Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme

Abstract1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H+-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 μM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H+-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme

Investigating volatile compounds in the Bacteroides secretome

Microorganisms and their hosts communicate with each other by secreting numerous components. This cross-kingdom cell-to-cell signaling involves proteins and small molecules, such as metabolites. These compounds can be secreted across the membrane via numerous transporters and may also be packaged in outer membrane vesicles (OMVs). Among the secreted components, volatile compounds (VOCs) are of particular interest, including butyrate and propionate, which have proven effects on intestinal, immune, and stem cells. Besides short fatty acids, other groups of volatile compounds can be either freely secreted or contained in OMVs. As vesicles might extend their activity far beyond the gastrointestinal tract, study of their cargo, including VOCs, is even more pertinent. This paper is devoted to the VOCs secretome of the Bacteroides genus. Although these bacteria are highly presented in the intestinal microbiota and are known to influence human physiology, their volatile secretome has been studied relatively poorly. The 16 most well-represented Bacteroides species were cultivated; their OMVs were isolated and characterized by NTA and TEM to determine particle morphology and their concentration. In order to analyze the VOCs secretome, we propose a headspace extraction with GC–MS analysis as a new tool for sample preparation and analysis of volatile compounds in culture media and isolated bacterial OMVs. A wide range of released VOCs, both previously characterized and newly described, have been revealed in media after cultivation. We identified more than 60 components of the volatile metabolome in bacterial media, including fatty acids, amino acids, and phenol derivatives, aldehydes and other components. We found active butyrate and indol producers among the analyzed Bacteroides species. For a number of Bacteroides species, OMVs have been isolated and characterized here for the first time as well as volatile compounds analysis in OMVs. We observed a completely different distribution of VOC in vesicles compared to the bacterial media for all analyzed Bacteroides species, including almost complete absence of fatty acids in vesicles. This article provides a comprehensive analysis of the VOCs secreted by Bacteroides species and explores new perspectives in the study of bacterial secretomes in relation the intercellular communication

Hypertrophy and Insulin Resistance of Epicardial Adipose Tissue Adipocytes: Association with the Coronary Artery Disease Severity

Changes in the structural and functional characteristics of the epicardial adipose tissue (EAT) are recognized as one of the factors in the development of cardiometabolic diseases. However, the generally accepted quantitative assessment of the accumulation of EAT does not reflect the size of adipocyte and presence of adipocyte hypertrophy in this fat depot. Overall contribution of adipocyte hypertrophy to the development and progression of coronary atherosclerosis remains unexplored. Objective: To compare the morphological characteristics of EAT adipocyte and its sensitivity to insulin with the CAD severity, as well as to identify potential factors involved in the realization of this relationship. The present study involved 24 patients (m/f 16/8) aged 53–72 years with stable CAD, who underwent coronary artery bypass graft surgery. Adipocytes were isolated enzymatically from EAT explants obtained during the operation. The severity of CAD was assessed by calculating the Gensini score according to selective coronary angiography. Insulin resistance of EAT adipocytes was evaluated by reactivity to insulin. In patients with an average size of EAT adipocytes equal to or exceeding the median (87 μm) the percentage of hypertrophic adipocytes was twice as high as in patients in whom the average size of adipocytes was less than 87 μm. This group of patients was also characterized by the higher rate of the Gensini score, lower adiponectin levels, and more severe violation of carbohydrate metabolism. We have revealed direct nonparametric correlation between the size of EAT adipocytes and the Gensini score (rs = 0.56, p = 0.00047). The number of hypertrophic EAT adipocytes showed a direct nonparametric correlation with the Gensini score (rs = 0.6, p = 0.002). Inverse nonparametric correlations were found between the serum adiponectin level and size (rs = −0.60, p = 0.001), hypertrophy of adipocytes (rs = −0.67, p = 0.00), and Gensini score (rs = −0.81, p = 0.00007). An inverse nonparametric correlation was found between the Gensini score and sensitivity of EAT adipocytes to insulin, estimated by the intracellular redox response (rs = −0.90, p = 0.037) and decrease in lipolysis rate upon insulin addition (rs = −0.40, p = 0.05). The intracellular redox response of adipocytes to insulin was directly correlated with fasting insulin and inversely with postprandial insulin. Our data indicate that the size and degree of hypertrophy of the epicardial adipocytes are related to the CAD severity. According to our results, insulin resistance of adipocytes may be considered as one of the factors mediating this relationship

Designing Stable Bacillus anthracis Antigens with a View to Recombinant Anthrax Vaccine Development

Anthrax is a disease caused by Bacillus anthracis that affects mammals, including humans. Recombinant B. anthracis protective antigen (rPA) is the most common basis for modern anthrax vaccine candidates. However, this protein is characterised by low stability due to proteolysis and deamidation. Here, for the first time, two modification variants leading to full-size rPA stabilisation have been implemented simultaneously, through deamidation-prone asparagine residues substitution and by inactivation of proteolysis sites. Obtained modified rPA (rPA83m) has been demonstrated to be stable in various temperature conditions. Additionally, rPA1+2 containing PA domains I and II and rPA3+4 containing domains III and IV, including the same modifications, have been shown to be stable as well. These antigens can serve as the basis for a vaccine, since the protective properties of PA can be attributed to individual PA domains. The stability of each of three modified anthrax antigens has been considerably improved in compositions with tobacco mosaic virus-based spherical particles (SPs). rPA1+2/rPA3+4/rPA83m in compositions with SPs have maintained their antigenic specificity even after 40 days of incubation at +37 °C. Considering previously proven adjuvant properties and safety of SPs, their compositions with rPA83m/rPA1+2/rPA3+4 in any combinations might be suitable as a basis for new-generation anthrax vaccines

