Search Results (14)

In this study, we present a method for ion-associated liquid phase (IALP) separation and concentration of analytes from an aqueous matrix into an IALP formed in situ by the charge neutralization reaction of organic cations and anions, without centrifugation. The effects of various factors on the extraction efficiency and other parameters are investigated, whereas no instrumental stirring, such as vortexing or ultrasonics, is required because the solvent (IALP) is formed in situ. The organic cation and anion used are ethylhexyloxypropylammonium and dodecyl sulfate, respectively. The developed in situ IALP microextraction method for phase separation without centrifugation is tested using the thymol blue dye and several endocrine disruptors. The tested endocrine disruptors (bisphenol A, 17β-estradiol, 17α-ethinylestradiol, and estrone) are analyzed via high-performance liquid chromatography/fluorescence detection, with respective detection limits of 0.02, 0.02, 0.02, and 0.4 μg L⁻¹, and the corresponding enrichment factor ranging from 47 to 71. This IALP microextraction method can be used to separate and concentrate environmental water samples of different matrices. The employed IALP is fast and easy to use, enables an approximately 100-fold analyte concentration, and has a high affinity for estrogens, thus holding promise for the separation, concentration, and quantitation of diverse trace analytes. Full article

Numerous studies indicate the involvemen of oxidative stress in the pathogenesis of schizophrenia. It has been shown that the serum pool of antibodies in patients with schizophrenia contains catalytically active antibodies (abzymes) that have a wide range of activities, including redox properties. In the present work, the effects of IgGs—having oxidoreductase activities—isolated from the serum of patients with schizophrenia and healthy individuals were studied in vitro. The IgGs were purified by affinity chromatography followed by an SDS-PAGE analysis of homogeneity in a 4–18% gradient gel. The catalase and superoxide dismutase (SOD) activities of the IgGs were measured spectrophotometrically using a kinetic module. Human neuroblastoma SH-SY5Y cells were cultured with IgG at a final concentration of 0.2 mg/mL for 24 h. In a parallel experiment, tert-butyl hydroperoxide was used as an oxidative stressor. The number of dead cells after incubation was determined with fluorescent dyes, propidium iodide and Hoechst, by high-throughput screening on the CellInsight CX7 platform. A cytotoxic effect of the IgG from the schizophrenia patients on SH-SY5Y cells was detected after 24 h incubation. A correlation was found between the SOD activity of the IgGs and IgG-induced cell death. Under the induced oxidative stress, the cytotoxic effect of the IgG from the patients with schizophrenia on the SH-SY5Y cell line was five times stronger. Meanwhile, the IgG from the healthy individuals exerted a cytoprotective effect on the cultured cells, accompanied by high catalase activity. Thus, the observed influence on cell viability depends on the catalytic properties of the abzymes. Full article

Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (K_d = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia. Full article

Food, drugs, dyes, extracts, and minerals are all made up of complex elements, and utilizing unidimensional chromatography to separate them is inefficient and insensitive. This has sparked the invention of several linked chromatography methods, each of them with distinct separation principles and affinity for the analyte of interest. Multidimensional chromatography consists of the combination of multiple chromatography techniques, with great benefits at the level of efficiency, peak capacity, precision, and accuracy of the analysis, while reducing the time required for the analysis. Various coupled chromatography techniques have recently emerged, including liquid chromatography–gas chromatography (LC–GC), gas chromatography–gas chromatography (GC–GC), liquid chromatography–liquid chromatography (LC–LC), GCMS–MS, LCMS–MS, supercritical fluid techniques with chromatography techniques, and electro-driven multidimensional separation techniques. In this paper, the different coupled chromatography techniques will be discussed, along with their wide spectrum of applications for food, flavor, and environmental analysis, as well as their usefulness for the pharmaceutical, color, and dyes industries. Full article

