ESKAPE bacteria are a major global threat due to their rapid antibiotic resistance acquisition and severe healthcare-associated infections. Effective countermeasures require epidemiological surveillance and resistance transmission studies, particularly for antimicrobial-resistant (AMR) colonization in intensive care unit (ICU) patients. Whole-genome sequencing (WGS) provides critical information on resistance spread and mechanisms. In the provided protocol, rectal and oropharyngeal swabs, or endotracheal aspirate/bronchoalveolar lavage for intubated patients, are collected at ICU admission and twice weekly. Patient interviews and medical records identify risk factors for resistant microflora. Samples undergo cultivation, species identification, antibiotic susceptibility testing, and DNA extraction. Sequencing is performed using second- and third-generation platforms, with selected isolates subject to hybrid genome assembly. Resistance genes, virulence factors, and typing profiles (MLST, cgMLST) are determined. This protocol characterizes the ICU patient colonization by AMR pathogens, including species distribution, phenotypic and genotypic resistance profiles, clonal structure, and temporal changes. It estimates detection frequency and colonization patterns at each locus, identifies key risk factors, including prior community or inter-facility exposure, and analyzes associations between risk factors and admission colonization. The study aims to estimate AMR infection risk and severity in ICU patients through the comprehensive analysis of colonization dynamics, resistance patterns, and clonal characteristics using WGS data on pathogen composition and AMR trends.

Phytases of the PhyD class according to their pH optimum (7.0–7.8) and high thermal stability can claim to be used in the production of feed supplements. However, today they have no practical application in feed production because there are no suitable producers sufficient for its biotechnological production compared to the PhyA and PhyC class ones. Moreover, in most cases, the technologies with the enzymes produced in secretory form are preferable for the production of phytases, though upon microencapsulation in yeast-producing cells, the phytase thermal stability increases significantly compared to the extracellular form, which improves its compatibility with spray drying technology. In this study, we assayed the intracellular heterologous expression of PhyD phytase from Bacillus species in the Yarrowia lipolytica yeast cells. While the technology has been successfully used to synthesize PhyC phytase from Obesumbacterium proteus, PhyD phytase tends to aggregate upon intracellular accumulation. Furthermore, we evaluated the prospects for the production of encapsulated phytase of the PhyD class of high enzymatic activity when it accumulates in the cell cytoplasm of the Y. lipolytica extremophile yeast, a highly effective platform for the production of recombinant proteins.

IQGAP3 (IQ Motif Containing GTPase Activating Protein 3) is member of the IQGAP family of scaffold proteins, which are essential for assembling multiprotein complexes that coordinate various intracellular signaling pathways. Previous research has shown that IQGAP3 is overexpressed in psoriatic skin lesions. Given its involvement in processes like cell proliferation and chemokine signaling, we sought to explore its molecular role in driving the psoriatic phenotype of keratinocytes. By conducting transcriptome profiling of HaCaT keratinocytes, we identified numerous psoriasis-associated pathways that were affected when IQGAP3 was knocked down. These included alterations in NFkB signaling, EGFR signaling, activation of p38/MAPK and ERK1/ERK2, lipid metabolism, cytokine production, and the response to inflammatory cytokine stimulation. Real-time analysis further revealed changes in cell growth dynamics, including proliferation and wound healing. The balance between cell proliferation and apoptosis was altered, as were skin barrier functions and the production of IL-6 and IFNγ. Despite these significant findings, the diversity of the alterations observed in the knockdown cells led us to conclude that IQGAP3 may not be the best target for the therapeutic inhibition to normalize the phenotype of keratinocytes in psoriasis.

The strategies of future medicine are aimed to modernize and integrate quality approaches including early molecular-genetic profiling, identification of new therapeutic targets and adapting design for clinical trials, personalized drug screening (PDS) to help predict and individualize patient treatment regimens. In the past decade, organoid models have emerged as an innovative in vitro platform with the potential to realize the concept of patient-centered medicine. Organoids are spatially restricted three-dimensional clusters of cells ex vivo that self-organize into complex functional structures through genetically programmed determination, which is crucial for reconstructing the architecture of the primary tissue and organs. Currently, there are several strategies to create three-dimensional (3D) tumor systems using (i) surgically resected patient tissue (PDTOs, patient-derived tumor organoids) or (ii) single tumor cells circulating in the patient’s blood. Successful application of 3D tumor models obtained by co-culturing autologous tumor organoids (PDTOs) and peripheral blood lymphocytes have been demonstrated in a number of studies. Such models simulate a 3D tumor architecture in vivo and contain all cell types characteristic of this tissue, including immune system cells and stem cells. Components of the tumor microenvironment, such as fibroblasts and immune system cells, affect tumor growth and its drug resistance. In this review, we analyzed the evolution of tumor models from two-dimensional (2D) cell cultures and laboratory animals to 3D tissue-specific tumor organoids, their significance in identifying mechanisms of antitumor response and drug resistance, and use of these models in drug screening and development of precision methods in cancer treatment.

The interaction between gut microbiota and the host immune system is a complex and understudied field, with cytokines like TNFα, IL-6, IL-8, and IL-10 playing pivotal roles. Commensal bacteria, including lactobacilli, respond to these cytokines through adaptive mechanisms that support their survival and function within the gut. While the influence of cytokines on pathogenic bacteria is well documented, their impact on commensal bacteria, particularly lactobacilli, remains underexplored. This study investigates the transcriptional responses of Lacticaseibacillus rhamnosus strains K32 and R19-3 to various cytokines using next-generation RNA sequencing (RNA-seq). Our findings reveal that cytokines, especially IL-8 and IL-10, significantly alter the L. rhamnosus transcriptome, affecting genes involved in carbohydrate metabolism, stress response, and transcriptional regulation. Notably, IL-8 and IL-10 induce a significant downregulation of genes related to the phosphotransferase system, suggesting a reduction in metabolic activity in response to inflammatory signals. This study unveils a previously unexplored aspect of L. rhamnosus adaptation, highlighting its intricate response to cytokine signals. By modulating gene expression, L. rhamnosus may mitigate the adverse effects of inflammation and promote gut health. These insights could inform the development of targeted probiotic therapies for inflammatory bowel disease (IBD) and other conditions with altered cytokine levels. Our results suggest that co-evolution between a host and gut microbiota enables bacteria to respond to specific cytokines through gene expression changes, revealing a unique and underexplored facet of the interaction between commensal bacteria and the host organism.

One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR-Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR-Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR-Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.

In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.

Mycelial fungi grow as colonies consisting of polar growing hyphae, developing radially from spore or inoculum. Over time, the colony develops, hyphae are subject to various exogenous or endogenous stimuli, and mycelium becomes heterogeneous in growth, gene expression, biosynthesis, and secretion of proteins and metabolites. Although the biochemical and molecular mechanisms of mycelium heterogeneity have been the subject of many studies, the role of lipids in colony development and zonality is still not understood. This work was undertaken to extend our knowledge of mycelium heterogeneity and to answer the question of how different lipid molecular species are distributed in the surface colony of the basidial fungus Flammulina velutipes and how this distribution correlates with its morphology. The heterogeneity in the lipid metabolism and lipid composition of the fungal mycelium was demonstrated. According to the real-time PCR and LC-MS/MS results, the expression of genes of PC metabolism, accumulation of phospholipid classes, and degree of unsaturation of PC and PE increased in the direction from the center to the periphery of the colony. The peripheral zone of the colony was characterized by a higher value of the PC/PE ratio and a higher level of phospholipids esterified by linolenic acid. Considering that the synthesis of phospholipids in fungi occurs in different ways, we also conducted experiments with deuterium-labeled phospholipid precursors and found out that the Kennedy pathway is the predominant route for PC biosynthesis in F. velutipes. The zonal differences in gene expression and lipid composition can be explained by the participation of membrane lipids in polar growth maintenance and regulation.

The development of genomic editing technologies has significantly intesificated the shortcomings of the legal regulation for genetic technologies as in general and as for new technologies of genetic engineering in particular. Because of imperfected legislation for genetic engineering, and in some cases because of its archaism, new genomic technologies, such as genetic editing technologies, which are growth drivers in science, could not be drivers in the economics. The outdated terms and concepts framework and the legal indetermination of using the products based on new genetic technologies (including genome editing) are barriers to achieving the goals to ensure the technological independence of Russia. The basis of the current legal regulation system of genetic engineering in Russia is a focus to the process and technologies, i.e. methods of the product producing. This means that the changes within genetic information is not important, but only the method of development is. Such way of regulation provides a priori chronic lag in legislation, which could finally lead to technological lag as whole country. The transition to a product-oriented system, when the analysis of base of genetic information changes makes possible to clearly answer to the question of the methods used for this, will allow us to avoid the shortcomings within development of synthetic biology methods indicated earlier, as now as in the future.

Based on the data presented in the public domain on the GEO-NCBI Platform for 21 projects to determine the genomewide DNA methylation profile using the Infinium Human Methylation 450K BeadChip (Illumina©), we calculated the prediction error for chronological age values in percentage terms and MAD using three different algorithms: linear regression, generalized regression neural networks and multilayer feedforward neural networks. For each algorithm, we have provided guidelines to select the samples for the study. We have also proved that the minimum and sufficient sample size, an increase in which does not lead to a significant decrease in the MAD value, is at least 200 people.

On the basis of bioinformatics and statistical analysis of GEO projects to determine the genome-wide profile of human DNA methylation, a list of 41 CpG dinucleotides with high predictive potential was formed to create models for predicting human age from biological samples. The methylation level was determined for 1208 samples of individuals from the Republic of Belarus (275—blood, 466—buccal epithelium, 467—semen). The correlation coefficients R were calculated, and mathematical models for determining the age of individual were constructed. The mean value of the accuracy of age prediction from blood samples using 12 CpG dinucleotides was 3.4 years (3.3 for men, 3.5 for women); from buccal epithelium samples using 6 CpG dinucleotides, it was 4.6 years (4.5 for men, 4.7 for women); from semen samples using 5 CpG dinucleotides, it was 3.0 years. The results obtained will be used as a basis for the development of calculators for predicting the age of an individual based on traces of biological character for forensic experts.

Background and aim Human evolution resulted from changes in our biology, behaviour, and culture. One source of these changes has been hypothesised to be our self-domestication (that is, the development in humans of features commonly found in domesticated strains of mammals, seemingly as a result of selection for reduced aggression). Signals of domestication, notably brain size reduction, have increased in recent times. Methods In this paper, we compare whole-genome data between the Late Neolithic/Bronze Age individuals and modern Europeans. Results We show that genes associated with mammal domestication and with neural crest development and function are significantly differently enriched in nonsynonymous single nucleotide polymorphisms between these two groups. Conclusion We hypothesise that these changes might account for the increased features of self-domestication in modern humans and, ultimately, for subtle recent changes in human cognition and behaviour, including language.

IQGAP proteins coordinate cell signaling cascades acting as scaffolds for assembly of multiprotein complexes. They mediate growth factor signaling, epithelial-mesenchymal transition, cell proliferation and migration. All of these pathways are important for the pathogenesis of psoriasis, so we suggested that the genes of the IQGAP family may play important roles in the development of this disease. qPCR analysis of the IQGAP1 , IQGAP2 , and IQGAP3 expression in skin of psoriasis patients showed the genes to be differentially expressed, with IQGAP1 being downregulated and IQGAP3 upregulated. Next we conducted a bioinformatic analysis of IQGAP protein partners in order to identify the protein interactions which would explain the difference between the lesional and visually nonlesional psoriatic skin. On the basis of the meta-analysis of the RNAseq data and the protein-protein interaction databases, we constructed IQGAP PPI graphs which highlighted the IQGAP protein partners involved in psoriasis. Therefore, our studies confirmed the hypothesis on the role of the IQGAP family in the pathogenesis of psoriasis.

Yersinia pestis , the causative agent of plague, has been prevalent among humans for at least 5000 years, being accountable for several devastating epidemics in history, including the Black Death. Analyses of the genetic diversity of ancient strains of Y. pestis have shed light on the mechanisms of evolution and the spread of plague in Europe. However, many questions regarding the origins of the pathogen and its long persistence in Europe are still unresolved, especially during the late medieval time period. To address this, we present four newly assembled Y. pestis genomes from Eastern Europe (Poland and Southern Russia), dating from the fifteenth to eighteenth century AD. The analysis of polymorphisms in these genomes and their phylogenetic relationships with other ancient and modern Y. pestis strains may suggest several independent introductions of plague into Eastern Europe or its persistence in different reservoirs. Furthermore, with the reconstruction of a partial Y. pestis genome from rat skeletal remains found in a Polish ossuary, we were able to identify a potential animal reservoir in late medieval Europe. Overall, our results add new information concerning Y. pestis transmission and its evolutionary history in Eastern Europe. This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.

Keratinocytes are immunocompetent cells important for the structural and barrier function of skin. Psoriatic keratinocytes are characterized by the enhanced proliferation and reduced differentiation rates as well as by the elevated production of proinflammatory cytokines and chemoattractants. In this research we have knocked-down a scaffold protein IQGAP3 using shRNA in HaCaT keratinocytes (HaCaT_shIQ3) in order to test the hypothesis if IQGAP3 mediates the psoriatic phenotype of keratinocytes. IQGAP interacts with cell adhesion molecules, with the cytoskeleton, with signaling molecules to regulate cell morphology, motility and kinase pathways. Earlier we have identified IQGAP3 to be overexpressed in skin of psoriatic patients and to be stimulated by the proinflammatory cytokines IL17, TNFa and IFNg. RNA-seq of the HaCaT_shIQ3 cells has shown that among the GO enriched by the downregulated genes were: positive regulation of MAPK cascade, negative regulation of inflammatory response, epidermis development, negative chemotaxis, cell adhesion, keratinization, keratinocyte differentiation, intracellular signal transduction. Among the GO enriched by the upregulated genes were positive regulation of macrophage cytokine production, extracellular matrix organization, positive regulation of I-kB/NF-kB signaling, calcium transport, cell adhesion, positive regulation of inflammatory response, lipid metabolic process, positive regulation of IL6 production, NO biosynthetic process. Almost all the onthologies mentioned above are involved in psoriatic process, highlighting the importance of the IQGAP3 for this disease. Using xCELLigence real time cell analysis system we have evaluated the growth rates of HaCaT_shIQ3. Compared to the control cells, HaCaT_shIQ3 demonstrated slower growing rates, delayed wound healing and were in a lesser degree growth-stimulated by psoriatic proinflammatory cytokines. Thus we concluded IQGAP3 to be an important mediator of the psoriatic phenotype of keratinocytes.

IQGAP proteins mediate many processes important for the development of hyperproliferative skin diseases, namely the EGF signaling, WNT and MAPK kinase cascades, they are required for cell adhesion and migration processes, tight junction and zonula occludens formation, cell cycle regulation. However, the possible contribution of the IQGAP3 in the development of hyperproliferative psoriatic plaques is unknown. We have shown earlier that IQGAP3 is overexpressed in the lesional skin of patients with psoriasis. Next we have performed the keratinocyte stimulation with proinflammatory cytokines associated with the development of psoriatic plaques (TNF alpha or a mix of IL-17+TNF alpha+IFN gamma in physiological concentrations) or with non-specific skin irritant 12-O-tetradecanoylphorbol-13-acetate (TPA) or with the EGF growth factor. We have shown that 24h later both the proinflammatory cytokines and EGF stimulation led to the moderate elevation of the IQGAP3expression (1,6 and 1,8 times respectively, p<0.05) unlike the TPA stimulation, which haven't led to any significant alterations in the IQGAP3 expression. As we have earlier identified FRA1, one of the AP1 transcription factors, to mediate keratinocyte activation in psoriasis, we were interested in the identification of a possible link to the IQGAP3 expression. Using a stable keratinocyte line overexpressing FRA1, we have shown that both the FRA1 overexpression itself or the overexpression under the proinflammatory conditions (IL-17+TNF alpha+IFN gamma stimulation) have led to the elevated expression of the IQGAP3 (3 and 4.8 times respectively, p<0.05). Thus the IQGAP3 overexpression in psoriatic plaques could be the consequence of the FRA1 overexpression under the proinflammatory conditions present in skin of patients with psoriasis. The research was supported by RSF (project 18-75-00126).