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Mohd Sukri, Siti Sabrina, i Mimi Sakinah A.M. "Effects of Support Matrix for XylanaseImmobilisationon Alginate Hydrogel Beads". International Journal of Engineering Technology and Sciences 4, nr 1 (30.06.2017): 28–35. http://dx.doi.org/10.15282/ijets.7.2017.1.4.1066.
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Enzymes serving as biocatalysts and play an important roles in many industrial field. However, the limitation of enzyme usage due to its high cost and unstable conditions of soluble enzyme to harsh conditions lead to findings an alternative to enhance the enzyme efficiency by immobilisation (insoluble enzyme). The present work reported a combination of immobilisation technique of xylanase by entrapment and covalent binding on alginate hydrogel beads. Xylanase enzyme was effectively immobilised within the support matrix, alginate hydrogel beads by entrapment and covalent binding on the surface of beads using glutaraldehyde as a cross-linked agent. The effects of support matrix comprised of sodium alginate concentration (% w/v) and calcium chloride, CaCl2 (M) were studied in order to obtain a better immobilisation yield. The suitable concentration of sodium alginate and CaCl2 to ensure a robust and stable hydrogel beads with higher immobilisation yield were formed as a support matrix for xylanase immobilisation. The analysis of xylanase activity was determined using dinitrosalicyclic (DNS) acid reagent method. Maximal enzyme immobilisation yield (>80 %) was achieved at 3.0 % w/v of sodium alginate concentration and 0.3 M of CaCl2. The study shows the support matrix of hydrogel beads gave a significant impact towards the immobilisation yield of xylanase.
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Groebe, Duncan R., Mary L. Maus, Terry Pederson, Jill Clampit, Stevan Djuric, James Trevillyan, Chun W. Lin, David J. Burns i Usha Warrior. "Putting Thought to Paper: A μARCS Protease Screen". Journal of Biomolecular Screening 8, nr 6 (grudzień 2003): 668–75. http://dx.doi.org/10.1177/1087057103258587.
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In micro-arrayed compound screening (μARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of μARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the μARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the μARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the μARCS format at a density of 384 compounds per sheet. IC50 values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.
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Thomas, W. A., A. W. Schaefer i R. M. Treadway. "Galactosyl transferase-dependence of neurite outgrowth on substratum-bound laminin". Development 110, nr 4 (1.12.1990): 1101–14. http://dx.doi.org/10.1242/dev.110.4.1101.
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The cell surface enzyme beta 1–4 galactosyl transferase (galtase) has been implicated in a number of cellular events involving adhesion and recognition, among them migration of neural crest and mesenchymal cells as well as initiation and elongation of neurites from PC12 cells. Results presented here demonstrate that reagents that specifically alter galtase activity modulate the rate of neurite outgrowth from chick dorsal root ganglia on substrata coated with the large extracellular matrix glycoprotein, laminin (LN), a known substrate for galtase activity. Not all neurites responded equally to reagent addition, and in every experiment a subset of neurites was ostensibly unaffected by reagent, even at the highest concentration tested. Those neurites that were affected demonstrated an ability to adapt to the continued presence of reagent and resume normal elongation. These results support the hypothesis that cell surface galtase activity plays an important role in mediating neurite elongation and suggest further that differential expression of galtase at the nerve growth cone might contribute to axonal guidance through glycoconjugate-rich environments in vivo.
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Sutrisno, Sutrisno, Anna Roosdiana, Sasangka Prasetyawan i Intan Permata Sari. "Determination of Immobilization Optimum Conditions of Trichoderma viride’s Xilanase on Acid-Activated Zeolite Matrix". JKPK (Jurnal Kimia dan Pendidikan Kimia) 2, nr 2 (7.09.2017): 97. http://dx.doi.org/10.20961/jkpk.v2i2.11912.
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<p>Xylanase is an enzyme that catalyzes the reaction of xylan hydrolysis into xylose. The free enzyme can be used only once, therefore it needs to be made in the form of immobilization. This study aims to determine the optimum conditions for immobilization of xylanase using an acid-activated zeolite. The xylanase immobilization was performed on variations of shaking time (1-5) hours, variation of xylanase concentration (2 - 4 mg / mL) using 0.1 g zeolite at room temperature and a shaking rate of 100 rpm. The amount of xylanase adsorbed on the zeolite was determined by spectrophotometry using the Biuret reagent and the immobilized xylanase activity formed was determined spectrophotometrically using a DNS reagent The results showed that the optimum condition of xylanase immobilization at zeolite was achieved at 3 hrs shaking time and xylanase concentration 3.5 mg / mL with xylanase adsorbed of 156.5 mg/g zeolite and activity 26.67 units.</p>
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Valkirs, G. E., i R. Barton. "ImmunoConcentration--a new format for solid-phase immunoassays." Clinical Chemistry 31, nr 9 (1.09.1985): 1427–31. http://dx.doi.org/10.1093/clinchem/31.9.1427.