Influence of Copper Oxide Nanoparticles on Gene Expression of Birch Clones In Vitro under Stress Caused by Phytopathogens

Recently, metal oxide nanoparticles (NPs) have attracted attention as promising components for the protection and stimulation of plant microclones in tissue culture in vitro. However, the effect of NPs on the genetic mechanisms underlying plant adaptive responses remains poorly understood. We studied the effect of column-shaped CuO NPs 50 nm in diameter and 70–100 nm in length at a concentration of 0.1–10 mg/L on the development of phytopathogenic fungi Alternaria alternata, Fusarium oxysporum, and Fusarium avenaceum in culture, as well as on the infection of downy birch micro-clones with phytopathogens and the level of genes expression associated with the formation of plant responses to stress induced by microorganisms. CuO NPs effectively suppressed the development of colonies of phytopathogenic fungi A. alternata and F. avenaceum (up to 68.42% inhibition at 10 mg/L CuO NPs) but not the development of a colony of F. oxysporum. Exposure to the NPs caused multidirectional responses at the level of plant genes transcription: 5 mg/L CuO NPs significantly increased the expression level of the LEA8 and MYB46 genes and decreased the expression of DREB2 and PAL. Infection with A. alternata significantly increased the level of MYB46, LEA8, PAL, PR-1, and PR-10 transcripts in birch micro-clones; however, upon exposure to a medium with NPs and simultaneous exposure to a phytopathogen, the expression of the MYB46, PR-1, and PR-10 genes decreased by 5.4 times, which is associated with a decrease in the pathogenic load caused by the effect of NPs and the simultaneous stimulation of clones in vitro. The results obtained can be used in the development of preparations based on copper oxide NPs for disinfection and stimulation of plant phytoimmunity during clonal micropropagation of tree crops

Association of Epicardial Adipose Tissue Adipocytes Hypertrophy with Biomarkers of Low-Grade Inflammation and Extracellular Matrix Remodeling in Patients with Coronary Artery Disease

The aim of the study was to compare the morphological features of epicardial adipose tissue (EAT) adipocyte with the circulating inflammatory biomarkers and parameters of extracellular matrix remodeling in patients with coronary artery disease (CAD). We recruited 42 patients with CAD (m/f 28/14) who were scheduled for coronary artery bypass graft surgery (CABG). EAT adipocytes were obtained by the enzymatic method from intraoperative adipose tissue samples. Concentrations of secreted and lipoprotein-associated phospholipase A2 (sPLA2 and LpPLA2), TNF-α, IL-1β, IL-6, IL-10, high-sensitive C-reactive protein (hsCRP), metalloproteinase-9 (MMP-9), MMP-2, C-terminal cross-linking telopeptide of type I collagen (CTX-I), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were measured in blood serum. Patients were divided into two groups: group 1—with mean EAT adipocytes’ size ≤ 87.32 μm; group 2—with mean EAT adipocytes’ size > 87.32 μm. Patients of group 2 had higher concentrations of triglycerides, hsCRP, TNF-α, and sPLA2 and a lower concentration of CTX-I. A multiple logistic regression model was created (RN2 = 0.43, p = 0.0013). Concentrations of TNF-α, sPLA2 and CTX-I appeared to be independent determinants of the EAT adipocyte hypertrophy. ROC analysis revealed the 78% accuracy, 71% sensitivity, and 85% specificity of the model, AUC = 0.82. According to our results, chronic low-grade inflammation and extracellular matrix remodeling are closely associated with the development of hypertrophy of EAT adipocytes, with serum concentrations of TNF-α, sPLA2 and CTX-I being the key predictors, describing the variability of epicardial adipocytes’ size

Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vaccination. However, the development of new subunit vaccines based on recombinant protective antigen (rPA) faces the problem of vaccine antigen instability. Here, the potential of simultaneous application of two different approaches to stabilize rPA was demonstrated. Firstly, we employed spherical particles (SPs) obtained from the tobacco mosaic virus (TMV). Previously, we had reported that SPs can serve as an adjuvant and platform for antigen presentation. In the current work, SPs were shown to increase the stability of the full-size rPA without loss of its antigenic properties. The second direction was site-specific mutagenesis of asparagine residues to avoid deamidation that causes partial protein degradation. The modified recombinant protein comprising the PA immunogenic domains 3 and 4 (rPA3 + 4) was stable during storage at 4 and 25°C. rPA3 + 4 interacts with antibodies to rPA83 both individually and as a part of a complex with SPs. The results obtained can underpin the development of a recombinant vaccine with a full-size modified rPA (with similar amino acid substitutions that stabilize the protein) and SPs

PERN : An EU-Russia initiative for rhizosphere microbial resources

Millions of microbial taxa inhabit the rhizosphere and could be used as biofertilizers, biopesticides, and/or for bioremediation. Only a fraction of these microbes have been described and/or are being utilized. Most are dispersed in collections, but coordination of their accessibility and availability is challenging. Here, we present the Pan-European Rhizosphere Resource Network (PERN), which is a transnational repository of microorganisms whose objectives are to facilitate access to rhizosphere resources and information and help users with technical and legal issues

Effect of MSCs and MSC-Derived Extracellular Vesicles on Human Blood Coagulation

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis