搜索结果：Acta naturae

 Metagenomic studies have revealed the taxonomic composition of the taiga tick (Ixodes persulca tus) microbiome, whereas metaproteomic data has provided information on the biochemically active fraction of the microbial community residing in the tick. The aim of this study was to characterize the biological pro cesses taking place within the microbiome of the taiga tick I. persulcatus using a metaproteomic approach. To expand the range of identifiable proteins, we used two trypsin concentrations in sample preparation for mass spectrometric analysis. The metaproteomes of unfed female and male ticks were analyzed, which ena bled identification of protein products encoded by 2,100 genes from microorganisms belonging to 203 bacteri al and fungal species. Increased abundance of proteins associated with Ascomycota fungi, particularly abun dant in females, were detected. Proteins from the pathogenic Rickettsia and Borrelia species were identified. These findings enable a transition from a taxonomic metagenomic description to a functional analysis of the microbial consortium role in the physiology of the vector tick, particularly given the identified microbiota differences related to the tick sex.

R-loops that contain a DNA:RNA hybrid and unpaired single-stranded DNA are important determinants of normal cell physiology and of the pathogenesis of numerous diseases. Although several different approaches to R-loop mapping in the genome have been developed, these techniques can produce conflicting results. In order to assess their robustness, a recent study by Chedin et al. compared the R-loop genomic distribution assessed using different methods in normal and cancer cell lines. Importantly, that study assumed a high degree of similarity between R-loop genomic distributions across different cellular types. Here, we compared DRIP datasets produced using the same protocol in different cell lines to show that only 26% of R-loop peaks are shared between chronic myeloid leukemia-derived HAP1 cells and human pluripotent stem cells. Meanwhile, HAP1-derived double knockout cell lines are characterized by much higher fractions of R-loop peaks that are identical both to each other (most of them) and to the R-loop peaks of their parental line (71 and 55%). We conclude that cellular type represents a major determinant of R-loop genomic distribution and, therefore, that only a systematic comparison of a large array of various cell/tissue type-derived R-loop datasets may address the inconsistencies between different R-loop mapping techniques.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for the stereoselective synthesis of chiral amines, which are key building blocks in pharmaceuticals and chemical manufacturing. Fundamental research on enzymatic transamination includes the classical works of Alexander Braunstein, who discovered the transamination reaction; David Metzler, who studied the spectral properties of PLP-dependent enzymes; Esmond Snell, who investigated the kinetics of PLP-dependent enzymes; as well as studies by other Russian and international researchers. Despite extensive studies on PLP-dependent transaminases, their practical application remains limited. In addition to the unfavorable equilibrium of the transamination reaction and the narrow substrate specificity of transaminases, their stability under manufacturing conditions is a major constraint. Transaminase stability encompasses not only the structural integrity of the protein globule, but also the enzyme's ability to retain the PLP cofactor. PLP dissociation leads to enzyme inactivation and termination of the reaction. Modern biocatalytic processes are predominantly designed for aqueous-organic media to increase the solubility of hydrophobic substrates to hundreds of grams per liter. Under these conditions, the stability of transaminases, as with other enzymes, decreases. In the context of these challenges, this work investigates the efficiency of PLP binding as a factor in stabilizing the active holoenzyme of the transaminase from Desulfomonile tiedjei in various aqueous-organic media. The study analyzes the transaminase stability and catalytic activity in the presence of methanol, DMSO, and Cyrene (up to 20% v/v), both in incubation mode and under reaction conditions. Particular attention is paid to the analysis of the effect of the amino acid substitution T199Q in the cofactor-binding region on the enzyme's resistance to organic solvents. The present study contributes to addressing the practical problem of stabilizing transaminases in aqueous-organic media. The results also deepen our understanding of the molecular basis of the stability of PLP-dependent enzymes.

Immune-mediated inflammatory diseases affect a substantial proportion of the global population, and tumor necrosis factor (TNF) plays a central role in their pathogenesis. The most common diseases in clude rheumatoid arthritis (RA), Crohn's disease, psoriasis, multiple sclerosis, and septic shock. All of these conditions are characterized by excessive production of TNF, which activates downstream signaling pathways contributing to disease development and progression. To improve the quality of life in patients with TNF overproduction, anti-TNF agents such as TNF receptors and monoclonal antibodies are used. However, the availability of these therapies is limited. Therefore, the development of novel, more affordable TNF inhibi tors with comparable efficacy and improved safety remains a pressing issue. This review summarizes recent advances in the development of promising TNF inhibitors, including those derived from orthopoxvirus im munomodulatory proteins.

BALB/C mice were subjected to vestibular loading (rotation in individual containers at a speed of 80 rpm) for 8 h. As a result of this loading, the animals exhibited a decrease in horizontal and vertical locomotor activity, which returned to the control levels after 5 days. An immunohistochemical study of mi croglia and astrocytes in the lateral vestibular nuclei (LVN) revealed elevated levels of protein markers for astrocytes (GFAP) and microglia (Aif1) one hour and 5 days after the stimulation. These changes were indic ative of a gradual development of neuroinflammation in the LVN, which lasted for at least 5 days. Microglia, which appeared in branched shape in control animals, acquired an amoeboid reactive shape after vestibu lar loading. Moreover, expression of the genes coding for these proteins remained at the control level one hour after the stimulation and showed a reduction after 5 days. It is assumed that such a decrease helps re solve the neuroinflammation, preventing it from becoming chronic. Neuroinflammation in the acute phase is known to play a protective role and is required for plastic rearrangements of neuronal and glial networks. Transition to the chronic phase results in neuronal damage. The results of this study would allow one to de termine the period when it is reasonable to use anti-inflammatory therapy to mitigate damage. The applied model of vestibular stimulation allows one to solve problems when studying plastic rearrangements in the brain structures of the vestibular system under high-intensity sensory load.

The influenza virus causes seasonal epidemics throughout the world. At the same time, the rapid mutation of the virus renders the use of seasonal vaccines less effective. One of the approaches sought to improve influenza prevention is the use of monoclonal antibodies that are active against a wide range of influenza virus strains. In this study, the virus-neutralizing activity of the monoclonal antibodies CR9114, MHAA4549A, MEDI8852, C585, and 1G01 against the influenza virus was assessed. To this end, recombinant vectors based on adeno-associated virus (rAAV) encoding these antibodies were used. The rAAV vectors were expressed in mice in vivo, and the virus-neutralizing activity of the sera against the H1N1 and H3N2 influenza virus strains was assessed. Administration of rAAV-C585, rAAV-MHAA4549A, and rAAV-1G01 conferred 100% protection to mice challenged with a lethal dose of the H3N2 influenza virus. The efficacy of rAAV-CR9114 and rAAV-MEDI8852 against this influenza virus strain was lower, at 80 and 75%, respectively.

Fatty acid-acylated cholines, a recently identified class of endogenous compounds, have been de tected in both human and animal organisms. Our prior work established that oleoylcholine (Ol-Chol), and other acylcholines, at micromolar levels, modulate the cholinergic system and are suitable as cationic lipids for introducing nucleic acids into human and animal cells. The present research examines the interaction with the nicotinic acetylcholine receptors (nAChR) of two ionic forms of Ol-Chol and two synthesized cat ionic lipids, each featuring a quaternary ammonium moiety and two oleic acid chains. A radioligand bind ing assay revealed that the affinity of acylcholines and synthetic cationic lipids for the muscle-type nAChR surpasses that for the human neuronal α7 nAChR by a factor of 2-5.5. Oleoylcholine iodide demonstrated a two-fold higher efficacy of mesylate in binding to the orthosteric site of muscle and α7 nAChR. In a func tional calcium imaging assay, both compounds exhibited superior inhibition of α7 nAChR by several orders of magnitude, suggesting potential interaction with allosteric binding sites. Compared to oleoylcholine, syn thetic cationic lipids demonstrated markedly reduced efficacy in binding to α7 nAChRs and, in contrast to oleoylcholine, induced a substantial cytotoxic impact on SH-SY5Y neuroblastoma cells, a phenomenon unaf fected by specific nAChR ligands. As a result, the nAChR-inhibitory properties are attributed to the quater nary ammonium group present in all studied compounds. However, the modification of the lipophilic moiety with two oleic acid residues curbs these properties but enhances cytotoxic activity through an alternative mechanism independent of nAChR.

Parkinson's disease (PD) is one of the most common chronic neurodegenerative diseases. PD is characterized by the dysfunction of multiple body functions caused by changes in the expression of a large number of genes. Current evidence suggests that changes in the innate immunity and neuroinflammation may play an important role in the pathogenesis of the disease. However, the exact mechanism through which immune dysfunction develops in the context of PD pathogenesis remains unclear. In this study, with the use of transcriptome sequencing (RNA-seq), followed by quantitative PCR, we managed to detect a differential expression of the genes involved in the immune activity in neural progenitors (NPs) and glial cells derived from induced pluripotent stem cells from healthy donors (HDs) and PD patients carrying mutations in the PARK2 gene. Expression of many of the genes involved in a number of innate immune signaling pathways (in particular, in the canonical NFκB, non-canonical NFκB, the TNFα/NFκB, IL6/STAT3, IL2/STAT5 pathways, as well as the IFNγ and IFNα response) in cells from PD patients was found to be reduced compared to that in the cells from healthy donors. A mechanism for regulating these signaling pathways in the neural precursors of PD patients carrying mutations in the PARK2 gene is proposed.

Finding the optimal combination of drugs for the effective inhibition of cancer cell growth is an extremely important task today, as the number of such drugs continues to grow. There are several approaches to determining the nature of drug interactions, allowing one to establish whether they are additive, synergis tic, or antagonistic. One such approach is described here, and it is demonstrated how to quantitatively meas ure the degree of interaction between two drugs. It is shown that human peripheral blood serum and EGF modulate the activity of HER2-targeted drugs in inhibiting the proliferation of HER2-positive BT474 and SK-BR-3 cells. We compared the effect of blood serum samples from breast cancer (BC) patients and healthy donors on the action of trastuzumab. Using the proposed method, it is possible to calculate the Combination Index (CI). For 17 serum samples from healthy donors, the mean CI was 0.396, while for 19 serum samples from patients with BC, the mean CI was 0.214. These results indicate a synergistic interaction between tras tuzumab and blood serum in both groups. We also found significant differences in CI values between healthy donors and breast cancer patients: blood serum samples from patients enhance the effect of trastuzumab to a greater extent.

Viral diseases represent an increasingly serious threat for potato production all around the world, including in the Russian Federation, which leads to a significant decrease in potato crop yield, quality, and shelf life. In this study, we carried out screening of potato leaf and tuber samples collected from commercial potato fields to determine the spread of potato viruses in 16 regions of the European part, and two regions in the Ural Federal District, of the Russian Federation. The samples were sequenced, and full-length viral genomes were subsequently assembled de novo. A phylogenetic analysis of the identified virus variants was performed to assess their genetic diversity and possible origin. It has been shown that the most dangerous and economically important potato virus Y (PVY) is widespread and is represented by recombinant variants, NTNa, NTNb, and N-Wi being the most common ones. The second most common virus was potato virus M (PVM), which was frequently encountered in conjunction with potato virus S (PVS). The presence of potato leafroll virus (PLRV), which is recognized as an economically detrimental potato pathogen, along with PVY, has been found in two Russian regions. Mixed infections were detected in at least half of the studied samples, many containing both PVY and PVM (about one-third of all the samples). The data on the evolutionary variability of virus populations lay the groundwork for developing innovative strategies meant to contain a broad range of viruses and their strains using specifically designed double-stranded RNA (dsRNA).

The advent of the T-cell engineering technology using chimeric antigen receptors (CARs) has revolutionized the treatment of hematologic malignancies and reoriented the direction of research in the field of immune cell engineering and immunotherapy. Regrettably, the effectiveness of CAR T-cell therapy in specific instances of hematologic malignancies and solid tumors is limited by a number of factors. These include (1) an excessive or insufficient CAR T-cell response, possibly a result of both resistance within the tumor cells or the microenvironment and the suboptimal structural and functional organization of the chimeric receptor; (2) a less-than-optimal functional phenotype of the final CAR T-cell product, which is a direct consequence of the manufacturing and expansion processes used to produce CAR T-cells; and (3) the lack of an adequate CAR T-cell control system post-administration to the patient. Consequently, current research efforts focus on optimizing the CAR structure, improving production technologies, and further developing CAR T-cell modifications. Optimizing the CAR structure to enhance the function of modified cells is a primary strategy in improving the efficacy of CAR T-cell therapy. Since the emergence of the first CAR T-cells, five generations of CARs have been developed, employing both novel combinations of signaling and structural domains within a single molecule and new systems of multiple chimeric molecules presented simultaneously on the T-cell surface. A well thought-out combination of CAR components should ensure high receptor sensitivity to the antigen, the formation of a stable immune synapse (IS), effective costimulation, and productive CAR T-cell activation. Integrating cutting-edge technologies - specifically machine learning that helps predict the structure and properties of a three-dimensional biopolymer, combined with high-throughput sequencing and omics approaches - offers new possibilities for the targeted modification of the CAR structure. Of crucial importance is the selection of specific modifications and combinations of costimulatory and signaling domains to enhance CAR T-cell cytotoxicity, proliferation, and persistence. This review provides insights into recent advancements in CAR optimization, with particular emphasis on modifications designed to enhance the therapeutic functionality of CAR T-cells.

Mitochondrial dysfunction is one of the pathogenetic mechanisms of neuronal damage during aging. The high energy dependence of neurons makes them particularly vulnerable to age-related changes accompanied by oxidative stress and impaired energy metabolism. The maintenance of a pool of functional mitochondria is regulated by mitophagy, which ensures the utilization of damaged organelles, thereby preventing the progression of mitochondrial dysfunction. Brain aging is accompanied by a reduced level of activity of metabolic processes, aggravated mitochondrial dysfunction, and an increased risk of developing neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review highlights the molecular and signaling pathways of mitophagy and its dysregulation during physiological and pathological aging, which is of particular interest for identifying pharmaceutical targets and developing potential therapies for neurodegenerative conditions.

Antibiotic resistance threatens global healthcare. In clinical practice, conventional antibiotics are becoming gradually less effective. Moreover, the introduction of new antimicrobial agents into clinical practice leads to the emergence of resistant pathogenic strains within just a few years. Hence, the development of platforms for massive creation and screening of new antimicrobial agents is of particular importance. Massive parallel screening will greatly reduce the time required to identify the most promising drug candidates. Meanwhile, DNA-encoded antimicrobial agents offer unique opportunities for the high-throughput development of new antibiotics. Here, the yeast Pichia pastoris was engineered to produce a panel of antimicrobial peptides (AMPs), followed by high-throughput screening of AMP producers that inhibit bacterial growth in situ. Yeast clones producing thanatin and protegrin-1 exhibited the highest level of antimicrobial activity among the panel of AMPs under investigation. The production level of recombinant thanatin was significantly higher than that of protegrin-1, which correlates with its low toxicity. The designed technique of massive assessment of the activity of DNA-encoded antimicrobial agents enables the identification of drug candidates with an increased therapeutic index. Further development of methods for a rational design of artificial diversity in AMPs, followed by deep functional profiling of antimicrobial activity, will yield new AMPs with improved therapeutic characteristics.

MicroRNAs are endogenous, small non-coding RNAs that regulate gene expression at the post-transcriptional level by cleaving target mRNAs. Mature microRNAs are products of the processing of their primary transcripts (pri-miRNAs). Now, it has been discovered that the products of the translation of some plant pri-miRNAs are peptide molecules (miPEP). These peptides have the capacity to physically interact with their open reading frames (ORFs) in the transcribed pri-miRNAs and, thus, positively regulate the accumulation of these RNAs and the corresponding mature microRNAs. Most conserved microRNAs play an important role in plants development and their response to stress. In this work, we obtained transgenic Physcomitrium patens moss plants containing Brassica oleracea miPEP156a ORF in the genome under the control of a strong 35S cauliflower mosaic virus promoter and analyzed the effect of the exogenous peptide on the transcription of this ORF in the protonemata of two transgenic moss lines. It turned out that the chemically synthesized peptide miPEP156a increases the accumulation of its own mRNA during moss culture growth, as was previously shown in studies by foreign researchers and in our own work for a number of peptides in monocotyledonous and dicotyledonous plants. These findings confirm that pri-miRNA regions that are located outside the coding region of the peptide are not required for transcriptional activation. Moreover, we have also succeeded in showing that the presence of a specific promoter of the microRNA gene does not affect the phenomenon of transcription activation; this phenomenon per se is not species-specific and is observed in transgenic plants, regardless of the origin of the miPEP.

Proprotein convertases (PCs) constitute an enzyme family that includes nine highly specific human subtilisin-like serine proteases. It is known that the PCs mRNA levels vary in tumors, and that these proteases are involved in carcinogenesis. Thus, PCs may be considered as potential markers for typing and predicting the course of the disease, as well as potential targets for therapy. We used quantitative real-time PCR to evaluate the expression levels of PC genes in the paired samples of tumor and adjacent normal tissues derived from 19 patients with esophageal squamous cell carcinomas. We observed a significant enrichment of PCSK6, PCSK9, MBTPS1, and FURIN mRNAs in the tumor tissue, which may be indication of the involvement of these PCs in the development and progression of esophageal cancers. Additionally, cluster analysis of PC expression alteration patterns in tumor compared to normal adjacent tissues (esophageal and previously analyzed lung tissue samples) revealed a limited set of scenarios for the changes in PC expression. These scenarios are implemented during malignant transformation of lung and esophagus cells, as well as, probably, the cells of other organs. These findings indicate that PC genes may be important markers of human cancers.

Boron neutron capture therapy (BNCT) is a rapidly developing field of radiation therapy for cancer that is based on the accumulation of the radiosensitive 10B isotope in cancer cells, followed by tumor irradiation with thermal neutrons. Widespread use of BNCT in clinical practice remains limited because of the poor accumulation of boron-containing (10B) drugs in the tumor or their high toxicity to the body. This study focuses on the engineering of tumor-specific liposomes loaded with 4-L-boronophenylalanine (4-L-10BPA) for application in boron neutron capture therapy. According to the spectrophotometry and ICP-mass spectroscopy data, the 4-L-10BPA-to-liposome molar ratio is ~ 120,000. Liposomal targeting of human epidermal growth factor receptor 2 (HER2) was determined by HER2-specific designed ankyrin repeat protein (DARPin)_9-29 on the outer surface of liposomes. DARPin-modified liposomes were found to bind to HER2-overexpressing cells and be effectively internalized into the cytoplasm. The ability of DARPin-functionalized liposomes to precision-deliver large quantities of 4-L-10BPA into cancer cells may open up new prospects for BNCT.

Red fluorescent proteins (RFPs) are often probes of choice for living tissue microscopy and whole-body imaging. When choosing a specific RFP variant, the priority may be given to the fluorescence brightness, maturation rate, monomericity, excitation/emission wavelengths, and low toxicity, which are rarely combined in an optimal way in a single protein. If additional requirements such as prolonged fluorescence lifetime and/or blinking ability are applied, the available repertoire of probes could dramatically narrow. Since the entire diversity of conventional single-component RFPs belongs to just a few phylogenetic lines (DsRed-, eqFP578- and eqFP611-derived being the major ones), it is not unexpected that their advantageous properties are split between close homologs. In such cases, a systematic mutagenetic analysis focusing on variant-specific amino acid residues can shed light on the origins of the distinctness between related RFPs and may aid in consolidating their strengths in new RFP variants. For instance, the protein FusionRed, despite being efficient in fluorescence labeling thanks to its good monomericity and low cytotoxicity, has undergone considerable loss in fluorescence brightness/lifetime compared to the parental mKate2. In this contribution, we describe a fast-maturing monomeric RFP designed semi-rationally based on the mKate2 and FusionRed templates that outperforms both its parents in terms of molecular brightness, has extended fluorescence lifetime, and displays a spontaneous blinking pattern that is promising for nanoscopy use.

This paper presents a new bioinformatics tool to meet the needs of researchers in the search for short (≥ 3) amino acid subsequences in protein sequences annotated in public databases (UniprotKB, SwissProt) and illustrates its efficacy with the example of a search for the EVHH tetrapeptide in the human proteome, which is a molecular determinant of amyloid beta and is involved in interactions that are crucial in Alzheimer's disease pathogenesis. The topicality of developing such a tool is, on the one hand, supported by experimental data on the role of short tetrapeptide motifs in the architecture of intermolecular interfaces. On the other hand, there are currently no software products for efficient search for short (≥3) amino acid sequences in public databases, which drastically limits researchers' ability to identify proteins with exact matches of short subsequences. This tool (PepString server, http://pepstring.eimb.ru/) allows one to use intuitive queries to retrieve information about all the proteins that contain sequences of interest, as well as their combinations.

Extracellular vesicles (EVs) are secreted by nearly all mammalian cells and play a major role in intercellular communication via the transport of various active biomolecules. In cancer, pathological EVs contribute to tumor progression by participating in metastasis, angiogenesis, and immune evasion. Recent advancements in EV research have revealed their potential as noninvasive biomarkers. This review addresses the latest advancements in EV isolation and characterization techniques, elucidates the molecular mechanisms underlying EV biogenesis, and examines their functional roles in cancer progression. Furthermore, we discuss emerging strategies that leverage EV profiling and molecular composition analysis, in conjunction with liquid biopsy technologies, offering possible breakthroughs in early cancer diagnosis and treatment monitoring. By synthesizing these insights, this review emphasizes the growing significance of EVs as versatile and powerful diagnostic tools in oncology.