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Abstract A new format for solid-phase immunoassays has been developed in which a monoclonal antibody-coated membrane, incorporated into a cylindrical, disposable device, regulates sample and reagent delivery. We illustrate the method with a two-site, immunoenzymometric assay that can detect human choriogonadotropin at less than 50 int. units/L (4 micrograms/L) in urine and less than 25 int. units/L (2 micrograms/L) in serum and takes less than 5 min to perform. The solid-phase antibody is located in a circular area in the center of the membrane so that in the presence of the hormone, after addition of substrate, a blue enzyme product is generated in this circular area. The high ratio of surface area to volume within the microporous matrix of the membrane assures short diffusion distances and therefore rapid binding of liquid-phase reagents to the solid phase. Pseudo-first-order reaction kinetics describe the binding of antigen to immobilized antibody and the binding of enzyme-labeled antibody to immobilized antigen. The speed and simplicity of this format may facilitate testing for many analytes, both soluble and particulate, as well as serological testing.
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Rathore, Rakesh, Jay Corr, George Scott, Pauline Vollmerhaus i Kenneth D. Greis. "Development of an Inhibitor Screening Platform via Mass Spectrometry". Journal of Biomolecular Screening 13, nr 10 (21.11.2008): 1007–13. http://dx.doi.org/10.1177/1087057108326143.
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Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC50 values for several known kinase inhibitors against cyclic AMP—dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS—based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. ( Journal of Biomolecular Screening 2008:1007-1013)
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Rasmussen, Fred H., Nolan Yeung, Laura Kiefer, Gillian Murphy, Carlos Lopez-Otin, Michael P. Vitek i Marcia L. Moss. "Use of a Multiple-Enzyme/Multiple-Reagent Assay System To Quantify Activity Levels in Samples Containing Mixtures of Matrix Metalloproteinases". Biochemistry 43, nr 11 (marzec 2004): 2987–95. http://dx.doi.org/10.1021/bi036063m.
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REHM, Bernd H. A., Qingsheng QI, Br Bernd BEERMANN, Hans-Jürgen HINZ i Alexander STEINBÜCHEL. "Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli". Biochemical Journal 358, nr 1 (8.08.2001): 263–68. http://dx.doi.org/10.1042/bj3580263.
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In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.
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Huber, S., S. Günther, E. Cambria, M. Leunig i SJ Ferguson. "Physiological stretching induces a differential extracellular matrix gene expression response in acetabular labrum cells". European Cells and Materials 44 (3.10.2022): 90–100. http://dx.doi.org/10.22203/ecm.v044a06.
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The acetabular labrum is a fibrocartilaginous ring surrounding the acetabulum and is important for hip stability and contact pressure dissipation through a sealing function. Injury of the labrum may contribute to hip-joint degeneration and development of secondary osteoarthritis. Understanding how extracellular matrix (ECM) production and remodelling is regulated is of key importance for successful tissue restoration. The present study hypothesised that physiological stretching enhanced the metabolic activity and altered the ECM gene expression in labrum cells. Primary bovine labrum cells were physiologically stretched for up to 5 d. 24 h after the last stretch cycle, changes in metabolic activity were measured using the PrestoBlue™ HS Cell Viability Reagent and ECM gene expression was examined using the quantitative polymerase chain reaction method. Targets of interest were further investigated using immunofluorescence and enzyme-linked immunosorbent assay. Metabolic activity was not affected by the stretching (0.9746 ± 0.0614, p > 0.05). Physiological stretching upregulated decorin (DCN) (1.8548 ± 0.4883, p = 0.002) as well as proteoglycan 4 (PRG4) (1.7714 ± 0.6600, p = 0.029) and downregulated biglycan (BGN) (0.7018 + 0.1567, p = 0.008), cartilage oligomeric matrix protein (COMP) (0.5747 ± 0.2650, p = 0.029), fibronectin (FN1) (0.5832 ± 0.0996, p < 0.001) and spondin 1 (SPON1) (0.6282 ± 0.3624, p = 0.044) gene expression. No difference in PRG4 and DCN abundance or release could be measured. The here identified mechanosensitive targets are known to play relevant roles in tissue organisation. Therefore, physiological stretching might play a role in labrum tissue homeostasis and regeneration.
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WOODS, Alan A., Stuart M. LINTON i Michael J. DAVIES. "Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques". Biochemical Journal 370, nr 2 (1.03.2003): 729–35. http://dx.doi.org/10.1042/bj20021710.
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Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine (o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for 83—96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The detection of elevated levels of DOPA and o-Tyr, which have been previously attributed to the occurrence of oxygen-radical-mediated reactions, by HOCl treatment, suggests an alternative route to the formation of these materials in plaques. This is believed to involve the formation and subsequent decomposition of protein chloramines.
