities of specific histone modifications, heterochromatin proteins (HP1) and specific small RNA including piRNAs. In case of cis-acting PEV the distribution of heterochromatic modifications (H3K9me2/3) and main heterochromatic protein (heterochromatin protein HP1a) from the heterochromatin to the euchromatin area is well studied and is associated with the expression inhibition of several genes. Heterochromatic trans-acting PEV has been thoroughly investigated only in a few cases including the invertion In(2)A4 and satellite DNA fragment insertion in brown gene (bwD). In both cases genomic rearrangements took place at the second chromosome of Drosophila melanogaster. Molecular mechanisms of trans-acting PEV are less studied than those of cis-acting PEV. It was shown that Su(Var)2-HP2, SAYP, SETDB1 participate in trans-acting PEV in case of the inversion In(2)A4. Perspective research in the field of cis- and trans-acting PEV includes the study of the role of prod and D1 mutations that influence the integrity of Drosophila melanogaster chromocenters.
The global aging of the population and the rising prevalence of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, pose a significant challenge to healthcare systems in developing countries. Non-human primates are regarded as the most relevant natural model for studying age-related neurodegeneration due to their high degree of similarity to humans in terms of brain anatomy, genetics, and cognitive functions. In the present study, brain specimens from 21 aged individuals (20—34 years old) representing several primate species were analyzed. Macroscopic examination revealed atrophy of the occipital lobes and ventriculomegaly. Histopathological assessment demonstrated consistent age-associated alterations across all specimens, including vascular sclerosis and hyalinosis, reduced neuronal density in the cerebral cortex, leukoaraiosis, vacuolar degeneration, and accumulation of lipofuscin — the so-called «aging pigment». These pathological features closely resemble those observed in human brain aging and neurodegenerative conditions. Therefore, aged non-human primates represent a valuable biological resource for investigating the pathogenesis of spontaneous neurodegeneration and for developing therapeutic strategies with high translational relevance to clinical medicine. This study constitutes the first systematic description in Russia of age-related neuropathological changes in non-human primates and paves the way for establishing translational models of neurodegenerative diseases based on natural aging.
The purpose of this review is to summarize current state of primate biobanks and their role in international scientific networks, as well as specialized genomic resources for research on hominids and other models. Modern methods of sample authentication and genetic passportization, such as microsatellite and SNP analysis, are presented, ensuring high-precision individual identification and quality control of biological materials. Special attention is given to the importance of standardization processes and international collaboration to enhance research efficiency and preserve genetic diversity. Conclusion. The authors decided that the integration of genetic information into digital ecosystems of primate biobanks leads to a transition from traditional collections to intelligent platforms utilizing advanced analysis and modeling technologies, opening new prospects for research and personalized medicine.
The aim. Determination of changes in the levels of circular RNAs (circRNAs) of the Mvp, Rgs9 and Dlgap4 genes in the frontal cortex of ischemic rats 24 h after transient occlusion of the right middle cerebral artery (tMCAO). CircRNAs are covalently closed biogenic RNA molecules that have increased metabolic stability, the ability to participate in the regulation of gene expression and act as potential biomarkers of diseases and therapeutic targets for treatment. Previously, we demonstrated the involvement of genes of the neurosignaling and inflammatory systems in the response to ischemic injury, and also revealed significant changes in the expression of circRNA of Rgs9 and Mvp genes in subcortical brain structures 24 h after tMCAO in rats. These genes encode proteins involved in the inflammatory response (Mvp) and nerve signal transmission (Rgs9). Here, the circRNA of the Mvp and Rgs9 genes were analyzed. Additionally, the circRNA of the Dlgap4 gene, which has been shown to play a role in ischemic stroke, was analyzed too. Materials and methods. Wistar rats, rat tMCAO model, real-time PCR, bioinformatics. Results. Changes in the levels of circRNAs of the Mvp, Rgs9, and Dlgap4 genes were studied in the frontal cortex, which contains predominantly regenerative penumbra cells, 24 h after tMCAO. We found that the circRNA of the Mvp gene demonstrated reliable increases in expression levels (fold change > 1.5; P < 0.05) in groups of ischemic rats relative to sham-operated animals, while the change in the level of circRNA of the Rgs9 and Dlgap4 genes was insignificant. Using bioinformatics, probable binding sites for microRNAs capable of controlling the work of genes of focal adhesion systems, neurotransmission and neurogenesis were identified in the circRNA sequences of the Dlgap4, Mvp and Rgs9 genes. Conclusions. The results allow us to assess the significance of circRNAs of Mvp, Rgs9 and Dlgap4 genes for the possibility of further creating promising diagnostic systems and choosing stroke treatment tactics on their basis.
