Agglutination processes of activated sludge cultures induced by extracellular lectins

We examine the agglutinating ability of five compounds, namely, A1, A2, A3, A4 and BS1, isolated from activated sludge on selective media typical of a number of dominant microbial cultures that contribute to the formation of microbial aggregates. The morphological properties of the isolates and their lectin activity, as well as the physiological and biochemical properties of individual isolates were studied; microorganisms in their composition were identified. We assessed the capacity of the isolates under study to synthesize an exopolysaccharide matrix, as well as the sedimentation of activated sludge under the action of the native solution and culture liquid of the BS1 isolate. Based on their capacity to agglutinate, the BS1 and A2 isolates were selected for further research as producers of extracellular lectins and objects of agglutination, respectively. The biophysiochemical properties and molecular-genetic identification of the BS1 isolate allowed the degree of identity with r. Bacillus to be defined (96.19%); for the A2 isolate, 92.93% identity with p. Shigella and p. Escherichia was determined. To assess the capacity to synthesize a biofilm matrix, the BS1 and A2 isolates were cultivated on an agar nutrient solution using Congo Red dye. According to the obtained results, the isolates are capable of synthesizing an exopolysaccharide matrix, the main component of bacterial biofilms. The research results on the sedimentation of activated sludge induced by the native solution and culture liquid of BS1 showed the following. The sedimentation rate of activated sludge increased significantly at the beginning of the process upon adding a BS1 cell suspension, while the introduction of the native solution of BS1 intensified the process following 5 minutes of contact. The obtained experimental data suggest that the media containing extracellular bacterial lectins can be effectively used as a coagulant (flocculant) for the sedimentation of activated sludge.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson’s disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry

When electrosprayed from native solution conditions, RNA hairpins and kissing complexes acquire charge states at which they get significantly more compact in the gas phase than their initial structure in solution. Here we show the limits of using force field molecular dynamics to interpret the gas-phase structures of nucleic acid complexes in the gas phase, and we suggest that higher-level calculation levels should be used in the future.<br>

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry

When electrosprayed from native solution conditions, RNA hairpins and kissing complexes acquire charge states at which they get significantly more compact in the gas phase than their initial structure in solution. Here we show the limits of using force field molecular dynamics to interpret the gas-phase structures of nucleic acid complexes in the gas phase, and we suggest that higher-level calculation levels should be used in the future.<br>

Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography

ABSTRACTImages of micron-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains’ shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been lacking, leading to an inability to answer key questions. For example, researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two new methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under native solution conditions. The first method is optimized for probe-free applications: domains are directly distinguished from the surrounding membrane by their thickness. This technique measures area fractions of domains with quantitative accuracy, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to the first direct and quantitative imaging of submicron membrane domains under native conditions.SIGNIFICANCE STATEMENTFluorescence micrographs that capture the sizes, shapes, and distributions of liquid domains in model membranes have provided high standards of evidence to prove (and disprove) theories of how micron-scale domains form and grow. Corresponding theories about smaller domains have remained untested, partly because experimental methods of identifying submicron domains in vesicles under native solvent conditions have not been available. Here we introduce two such methods. Both leverage cryo-electron tomography to observe membrane features far smaller than the diffraction limit of light. The first method is probe-free and identifies differences in thicknesses between liquid domains and their surrounding membranes. The second method identifies membrane regions labeled by an electron-dense, fluorescent protein, which enables direct comparison of fluorescence micrographs with cryo-electron tomograms.

A microfluidic strategy for the detection of membrane protein interactions

Membrane proteins are gatekeepers for exchange of information and matter between the intracellular and extracellular environment. This paper opens up a route to probe membrane protein interactions under native solution conditions using microfluidics.

DETERMINATION OF SOME INDICATORS OF IMMUNITY AND LIPOPEROXIDATION IN THE ORAL FLUID IN PERSONS WITH A LOW VITAMIN D LEVEL

Oral fluid is a unique biological environment, containing a wide range of substances, coming from local and systemic sources, which makes it possible to use it as an object for assessing pathological changes in the body both at the local and systemic levels. In comparison with the traditional method of blood analysis, the advantage of evaluating the parameters of the oral fluid is the non-invasive of this method of obtaining material. All patients underwent oral fluid sampling using special plastic containers with a swab, which facilitate the selection of material, eliminating the penetration of mucin into a clean test sample, which helps to obtain more accurate analysis results.The amount of secretory IgA, lipopolysaccharide-binding protein (LBP), TBA-active products, the level of total antioxidant activity in the oral fluid in individuals with a low level of 25(OH)D before and after taking the native solution of vitamin D “Aqua Trim” were determined. The concentrations of secretory immunoglobulin A, lipopolysaccharide, binding protein and the level of total antioxidant activity are reduced in the oral fluid of people with vitamin D deficiency, but the number of intermediate products of lyoperoxidation increases. The course intake of the native solution of vitamin D (International Nonproprietary Name - Colecalciferol) normalizes the functioning of the immunity of the oral cavity and restores the balance of the “lipid peroxidation-antioxidants” system.

Features of low-temperature storage of streptomyces strains - producers of glycosidaz inhibitors

The aim of the work is to study the inhibitory activity of the Streptomyces lucensis VKPM Ac-1743 and Streptomyces violaceus VKPM Ac-1734 strains stored at –18 °C in a 15 % glycerol solution and in a 0.9 % sodium chloride solution.The object of the study was the actinomycete strains of S. lucensis VKPM Ac-1743 and S. violaceus VKPM Ac-1734 producers of glycosidase inhibitor, which is a micro-ingredient for creating products with a low glycemic index. The storage time was nine months. Bookmark storage was performed by flushing with a taped agar starchcontaining environment of Capek. For comparison, we studied the properties of cultures stored at +4 °C without cryoprotectant.Deep cultivation of Streptomyces strains was carried out in a periodic manner on a medium containing corn starch hydrolyzate under the Multitron incubator shaker (INFORS,Switzerland).Inhibitory activity was determined in inactivated native solutions by a colorimetric method with respect to pancreatic α-amylase, a test glycosidase involved in carbohydrate metabolism, and whose activity was chosen as a criterion for assessing the hypoglycemic action of the inhibitor. The proteinase activity of the inactivated native solution was determined by a modified method using a casein substrate.The experimental data were processed using the methods of mathematical statistics and Excel XP programs.As a result of the research, it was established that the actinomycete strains of S. lucensis and S. violaceus, the producers of glycosidase inhibitors, can maintain inhibitory activity during low-temperature storage for nine months.For the S. lucensis actinomycete strain, stored in a 15 % glycerol solution at – 18 °C, the maximum inhibitory activity is 48 hours in the biotechnological process and is (3686 ± 300) IU/cm3 native solution, and for theviolaceus strain – (3150 ± 200) IU/cm³of the native solution, respectively.For the strain of actinomycete S. lucensis, stored in 0.9 % sodium chloride solution at a temperature of – 18 °C, the maximum inhibitory activity accounts for 72 h of the biotechnological process and is (2600 ± 200) IU/cm³ of the native solution, and for the strain S. violaceus the maximum inhibitory activity accounts for 24 hours of the biotechnological process and is (3530 ± 200) IU/cm³ of the native solution.At a storage temperature of +4 °C, the inhibitory activity for the strain of actinomycete S. lucensis is (560 ± 20) IU/cm³of native solution, and for the strain of S. violaceus – (1747 ± 100) IU/cm³ of native solution, respectively. On the basis of the data obtained, it can be concluded that a temperature of –18 °C is preferred for long-term storage.During the cultivation of Streptomyces strains, proteinase activity ranged from (0,012 ± 0,001) U/cm³ to (0,072 ± 0,002) U/cm³.The obtained data can be applied in further studies to develop conditions for long-term storage of collection crops.