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Tumurjav, Buyannemekh, Mohamad Alaa Terkawi, Houshuang Zhang, Guohong Zhang, Honglin Jia, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Chihiro Sugimoto i Xuenan Xuan. "Optimization and validation of an ELISA using recombinant Toxoplasma gondii matrix antigen 1 for serodiagnosis of the infection". Mongolian Journal of Agricultural Sciences 15, nr 2 (30.09.2015): 56–60. http://dx.doi.org/10.5564/mjas.v15i2.546.
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Toxoplasma gondii infection can be diagnosed directly by polymerase chain reaction (PCR), hybridization and isolation of parasites and indirectly with serological methods[4; 5; 18].Although all these tests have shortcomings, serological tests, particularly the enzyme-linked immunosorbent assay (ELISA), seem to be the most practical and economical. The crude antigen prepared from tachyzoites has been traditionally utilized for commercially serological detection kits. However the use of recombinant antigens can be alternative sources of antigens allowing better standardization of the tests and reducing the costs of production requires mass production of the parasite either from the peritoneal fluids of infected mice or from tissue cultures. In spite of the potential advantages of using recombinant antigens in serology tests, their sensitivities have not yet achieved perfect result; therefore, further research on new antigensis extremely desirable [10; 16; 17; 3]. In this context, the Toxoplasma gondii matrix antigen 1 (TgMAG1) known as 65-kDa protein abundantly expressed within the cyst and in the cyst wall surrounding the bradyzoites [15], has documented to be immunogenic during the infection with T. gondii in mouse model and promising reagent for serodiagnosis of toxoplasmosis in humans [15;12; 6]. However, its usefulness has not yet been confirmed in animal toxoplasmosis.In this study, the optimization and validation of E.coli-expressed rTgMAG1as ELISA antigen were describedMongolian Journal of Agricultural Sciences Vol.15(2) 2015; 56-60
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HEFLE, SUSAN L., ELIZABETH JEANNITON i STEVE L. TAYLOR. "Development of a Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Egg Residues in Processed Foods†". Journal of Food Protection 64, nr 11 (1.11.2001): 1812–16. http://dx.doi.org/10.4315/0362-028x-64.11.1812.
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Chicken eggs are used extensively as an excellent source of dietary proteins. These proteins have many functional properties, making them valuable food ingredients. However, eggs are a frequent cause of food hypersensitivity, especially in children. Of major concern to food processors is the inadvertent cross-contact of food products with allergenic residues, which could result in potentially life-threatening reactions in those with a food allergy. The aim of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of undeclared egg residues in foods. Commercially purified ovalbumin (OVA) and dehydrated egg white solids were used as antigens to induce antibodies in rabbits and goats. Reference pasta standards and various food samples were extracted, then clarified by centrifugation. Goat anti-egg white antibodies were used as the capture reagent, nonspecific sites were blocked with gelatin, then standard and sample extracts were added. Rabbit anti-OVA antibodies were used as detector antibodies, followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. Twenty brands of egg-free pasta (two lots each) were analyzed using the ELISA. Fourteen common pasta ingredients were also evaluated for cross-reactivity problems in the method. The detection limit of the assay was 1 ppm spray-dried whole egg. Fifty-five percent (22 samples) of the egg-free pasta samples tested positive for the presence of undeclared egg residues, with values ranging from 1 to >100,000 ppm. Minimal cross-reactivity was encountered in general, but portobello mushrooms and basil caused some minor matrix effects. This sandwich-type ELISA method can be used to detect undeclared egg residues in processed foods and to evaluate industrial clean-up operations.
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Kuzushima, Kiyotaka, Naomi Hayashi, Hiroshi Kimura i Tatsuya Tsurumi. "Efficient identification of HLA-A*2402–restricted cytomegalovirus-specific CD8+ T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay". Blood 98, nr 6 (15.09.2001): 1872–81. http://dx.doi.org/10.1182/blood.v98.6.1872.
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Abstract Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)–specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8+ T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-γ production revealed 0.04% to 0.27% of CD8+ T cells in peripheral blood of HLA-A24+ and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8+ T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.
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Cameron, Mark. "Rapid and cost effective immunoassay development and fit-for-purpose validation of a biomarker assay using Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL) (TECH2P.760)". Journal of Immunology 194, nr 1_Supplement (1.05.2015): 206.6. http://dx.doi.org/10.4049/jimmunol.194.supp.206.6.
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Abstract A novel detection technology was used for rapid immunoassay development and validation. SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) is a proximity dependent and homogeneous chemiluminescent detection method. In a SPARCL assay, a chemiluminescent substrate (acridan) is brought into the proximity of an oxidative enzyme (horseradish peroxidase) through the specific antigen/antibody interaction. A flash of light is generated and is proportional to the amount of IL-8 bound. Assay development was completed in less than 3 days. The validated assay features a wide dynamic range (2 - 4,000 pg/mL), low MRD, short assay run time (60 min.), and sensitivity (2 pg/mL). Quality control samples (QC’s) run in plasma resulted in statistics that show the assay is precise and accurate, with a total error of all QC levels of under 12%. Matrix spiked with IL-8 diluted in a linear manor. The SPARCL technology enables rapid assay development, delivers an assay with desirable performance characteristics and allows for considerable savings in labor, disposables and capital equipment compared to popular platforms. Existing software and luminometers may be used for a SPARCL data acquisition. As a result, there may not be a need to validate new software or plate readers already running in existing Laboratory Information Management Systems or in a regulated environment. The SPARCL assay is microtiter plate-based and is suitable for use with robotics and in HTS applications.
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Vadalà, G., G. Di Giacomo, L. Ambrosio, C. Cicione, V. Tilotta, F. Russo, R. Papalia i V. Denaro. "IRISIN STIMULATES ANABOLISM AND MATRIX SYNTHESIS IN HUMAN NUCLEUS PULPOSUS CELLS IN VITRO: NEW INSIGHTS INTO A CROSS-TALK BETWEEN THE MUSCLE AND THE INTERVERTEBRAL DISC". Orthopaedic Proceedings 105-B, SUPP_8 (11.04.2023): 53. http://dx.doi.org/10.1302/1358-992x.2023.8.053.
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This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.
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Zusfahair, Zusfahair, Dian Riana Ningsih, Amin Fatoni, Bilalodin Bilalodin i Aprilia Nafi Nuraini. "The Isolation, Immobilization, and Characterization of Urease from The Seeds of Winged Bean (Psophocarpus tetragonolobus (L.) DC." Molekul 18, nr 1 (20.03.2023): 70. http://dx.doi.org/10.20884/1.jm.2023.18.1.5932.
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Urease has been utilized in the field of health and industry. Urease is commonly used in the form of free enzyme, so that the utilization is limited. Urease efficiency can be improved using immobilization enzyme. This research aimed to do the urease isolation, immobilization, and characterization from the winged bean seeds. This research was started by determining the amino-acid content of winged bean seeds using the Liquid chromatography-mass spectrometry (LCMS). The winged bean seeds were germinated and extracted. The obtained crude extract’s activity was determined using Nessler reagent and measured using UV-Vis spectrophotometer with the wavelength of 500 nm. The urease of winged bean seeds was immobilized using the alginate matrix. The optimization of urease-immobilized beads could be made through the variations of natrium alginate concentration and beads formation periods in solution CaCl2. Characterization free and immobilized urease were made using the variations of urea substrate concentration, pH, temperature, and also the repeated utilization of immobilized urease. Winged bean seeds are rich with essential amino acid, such as leucine, isoleucine, histidine, phenylalanine, and valine. The urease obtained from the winged bean seeds had the optimum activity in the germination period of 8 days. The urease immobilization showed the optimum condition in the natrium alginate concentration of 5% (w/v) and beads formation period in solution CaCl2 for 60 minutes. The characterization results of free urease and immobilization had the optimum condition at the urea substrate of 0.2 M, and pH 7. Free urease had the optimum temperature of 35 oC, while the immobilized urease had the optimum temperature of 40 oC. The immobilized urease had the utilization stability up to 5 times with the relative activity of 48%. The EDX analysis results showed that the alginate did not contain N, while alginate urease beads contained N as much as 12%.
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Scholle, Michael D., Doug McLaughlin i Zachary A. Gurard-Levin. "High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease". SLAS DISCOVERY: Advancing the Science of Drug Discovery 26, nr 8 (19.06.2021): 974–83. http://dx.doi.org/10.1177/24725552211023211.
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Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.
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Balkian, Garo K., Kathleen M. Flores, Ramon Llull, Harry S. Ko, Kenneth W. Walker, Kirby S. Black i Charles W. Hewitt. "A Rapid and Sensitive Cellular Enzyme-Linked Immunoabsorbent Assay (CELISA) for the Detection and Quantitation of Antibodies against Cell Surface Determinants II. Optimal Reagent Concentrations and Predictive Analysis". Cell Transplantation 6, nr 4 (lipiec 1997): 431–37. http://dx.doi.org/10.1177/096368979700600411.