The presence of contaminants in water may involve a risk to human and animal health. Conventional water treatment methods such as coagulation, flocculation, and sedimentation are ineffective for cyanotoxin removal. In addition, high amounts of cyanotoxins can be released during those processes if cells lyse. Thus, new mitigation strategies must be developed to ameliorate the consequences of harmful algal blooms. In this sense, nanotechnology has become a promising tool for the treatment of contaminated water. Several nanomaterials with specific chemical affinities can be combined into hybrid structures, leading to nanostructured agents with a large surface area and with the ability to absorb different contaminants. In addition, these structures can include magnetite, which enables separation from the detoxified substance by magnetic extraction, which is considered a green technique. This approach has been successfully applied to the removal of dyes, endocrine disruptors, and heavy metal ions. Recently, we have described the use of carbon nanoparticles to remove around 60% of microcystins from contaminated solutions, but with a low efficiency in the adsorption of anatoxin-a and cylindrospermopsin. In this work, a new set of biocompatible magnetic nanocomposites were tested using artificially contaminated water. The toxin content in solutions was determined before and after treatment by ultra-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). With these new nanocomposites, cyanotoxin elimination was highly improved, reaching toxin removal rates of up 80%. Therefore, the implementation of the nanotechnology in water treatment could be a promising approach to reduce the presence of natural toxins in the water. Full article

Targeted immunotherapy has expanded to simultaneous delivery of drugs, including chemotherapeutics. The aim of the presented research is to design a new drug carrier system. Systems based on the use of proteins as natural components of the body offer the chance to boost safety and efficacy of targeted drug delivery and excess drug removal. Congo red (CR) type supramolecular, self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin and antibodies complexed with antigen. CR can intercalate some chemotherapeutics including doxorubicin (Dox). The goal of this work was to describe the CR-Dox complexes, to analyze their interaction with some proteins, and to explain the mechanism of this interaction. In the present experiments, a model system composed of heated immunoglobulin light chain Lλ capable of CR binding was used. Heat aggregated immunoglobulins (HAI) and albumin were chosen as another model system. The results of experiments employing methods such as gel filtration chromatography and dynamic light scattering confirmed the formation of the CR-Dox complex of large size and properties different from the free CR structures. Electrophoresis and chromatography experiments have shown the binding of free CR to heated Lλ while CR-Dox mixed structures were not capable of forming such complexes. HAI was able to bind both free CR and CR-Dox complexes. Albumin also bound both CR and its complex with Dox. Additionally, we observed that albumin-bound CR-Dox complexes were transferred from albumin to HAI upon addition of HAI. DLS analyses showed that interaction of CR with Dox distinctly increased the hydrodynamic diameter of CR-Dox compared with a free CR supramolecular structure. To our knowledge, individual small proteins such as Lλ may bind upon heating a few molecules of Congo red tape penetrating protein body due to the relatively low cohesion of the dye micelle. If, however, the compactness is high (in the case of, e.g., CR-Dox) large ribbon-like, micellar structures appear. They do not divide easily into smaller portions and cannot attach to proteins where there is no room for binding large ligands. Such binding is, however, possible by albumin which is biologically adapted to form complexes with different large ligands and by tightly packed immune complexes and heat aggregated immunoglobulin-specific protein complex structures of even higher affinity for Congo red than albumin. The CR clouds formed around them also bind the CR-Dox complexes. The presented research is essential in the search for optimum solutions for SRLS application in immuno-targeting therapeutic strategies, especially with the use of chemotherapeutics. Full article

Fluorophore (FD) labeling is widely used for detection and quantification of various compounds bound to nanocarriers. The systems, composed of gold nanoparticles (GNPs) and oligonucleotides (ONs) labeled with FDs, have wide applications. Our work was aimed at a systemic study of how FD structure (in composition of ON-FDs) influenced the efficiency of their non-covalent associates’ formation with GNPs (ON-FD/GNPs). We examined ONs of different length and nucleotide composition, and corresponding ON-FDs (FDs from a series of xanthene, polymethine dyes; dyes based on polycyclic aromatic hydrocarbons). Methods: fluorometry, dynamic light scattering, high performance liquid chromatography, gel electrophoresis, molecular modeling and methods of thermodynamic and statistical analysis. We observed significant, differing several times, changes in surface density and Langmuir constant values of ON-FDs vs. ONs, evidence for the critical significance of FD nature for binding of ON-FDs with GNPs. Surface density of ON-FD/GNPs; hydrophobicity and total charge of ON or ON-FD; and charge and surface area of FDs were revealed as key factors determining affinity (Langmuir constant) of ON or ON-FDs for GNPs. These factors compose a specific set, which makes possible the highly reliable prediction of efficiency of ONs and ON-FDs binding with GNPs. The principal possibility of creating an algorithm for predictive calculation of efficiency of ONs and GNPs interaction was demonstrated. We proposed a hypothetical model that described the mechanism of contact interaction between negatively charged nano-objects, such as citrate-stabilized GNPs, and ONs or ON-FDs. Full article

The detection of viruses, disease biomarkers, physiologically active substances, drugs, and chemicals is of great significance in many areas of our lives. Immunodetection technology is based on the specificity and affinity of antigen–antibody reactions. Compared with other analytical methods such as liquid chromatography coupled with mass spectrometry, which requires a large and expensive instrument, immunodetection has the advantages of simplicity and good selectivity and is thus widely used in disease diagnosis and food/environmental monitoring. Quenchbody (Q-body), a new type of fluorescent immunosensor, is an antibody fragment labeled with fluorescent dyes. When the Q-body binds to its antigen, the fluorescence intensity increases. The detection of antigens by changes in fluorescence intensity is simple, easy to operate, and highly sensitive. This review comprehensively discusses the principle, construction, application, and current progress related to Q-bodies. Full article

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial. Full article

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4. Full article

Nowadays, fluoroquinolones (FQs) constitute one of the most important classes of antibiotics. FQs are used to treat infections caused by Gram-positive and Gram-negative species. A set of fluoroquinolone–Safirinium dye hybrids has been synthesized in our laboratory as potential new dual-action antibacterial agents. In the present study we have evaluated how such a modification influences the affinity of FQs to phospholipids. The immobilized artificial membrane (IAM) high-performance liquid chromatography (IAM-HPLC) was used as a tool for the determination of phospholipids partitioning. The obtained results indicate that the fluoroquinolone–Safirinium dye hybrids, especially the SafiriniumP conjugates, display significantly lower affinity to phospholipids than the parent FQs. Despite the fact that the hybrid structures comprise a quaternary nitrogen atom and hence are permanently charged, the attractive electrostatic interactions between the solutes and negatively charged phospholipids do not occur or occur at a lesser extent than in the case of the unmodified FQs. Since affinity of FQs to phospholipids involves molecular mechanism, which is not entirely determined by lipophilicity, assessment of phospholipid partitioning should be considered at the early stage of the development of new FQ antibiotics. Full article

In the present study, we report the development of a cellulose-based affinity adsorbent and its application for the purification of proteases from fish by-products. The affinity adsorbent was synthesized using cellulose microfibers as the matrix, isolated from recycled newspapers using the acid precipitation method. As an affinity ligand, the triazine dye Cibacron Blue 3GA (CB3GA) was used and immobilized directly onto the cellulose microfibers. Absorption equilibrium studies and frontal affinity chromatography were employed to evaluate the chromatographic performance of the adsorbent using as model proteins bovine serum albumin (BSA) and lysozyme (LYS). Absorption equilibrium studies suggest that the adsorption of both proteins obeys the Langmuir isotherm model. The kinetics of adsorption obey the pseudo-second-order model. The affinity adsorbent was applied for the development of a purification procedure for proteases from Sparus aurata by-products (stomach and pancreas). A single-step purification protocol for trypsin and chymotrypsin was developed and optimized. The protocol afforded enzymes with high yields suitable for technical and industrial purposes. Full article

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL⁻¹, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC₅₀ of 2.41 µg·mL⁻¹. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. Full article

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoA when NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source. Full article