Competitive ability of <i>Escherichia coli</i> strains in the intestinal microbiota of patients with Crohn's disease and healthy volunteers: physiological, biochemical and genetic characteristics

Introduction. Crohn's disease (CD) is a chronic inflammation of various parts of the gastrointestinal tract with an increased proportion of Escherichia coli. However, the role of E. coli in disease remains unclear. This study aims to evaluate the competitive abilities of E. coli strains from CD patients and healthy volunteers, and to identify the biochemical and genetic determinants underlying these features. Materials and methods. The antagonistic activity was assessed by co-cultivation of 11 clinical E. coli strains inhibiting the growth of the K-12, with Enterobacter cloacae, Klebsiella pneumonia and Salmonella enterica. To elucidate the mechanism of antagonistic activity, the evaluation of biochemical properties and a comparative genomic analysis were used. Results and discussion. Genes of bacteriocin production systems were identified in genomes of 11 strains from CD patients and healthy volunteers active against the E. coli K-12 strain. Three strains from healthy individuals demonstrated activity against several Enterobacteriaceae bacteria. The strains biochemical properties were typical of representatives of E. coli. Strains 1_34_12, active against E. cloacae, and 1_45_11, inhibiting all tested enterobacteria, are phylogenetically related to the laboratory strain K-12. Strain 1_39_1, active against K. pneumonia and S. enterica, is phylogenetically close to the Nissle1917, contains the genes for colibactin biosynthesis and a variant of the fimH gene that increases the adhesive ability of bacteria. Conclusion. The identified E. coli strains are able to displace Enterobacteriaceae bacteria and can be used to study the bacteria-bacteria and host-bacteria interactions, to understand their role in gut homeostasis and intestinal inflammation

Self-assembled nanoformulation of methylprednisolone succinatewith carboxylated block copolymer for local glucocorticoid therapy

A new self-assembled formulation of methylprednisolone succinate (MPS) based on a carboxylatedtrifunctional block copolymer of ethylene oxide and propylene oxide (TBC-COOH) was developed. TBC-COOH and MPS associated spontaneously at increased concentrations in aqueous solutions to form almostmonodisperse mixed micelles (TBC-COOH/MPS) with a hydrodynamic diameter of 19.6 nm, zeta potentialof −27.8 mV and optimal weight ratio ∼1:6.3. Conditions for the effective formation of TBC-COOH/MPSwere elucidated by comparing copolymers and glucocorticoids with different structure. The micellarstructure of TBC-COOH/MPS persisted upon dilution, temperature fluctuations and interaction with bloodserum components. TBC-COOH increased antiradical activity of MPS and promoted its intrinsic cytotoxi-city in vitro attributed to enhanced cellular availability of the mixed micelles. Intracellular transportationand hydrolysis of MPS were analyzed using optimized liquid chromatography tandem mass spectrometrywith multiple reaction monitoring which showed increased level of both MPS and methylprednisolonein neuronal cells treated with the formulated glucocorticoid. Our results identify TBC-COOH/MPS as anadvanced in situ prepared nanoformulation and encourage its further investigation for a potential localglucocorticoid therapy

Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors

Skin Microbiota in Contact Sports Athletes and Selection of Antiseptics for Professional Hygiene

Background. The aim of this study was to assess changes in skin microbiota of wrestlers during training sessions and to determine the sensitivity of hemolytic bacterial isolates to antiseptics. Methods. The main skin bacterial isolates obtained from the skin of 15 wrestlers were identified by cultivation method, with the following MALDI Biotyper and 16S rRNA gene sequencing methods. The sensitivity of hemolytic isolates to antiseptics (Veltosept-2, Cutasept F, Chlorhexidine, Miramistin, and Hydrogen Peroxide) was evaluated by measuring the size of bacterial growth inhibition zone on agar plates. Results. Opportunistic bacteria of the species Bacillus cereus, Staphylococcus aureus, S. epidermidis, and S. saprophyticus were the most commonly found species in skin microbiota of wrestlers before and after training sessions. Representatives of all these species mostly had a hemolytic activity. An alcohol-containing antiseptic Veltosept-2 showed the strongest inhibitory effect on the bacterial isolates of athletes’ skin microbiota most frequently detected in this study. Conclusions. The general increase in the bacterial colonization of wrestlers’ skin, as well as the presence of hemolytic forms of opportunistic bacteria in cutaneous microbiota, indicates dysbiotic changes and a decrease in the protective features of the host organism. Veltosept-2 application can reduce the incidence of skin infections in contact sports athletes with the highest efficiency

Diversity and Adaptations of Escherichia coli Strains: Exploring the Intestinal Community in Crohn’s Disease Patients and Healthy Individuals

Crohn’s disease (CD) is characterized by a chronic, progressive inflammation across the gastrointestinal tract with a series of exacerbations and remissions. A significant factor in the CD pathogenesis is an imbalance in gut microbiota composition, particularly the prevalence of Escherichia coli. In the present study, the genomes of sixty-three E. coli strains from the gut of patients with CD and healthy subjects were sequenced. In addition, eighteen E. coli-like metagenome-assembled genomes (MAGs) were reconstructed from the shotgun-metagenome sequencing data of fecal samples. The comparative analysis revealed the similarity of E. coli genomes regardless of the origin of the strain. The strains exhibited similar genetic patterns of virulence, antibiotic resistance, and bacteriocin-producing systems. The study showed antagonistic activity of E. coli strains and the metabolic features needed for their successful competition in the human gut environment. These observations suggest complex bacterial interactions within the gut which may affect the host and cause intestinal damage

Angiogenic Activity of Cytochalasin B-Induced Membrane Vesicles of Human Mesenchymal Stem Cells

The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. Objectives: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. Methods: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. Results: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-&alpha;, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFN&alpha;2, IFN-&gamma;, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-&alpha;, TNF-&beta;, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. Conclusions: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases

Serum Cytokine Profile, Beta-Hexosaminidase A Enzymatic Activity and GM2 Ganglioside Levels in the Plasma of a Tay-Sachs Disease Patient after Cord Blood Cell Transplantation and Curcumin Administration: A Case Report

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder that occurs due to a deficiency of a β hexosaminidase A (HexA) enzyme, resulting in the accumulation of GM2 gangliosides. In this work, we analyzed the effect of umbilical cord blood cell transplantation (UCBCT) and curcumin administration on the course of the disease in a patient with adult TSD. The patient’s serum cytokine profile was determined using multiplex analysis. The level of GM2 gangliosides in plasma was determined using mass spectrometry. The enzymatic activity of HexA in the plasma of the patient was assessed using a fluorescent substrate assay. The HexA α-subunit (HexA) concentration was determined using ELISA. It was shown that both UCBCT and curcumin administration led to a change in the patient’s cytokine profile. The UCBCT resulted in an increase in the concentration of HexA in the patient’s serum and in an improvement in the patient’s neurological status. However, neither UCBCT nor curcumin were able to alter HexA activity and the level of GM2 in patient’s plasma. The data obtained indicate that UCBCT and curcumin administration can alter the immunity of a patient with TSD, reduce the level of inflammatory cytokines and thereby improve the patient’s condition