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A cellular enzyme-linked immunosorbent assay (CELISA) was developed for the detection and quantification of antibodies elicited against allogeneic cell surface determinants. The technique uses a solid-phase cell matrix created by fixing cells with a mild formalin solution onto the bottom of a 96-well microtiter plate. A primary layer of alloantisera is first reacted against rat target cells. The secondary antibody, peroxidase conjugated antirat IgG, is then added to each well and serves as the second sandwich layer. Optimal reagent concentrations were determined by serial dilution analysis of various cell concentrations and secondary antibody dilutions. It was found that 200,000 cells per well was the optimal target cell concentration. However, 100,000 cells per well was also sufficient to run the assay with acceptable performance characteristics. Even lower cell concentrations of 10,000 and 20,000 cells/well, although not optimal, also produced acceptable results. Secondary antibody concentration with respect to the optimal cell concentration was determined to be 1:500. At 200,000 cells per well and a 1:500 secondary antibody dilution, the assay presented excellent coefficients of determination and high positive to negative ratios. The reaction was found to be very sensitive in yielding high antibody titers with low background levels and could be defined mathematically as a linear-log function. Titers of multiple unknown alloantibody samples were easily and accurately predicted in an automated manner by regression analysis form known standards. This immunoassay will be useful in studies of cell surface determinant expression and quantitation of antibodies reactive to such markers.
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Haroon, Zishan A., Thung S. Lai i Charles S. Greenberg. "Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation by Altering Gene Expression during Wound Healing." Blood 104, nr 11 (16.11.2004): 2626. http://dx.doi.org/10.1182/blood.v104.11.2626.2626.
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Abstract Fibrin deposition triggers an injury response that involves the migration of inflammatory cells formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TTG) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TTG is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGF beta-1) and expressed during wound healing response. In this study, we investigated how TTG modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin), implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TTG into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p<0.001), an effect similar in magnitude to 25 ng/ml of TGFbeta1 administered in the F-ZC. The pro-healing effect of TTG was associated with a 2-fold increase in microvessel density in granulation tissue at day 6 of wound healing response (p<0.001). As a negative control, inactive recombinant TTG mutant did not exhibit increased wound healing response or pro-angiogenic effect. The data suggested that TTG enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TTG enhanced wound healing were 1) angiogenesis and 2) deposition of matrix. To investigate TTG-induced gene expression, total RNAs were isolated from control- and TTG-treated F-ZCs (at Day 6) using Trizol reagent (Invitrogen, CA). Biotin-labeled cDNA probes were synthesized, and hybridized to nylon membranes containing angiogenesis-related gene arrays (Superarray, MD). The signals were detected using streptavidin-peroxidase and quantitated using Superarray’s software. We identified increased expression of VEGF receptors Flk-1, Flt1 and neuropilin, suggesting increased responsiveness to the potent angiogenic factor VEGF. In addition, increased levels of angiopoietin-1 and ephrin B2 were observed which are involved in vascular development and stabilization. For matrix enhancing effects, considerably decreased levels (5-fold) of matrix metalloproteinases (MMPs) coupled with increased TGFbeta receptors and connective tissue growth factor (CTGF) were observed. The gene expression profile suggests that TTG alters the balance between matrix production and destruction in favor of production resulting in increased deposition of ECM in granulation tissue. In conclusion, we have identified that TTG 1) enhances fibrin-dependent wound healing response, 2) increases angiogenesis through enhanced VEGF receptors, angiopoietin-1 and ephrin B2 expression, and 3) promoted matrix deposition by simultaneously reducing MMPs and increasing CTGF and TGFbeta receptors expression.
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MCGARRY, J. Denis, i Nicholas F. BROWN. "Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity". Biochemical Journal 349, nr 1 (26.06.2000): 179–87. http://dx.doi.org/10.1042/bj3490179.
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Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of β-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6×His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200-400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 °C compared with 4 °C (IC50 ≈ 11 μM versus 30 μM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver > fasted liver≈ liposomes prepared at 22 °C≈ P. pastoris mitochondria > liposomes prepared at 4 °C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6×His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.
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Rutten, Iene, Devin Daems, Karen Leirs i Jeroen Lammertyn. "Highly Sensitive Multiplex Detection of Molecular Biomarkers Using Hybridization Chain Reaction in an Encoded Particle Microfluidic Platform". Biosensors 13, nr 1 (6.01.2023): 100. http://dx.doi.org/10.3390/bios13010100.
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In the continuous combat against diseases, there is the need for tools that enable an improved diagnostic efficiency towards higher information density combined with reduced time-to-result and cost. Here, a novel fully integrated microfluidic platform, the Evalution™, is evaluated as a potential solution to this need. Encoded microparticles combined with channel-based microfluidics allow a fast, sensitive and simultaneous detection of several disease-related biomarkers. Since the binary code is represented by physically present holes, 210 different codes can be created that will not be altered by light or chemically induced degradation. Exploiting the unique features of this multiplex platform, hybridization chain reaction (HCR) is explored as a generic approach to reach the desired sensitivity. Compared to a non-amplified reference system, the sensitivity was drastically improved by a factor of 104, down to low fM LOD values. Depending on the HCR duration, the assay can be tuned for sensitivity or total assay time, as desired. The huge potential of this strategy was further demonstrated by the successful detection of a multiplex panel of six different nucleic acid targets including viruses and bacteria. The ability to not only discriminate these two categories but, with the same effort, also virus strains (human adenovirus and human bocavirus), virus subtypes (human adenovirus type B and D) and antibiotic-resistant bacteria (Streptococcus pneumonia), exemplifies the specificity of the developed approach. The effective, yet highly simplified, isothermal and protein-enzyme-free signal amplification tool reaches an LOD ranging from as low as 33 ± 4 to 151 ± 12 fM for the different targets. Moreover, direct detection in a clinically relevant sample matrix was verified, resulting in a detection limit of 309 ± 80 fM, approximating the low fM levels detectable with the gold standard analysis method, PCR, without the drawbacks related to protein enzymes, thermal cycling and elaborate sample preparation steps. The reported strategy can be directly transferred as a generic approach for the sensitive and specific detection of various target molecules in multiplex. In combination with the high-throughput capacity and reduced reagent consumption, the Evalution™ demonstrates immense potential in the next generation of diagnostic tools towards more personalized medicine.
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Naghneh, Ehsan, Es'hagh Pourmaleki i Azam Rahimpour. "Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells". Pharmaceutical Sciences 26, nr 4 (25.12.2020): 393–98. http://dx.doi.org/10.34172/ps.2020.37.
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Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.
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Zakhvatov, Aleksey Nikolaevich, Dalila Ali Khaydar, Il'ya Aleksandrovich Zakharkin, Tat'yana Viktorovna Kurmysheva, Sergey Aleksandrovich Tambovtsev, Andrey Sergeevich Kurmyshev, Alina Yur'evna Parshina i Irina Yur'evna Zhuravleva. "CYTOKINE-MEDIATED REACTIONS IN THE PATHOGENESIS OF EXPERIMENTAL PERIODONTITIS". Ulyanovsk Medico-biological Journal, nr 4 (26.12.2022): 139–48. http://dx.doi.org/10.34014/2227-1848-2022-4-139-148.
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Determining cytokinemia role in the destructive processes of the periodontal complex gives new opportunities to the development and subsequent implementation of practical methods for chronic periodontitis diagnosis, justifies including pathogenetic drugs that inhibit the secretion of pro-inflammatory mediators in treatment regimens.
The aim of the study is to evaluate the dynamics of cytokine profile indicators and identify their correlation with the local status of periodontal tissues in animals with experimental periodontitis.
Materials and methods. The study enrolled 65 white outbred rats weighing 180±20 g. The authors reproduced the model of experimental periodontitis according to K.D. Shkolnaya, V.G. Atrushkevich method (patent RU No. 2625295, December 07, 2017). The cytokine profile was assessed by pro-inflammatory and anti-inflammatory cytokine levels, determined by enzyme-linked immunosorbent assay, Bender MedSystems reagent kit. Local status of periodontal tissues was assessed according to the degrees of gingival hemorrhage, dental mobility and depth of periodontal pockets with a button-shaped and modified periodontal probes.
Results. During the experimental periodontitis modeling, a high level of cytokinemia was determined. It destructively effects the periodontal connective tissue matrix, which was confirmed by an increase in the depth of periodontal pockets, gingival hemorrhage and dental mobility. A strong correlation between a high level of cytokinemia and local destructive changes in periodontal tissues was determined. This fact emphasized the conjugation of these pathophysiological mechanisms.
Conclusion. Defined disorders of physiological balance in the cytokine balance necessitate the use of pathogenic drugs, as they have an inhibitory effect on the synthesis of pro-inflammatory cytokines and prevent further destruction of the periodontal complex. Changes in the cytokine profile indicate inflammation process, and a decrease in the level of cytokinemia indicate the resolution of infection and can be considered a criterion for the effective treatment.
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Kumar, Sujatha, Ryan Gallagher, Josh Bishop, Enos Kline, Joshua Buser, Lisa Lafleur, Kamal Shah, Barry Lutz i Paul Yager. "Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices". Analyst 145, nr 21 (2020): 6875–86. http://dx.doi.org/10.1039/d0an01098g.
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Mičková, B., P. Rauch, A. Montoya, E. Ferri, F. Fini i S. Girotti. "The determination of N-methylcarbamate pesticides using enzyme immunoassays with chemiluminescent detection". Czech Journal of Food Sciences 22, SI - Chem. Reactions in Foods V (1.01.2004): S280—S282. http://dx.doi.org/10.17221/10681-cjfs.
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In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. The sensitivity of immunochemical methods was expressed as detection limit, linear working range, and I<sub>50</sub> value. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of HRP allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing simply diluted fruit juices, spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more.
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Bibikov, S. O., i S. O. Shapovalov. "Application of microscopy methods for evaluation of feed digestibility". Kormlenie sel'skohozjajstvennyh zhivotnyh i kormoproizvodstvo (Feeding of agricultural animals and feed production), nr 10 (1.10.2020): 61–69. http://dx.doi.org/10.33920/sel-05-2010-07.
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The results of applying microscopic methods of analysis of pig feces to assess the digestibility of feed and diagnose various diseases of the gastrointestinal tract have been presented in the article. The microscopic method allows you to identify detritus, undigested fi ber, raw fat, fragments of animal feed ingredients, starch in the feces, gives an idea of the presence of foreign impurities (due to perverted appetite, contamination of feed), helminths, their eggs and other intestinal parasites. A number of preparations have been prepared to identify feed nutrients and feed components of the diet, and a number of coloring reagents were also used: Lugol’s solution for recognizing starch and its cleavage products (amylodextrin and erythrodextrin), Saathof’s reagent for detecting fat, and Hecht’s reagent for diff erentiating fat elements. The researches have been carried out under various magnifi cations of the microscope. Diff erent levels of detritus and undigested elements of feed in animals under the same conditions of rearing and feeding can identify individual features of digestion. The microscopic method of analysis does not require expensive equipment, reagents, and allows you to get the results of assessing the digestibility much faster than the methods of classical “wet” chemistry. This research method can be used by nutritionists to correct diets when using exogenous feed enzymes: phytase, xylanase, gluconase, amylase, mannanase, lipase, protease, etc., and provide results for discussion about the presence or absence of matrix eff ects from their use. The results of feces microscopy can give then idea of the quality of feed ingredients used: the content of non-starchy polysaccharides and the level of grain viscosity, the quality of animal ingredients (meat-bone, feather, fi sh meal), the level of indigestible fi ber in sunfl ower and soybean meals.
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Bagherbaigi, Saeedeh, Emma P. Córcoles i Dedy H. B. Wicaksono. "Cotton fabric as an immobilization matrix for low-cost and quick colorimetric enzyme-linked immunosorbent assay (ELISA)". Anal. Methods 6, nr 18 (2014): 7175–80. http://dx.doi.org/10.1039/c4ay01071j.
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Litvynova, A., i L. Pasiieshvili. "Pathogenetic and diagnostic role of osteoprotegerin in the combined course of osteoarthritis and obesity". Journal of Education, Health and Sport 12, nr 5 (31.05.2022): 284–91. http://dx.doi.org/10.12775/jehs.2022.12.05.022.
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In recent years, it has been proven that the formation of OA can occur against the background of impaired bone metabolism.
Bone remodeling is considered as a continuous complex process aimed at eliminating microdamages and updating the bone matrix. A key link in the regulation of this process is the system RANK / RANKL / OPG (osteoprotegerin), which provides a balance of activity of osteoblasts and osteoclasts. It blocks the interaction between RANK and RANKL by intercepting the RANKL ligand, which inhibits osteoclast development and reduces bone resorption.
Thus, these properties of OPG can be considered as one of the pathogenetic factors in patients with dystrophic joint lesions.
Objective: to investigate the content and role of osteoprotegerin in young patients with osteoarthritis, which occurs against the background of changes in body weight.
Materials and methods. The study involved 75 young patients (mean age - 30.92 ± 0.546 years) with overweight or obesity, who in the previous stages of the study was diagnosed with osteoarthritis (main group). The comparison group was represented by 50 patients with osteoarthritis and normal body weight. The age of patients was 30.95 ± 0.545 years; as in the previous group, men predominated - 64% and 36% respectively.
Benchmarks of osteoprotegerin were obtained in a study of 37 relatively healthy individuals of the same age and sex.
The level of osteoprotegerin was determined by enzyme-linked immunosorbent assay using the FineTest EH0247 reagent kit, China.
The outcomes were processed by the methods of variation statistics using the computer program STATISTICA.Results and discussion. When determining the content of OPG in the serum of patients of the main group was found to increase to 124.03 pg / ml, against control - 65.64 pg / ml. In the group of patients with isolated OA, this value was 92.29 pg / ml. Meaning that, in OA, running on the background of altered BMI, the content of OPG is likely to increase, both in relation to the norm and the results of the comparison group (p <0,001).
The formation and course of osteoarthritis is accompanied by an increase in osteoprotegerin, the content of which depends on the degree of obesity and the radiological stage of the process.
Conclusions: The course of osteoarthritis is accompanied by an increase in osteoprotegerin, which is considered a negative regulator of bone resorption.
In osteoarthritis, which occurs against the background of changes in body mass index, a direct correlation with the degree of obesity.
Changes in osteoprotegerin in patients with osteoarthritis depend on the radiological stage of joint damage and are most pronounced in individuals with stage 2 obesity and stage 2 radiological changes
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Hollinshead, Michael, Jeremy Sanderson i David J. Vaux. "Anti-biotin Antibodies Offer Superior Organelle-specific Labeling of Mitochondria over Avidin or Streptavidin". Journal of Histochemistry & Cytochemistry 45, nr 8 (sierpień 1997): 1053–57. http://dx.doi.org/10.1177/002215549704500803.
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The mitochondrial matrix contains endogenously biotinylated proteins. These proteins can cause unexpected background signal when biotin–avidin- or biotin–streptavidin-based detection systems are used in immunocytochemistry. Here we show that this reactivity can be deliberately exploited, using a simple anti-biotin reagent, to obtain strong and highly specific labeling of mitochondria by both light and electron microscopy. The signal is substantially stronger than when either avidin or streptavidin is used to detect the endogenous biotin. These results confirm the accessibility of protein-bound endogenous biotin to exogenous probes, and localize the biotinylated enzymes to the mitochondrial matrix. (J Histochem Cytochem 45:1053–1057, 1997)
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Shulyatnikova, Oksana, Anatoliy Godovalov, Gennadiy Rogozhnikov, Mihail Yakovlev, Kirill Batog i Elena Leushina. "ACTIVITY OF SALIV ALPHA AMYLASES AND LEVEL OF ORAL CAVITY HYGIENE UNDER DIFFERENT CLINICAL CONDITIONS". Actual problems in dentistry 17, nr 1 (6.05.2021): 172–76. http://dx.doi.org/10.18481/2077-7566-20-17-1-172-176.
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Subject. Changes in the activity of certain saliva enzymes, due to certain factors and in certain clinical conditions of the human body, can affect oral hygiene, as well as indirectly on the activity of the carious process. Of particular interest is the enzyme — alpha-amylase, which is due to its ability to cleave dextrans and levans, which are the basis of the matrix of the bacterial film. The article presents data on changes in the activity of saliva alpha-amylase in microecological disorders in the intestine and acute respiratory viral diseases, as well as its effect on the level of oral hygiene.
The goal — the assessment of changes in the activity of saliva alpha-amylase in microecological disorders in the intestine and acute respiratory diseases, taking into account the level of oral hygiene.
Methods. To determine the activity of alpha-amylase, a set of reagents "Amylase-Vital" was used according to the manufacturer's instructions, proportionally reducing the volume of reagents for the procedure in titration plates. The volunteers, divided into groups with severe acute respiratory syndrome, microecological intestinal disorders, and a control group, received mixed saliva and evaluated their oral hygiene status.
Results. The inverse relationship between the severity of intoxication syndrome in severe acute respiratory syndrome and the level of saliva alpha-amylase activity is shown. In addition, a correlation was established between an increase in human body temperature and a decrease in the activity of alpha-amylase. There is also a link between the presence of decompensated intestinal dysbiosis in humans and the activity of the enzyme. The dependence between the decrease in the activity of the studied enzyme and the increase in biofilm formation on the organs of the oral cavity was revealed.
Conclusions and Relevance. The conducted studies open up a promising direction for the development of additional diagnostic criteria based on the determination of the activity of saliva alpha-amylase. In addition, the data obtained on the deterioration of oral hygiene indicate the need for a comprehensive approach to the treatment of patients in this category with the mandatory involvement of a dentist.
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Cottone, Grazia, Sergio Giuffrida, Stefano Bettati, Stefano Bruno, Barbara Campanini, Marialaura Marchetti, Stefania Abbruzzetti i in. "More than a Confinement: “Soft” and “Hard” Enzyme Entrapment Modulates Biological Catalyst Function". Catalysts 9, nr 12 (4.12.2019): 1024. http://dx.doi.org/10.3390/catal9121024.
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Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different phase with respect to the reagents’ phase, requires either its physical confinement in an immobilization matrix or its physical adsorption on a surface. In this review, we will focus on the immobilization of biological catalysts, i.e., enzymes, by comparing hard and soft immobilization matrices and their effect on the modulation of the catalysts’ function. Indeed, unlike smaller molecules, the catalytic activity of protein catalysts depends on their structure, conformation, local environment, and dynamics, properties that can be strongly affected by the immobilization matrices, which, therefore, not only provide physical confinement, but also modulate catalysis.
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George, Jeanette, Michelle L. Teear, Christopher G. Norey i D. Dougal Burns. "Evaluation of an Imaging Platform during the Development of a FRET Protease Assay". Journal of Biomolecular Screening 8, nr 1 (styczeń 2003): 72–80. http://dx.doi.org/10.1177/1087057102239778.
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Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from bi-ologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70,000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMTbased fluorescence plate reader and at the same time evaluated a charged couple device (CCD)—based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens. (Journal of Biomolecular Screening 2003:72-80)
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Dhar, Manjima, Jeffrey Nam Lam, Tonya Walser, Steven M. Dubinett, Matthew B. Rettig i Dino Di Carlo. "Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay". Proceedings of the National Academy of Sciences 115, nr 40 (17.09.2018): 9986–91. http://dx.doi.org/10.1073/pnas.1803884115.
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Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.
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Chaubal, