The Clostridioides difficile infection (CDI) is one of the most common nosocomial infections around. The key pathogenicity factors of this bacterium include the toxins A and B that cause the disease symptoms. Neutralization of these toxins is one of the promising strategies for CDI treatment. Here, we isolated a panel of single-domain antibodies to the toxin B CROPs domain. Two antibodies, TB5A7 and TB4A8, exhibiting potent neutralizing activity were modified to produce homodimeric forms. The TB5A7 and TB4A8 dimers were characterized by enhanced neutralizing activity and protected animals from a toxin B lethal challenge. The obtained antibodies may be used to develop new agents for CDI treatment.
The nonsense-mediated mRNA decay (NMD) pathway is a mRNA quality control mechanism which not only degrades deleterious transcripts but also orchestrates a large number of post-transcriptional regulatory programs through unproductive splicing. We have developed a robust metric derived from splicing quantification in the RNA-seq data to measure NMD efficiency at a sample level. We demonstrate that NMD efficiency varies substantially both between and within tissues, with the magnitude of the variation comparable to that observed upon knockdown of the core NMD factor UPF1. By analyzing TCGA cancer cohorts, we further show that, in many tumors, unproductive splicing events undergo coordinated changes towards either collective suppression or collective activation of NMD isoforms, which is indicative of global deregulation of the activity of the NMD pathway. Consistently, we observed a striking divergence of NMD efficiency in cancers from the tissue-specific baseline level, suggesting that tumors partially erase the NMD signature of their tissue of origin. The application of the developed metric to RNA-binding protein knockdowns made it possible to identify several novel potential regulators of NMD efficiency. In sum, this study provides a solid framework for quantifying NMD efficiency, describes its biological and clinical relevance, and opens new avenues for dissecting mechanisms of post-transcriptional gene expression regulation by the NMD pathway.
Macrophage dysfunction is a key pathogenetic mechanism in the progression of a wide range of human chronic inflammation conditions, including atherosclerosis, rheumatoid arthritis, and metabolic disorders. The CDK8 and CDK19 paralogous kinases, the subunits of the Mediator complex acting as transcription regulators, are essential modulators of the inflammatory response. This study addresses the role of CDK8/19 kinases in the macrophage inflammatory response via multiple mechanisms: activation of pro- and anti-inflammatory genes and surface markers, STAT1 pathway modulation, activation of glycolytic cascade genes, regulation of the dynamics of lipid inclusions, and phagocytic activity. Experiments have shown that CDK8 and CDK19 may exhibit functional divergence. Cdk19 knockout reveals that CDK19 plays a role in the suppression of the M1 response, as well as the regulation of lipid homeostasis and phagocytosis. Double Cdk8/19 knockout macrophages are characterized by an exacerbated anti-inflammatory response while preserving normal lipid accumulation and phagocytosis levels. Inhibition of CDK8/19 kinase activity reproduces these effects only partially, suggesting that both kinase-dependent and kinase-independent mechanisms of action exist. The identified effects reveal previously unknown regulation mechanisms of macrophage immunometabolism. Modulation of CDK8 and CDK19 activity can become a novel therapeutic target for a wide range of chronic inflammatory conditions and metabolic disorders associated with macrophage dysfunction.
Cancer remains a critical public health challenge, and developing novel approaches to cancer therapy is highly relevant. Targeted tumor therapy using antibody-drug conjugates (ADCs) has already demonstrated its efficacy in multiple tumors. Ganglioside GD2 is a promising target for ADC development. However, its functional properties, which are important for the cytotoxicity of ADCs, remain poorly understood. This study focuses on the mechanisms of receptor-mediated endocytosis for the conjugate formed between the anti-GD2 antibody ch14.18 and monomethyl auristatin E (MMAE), as well as the approaches for modulating this process using endocytosis inhibitors. Our findings demonstrate that anti-GD2 ADCs are efficiently internalized into tumor cells through various endocytic pathways, including the clathrin- and caveolin-mediated pathways, as well as macropinocytosis. The efficiency of ADC accumulation directly correlates with the level of GD2 expression on the tumor cell surface and is determined by the properties of the parental antibody, independent of the cytotoxic payload. Endocytosis inhibitors can modulate the functional properties of anti-GD2 ADCs, either decreasing or increasing the cytotoxic effects of the conjugates in GD2-positive cells. Nystatin, a specific inhibitor of caveolin-mediated endocytosis, increased ADC accumulation in cells and reduced the IC50 1.5- to 2.5-fold depending on the cell line. Although administration of nystatin together with anti-GD2 ADCs did not enhance tumor growth inhibition compared to ADC monotherapy in the GD2- positive mouse tumor model, further optimization of combination therapy conditions aimed at enhancing receptor-mediated endocytosis is a promising strategy for potentiating the antitumor effects of anti-GD2 ADCs.
Genetic variation in the genes involved in the maintenance of cellular homeostasis may influence susceptibility to type 2 diabetes (T2D). However, the nature of the interplay among these loci and their prognostic value remains unclear. In this study, we analyzed variants in the FOXO3A, FOXO3, FOXO1, HMOX1, and SIRT1 genes. The HMOX1 rs2071746*T/T genotype was associated with an increased risk of T2D (OR = 1.36, PFDR = 0.030), whereas the FOXO1 rs9549240 (OR = 0.52, PFDR = 0.002) and SIRT1 rs3758391 (OR = 0.80, PFDR = 0.015) genotypes exhibited a protective effect. We detected nonlinear interactions, including FOXO1 rs9549240*G + SIRT1 rs3758391*T + SIRT1 rs7895833*A and HMOX1 rs2071746*A + SIRT1 rs3758391*T + SIRT1 rs7895833*A, with significant synergistic effects (SF = 3.19 and 2.56, P < 0.03). Models incorporating these interactions achieved an AUC of 69.8%, which increased to 86.2% when combined with age, sex, and the body mass index. These findings suggest that interactions between the pathways regulating oxidative stress and metabolism may contribute to the genetic predisposition to T2D.
Cysteine-rich peptides belonging to the EPF/EPFL (epidermal patterning factor/epidermal patterning factor-like) family are common in many plants, from mosses to angiosperms. EPF/EPFL play an important role in morphogenesis: they regulate stomatal patterning, the functioning of the shoot apical and lateral meristems, inflorescence architecture, vascular development, growth of leaf margin, as well as the development of flowers and fruits. Recent studies have indicated that EPFL may be involved in plant adaptation to biotic and abiotic stress. This review examines the structure, phylogenetic distribution, mechanisms of signal transduction, and functions of the EPF/EPFL peptide family.
The ProTx-I toxin from Thrixopelma pruriens tarantula venom inhibits voltage-gated sodium (NaV), potassium, and calcium channels, as well as the chemosensitive TRPA1 ion channel, affecting the activating processes of these channels. Due to its action at the NaV1.7, NaV1.8, and TRPA1 channels involved in pain perception and propagation, ProTx-I may be used as a model for the development of next-generation analgesics. ProTx-I consists of 35 amino acid residues, with three disulfide bonds forming an inhibitor cystine knot (ICK) motif, which challenges its heterologous production. An efficient ProTx-I production system is necessary to study, at the molecular level, the mechanism by which the toxin acts. In this study, we tested several approaches for bacterial production of disulfide-containing toxins. Cytoplasmic expression of ProTx-I fused with either thioredoxin or glutathione-S-transferase failed to yield a correctly folded toxin. However, the natively folded ProTx-I was successfully obtained by "direct" expression in the form of cytoplasmic inclusion bodies, followed by renaturation, as well as by secretion into the periplasmic space via fusion with maltose-binding protein. The activity of the recombinant ProTx-I was studied by electrophysiology in X. laevis oocytes expressing rat and human TRPA1 channels. The toxin proved to be more active on the rat channel than on the human channel (IC50 = 250 ± 85 and 840 ± 190 nM, respectively). The presence of an additional N-terminal methionine residue in the toxin obtained through "direct" expression significantly attenuated its activity.
The development of effective and accessible methods for the chiral analysis of amino acids is an important scientific and practical necessity. One of the most common and convenient techniques is the chromatographic determination of individual enantiomers of amino acids with preliminary conversion of enantiomers into diastereomers, which can then be separated on conventional achiral columns. We have shown that by adding ion-pair reagents to the eluent and varying their structure, one can regulate the efficiency of a chiral amino acid analysis based on the chromatographic determination and resolution of the diastereomeric isoindoles obtained upon pre-column derivatization of amino acids by o-phthalaldehyde in the presence of N-acetyl-L-cysteine. The use of ion-pair reagents allows one to achieve a better resolution of diastereomeric isoindole peaks, while the analysis time can be reduced by increasing the ionic strength. Hence, adding ionpair reagents and optimizing the mobile phase composition are useful approaches in the engineering of an amino acid chiral analysis, along with the synthesis of new chiral SH compounds and the choice of stationary phases.
This review examines the potential applications of affibody molecules in various fields of biotechnology and clinical medicine. Consideration is given to the high affinity and specificity of affibody molecules for selected molecular targets, as well as their potential for the in vivo visualization of various malignant tumors. Significant attention is paid to preclinical and clinical studies of affibody conjugates with various radioisotopes for targeted radionuclide tumor imaging, which is particularly relevant in addressing challenges encountered during the diagnosis and treatment of these patients. Clinical trials demonstrate that radiopharmaceuticals are well-tolerated and effective for the assessment of tumor process prevalence and the determination of HER2/neu status in breast cancer patients, supporting further research.
The development of a symbiotic gut ecosystem is a crucial step in postnatal adaptation. The gut microbiome of very preterm infants is characterized by an overall instability, reduced microbial diversity, and a predominance of Gram-negative Proteobacteria, all factors associated with an increased risk of necrotizing enterocolitis (NEC). Short-chain fatty acids (SCFAs) are the key bacterial metabolites that are essential for maintaining intestinal homeostasis, supporting immune development, enhancing intestinal barrier integrity, and reducing inflammation. This review examines the role of gut microbiota and SCFAs in neonatal NEC, with a focus on potential diagnostic and therapeutic strategies. Clinical studies have consistently demonstrated a significant decrease in total SCFA levels and individual bacterial metabolites in preterm infants with NEC. This finding has been corroborated by various experimental models. Clarification of the role of SCFAs in NEC pathogenesis, determination of their diagnostic utility, and assessment of the feasibility of developing comprehensive pro- and postbiotic formulations require multi-center, multi-omics investigations that include a large cohort of very preterm infants.
Alzheimer's disease, first described over a century ago, is currently among the most common neurodegenerative diseases whose significance is increasingly growing with the aging of populations. Throughout the entire period of its study, no remedies have been found that would be effective in treating - or at least significantly slowing - the pathological process, while being sufficiently safe. In this regard, significant attention is paid to the development and application of natural peptide drugs lacking side effects. The present study assessed the effect of the known neuroprotective peptide Semax and its derivative on the behavioral characteristics and development of amyloidosis in transgenic APPswe/PS1dE9/Blg mice acting as a model of Alzheimer's disease. The open field, novel object recognition, and Barnes maze tests demonstrated that both Semax and its derivative improved cognitive functions in mice. Histological examination showed that these peptides reduced the number of amyloid inclusions in the cortex and hippocampus of the animals' brains. These findings demonstrate the high potential of Semax and its derivatives when used to develop therapeutic and corrective strategies for Alzheimer's disease.
mRNA-based cancer vaccines represent an innovative approach to cancer treatment. Cancer mRNA vaccines are structurally based on specific tumor antigens, a technique which enables the patient's immune system to become activated against cancer cells. Clinical trials of mRNA vaccines against various types of tumors, including melanoma, lung cancer, pancreatic carcinoma, breast cancer and others, are currently underway. Because of their favorable safety profile and adaptability, these therapeutics hold considerable promise in efforts to enhance cancer treatment efficacy and prolong patient life. This review outlines steps in the development of manufacturing technologies for mRNA-based therapeutics, describes the algorithm used to design personalized anti-tumor mRNA vaccines, discusses their practical implementation, and summarizes current clinical trials in cancer immunotherapy.
Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.
The efficiency of dairy product fermentation directly depends on the properties of the lactic acid bacteria used, particularly on their metabolic activity and resistance to bacteriophages. Therefore, an understanding of the relationships between the genetic and phenotypic traits of industrial strains is of elevated importance. In this study, we comprehensively analyzed five Lactococcus strains widely used in the Russian dairy industry, combining whole-genome sequencing with phenotypic profiling. Despite the fact of genetic similarity among four of the L. lactis strains, we still identified significant differences in their metabolic activity. Comparative structural analysis of previously published genomes of 337 L. lactis and 147 L. cremoris strains revealed species-specific features of the lactose metabolism; in particular, the absence of the lacZ gene in L. cremoris. Notably, prophages were found in three of the studied strains, which was in correlation with their reduced acidification activity. L. lactis FNCPS 51n and 73n strains displayed resistance to all 50 tested bacteriophages, which may be associated with the presence of the AbiB abortive infection system. These findings underscore the importance of integrating genomic and phenotypic analyses when selecting efficient and phage-resistant Lactococcus starters in the dairy industry.
Radiation therapy is a commonly used cancer treatment modality. However, its application is limited because of its toxicity to healthy tissue. The search for effective radioprotective agents remains one of the key goals of radiation oncology and radiobiology. This study focuses on experimental modeling of radiation injury in animals and the investigation of Dimephosphon radioprotective properties, a drug exhibiting anti-acidotic, antitumor, and antioxidant activities. It was shown that 14-day administration of the drug at a dose of 750 mg/kg after single-dose (5 Gy) irradiation of CD-1 mice resulted in a local radioprotective effect, reducing the severity of the radiation-induced injury to the intestinal epithelium and splenic capsule. The results of metabolomic screening revealed that the levels of the key metabolites responsible for antioxidant properties such as alpha-tocopherol, nicotinamide riboside, N-carbamoyl-L-aspartate, and adenylosuccinate were significantly increased, indicating that the Dimephosphon drug provides enhanced antioxidant protection.
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.
Widespread environmental contamination with polychlorinated biphenyls (PCBs) leads to serious health problems for humans and animals. Our main focus should be on studying the negative effects of exposure to medium- and highly chlorinated PCBs in the human body. There is limited information on the impact of low-chlorinated biphenyls containing 1-2 substituents per molecule on the functions of mammalian organs and systems. Under natural conditions, PCBs can undergo bacterial degradation; the resulting compounds belong to a group of secondary pollutants and are considered hazardous to the environment. Because of limited research, the question regarding the impact of mono-substituted chlorobiphenyl congeners, as well as the products of their biotransformation, remains open. In the presented work, the effects of ortho- and meta- substituted monochlorinated biphenyls on the functions of immune system cells and the morphofunctional state of the liver of mammals in vivo are revealed for the first time. PCB 1 and PCB 2 were found to suppress humoral immunity and induce a productive inflammatory response, as well as widespread protein dystrophy with necrotic foci in the liver. The products of a aerobic bacterial transformation of PCB 1 and PCB 2 were shown to not have a negative effect on the mammalian immune system but proved toxic to hepatocytes, although to a lesser extent than the original chlorobiphenyls.
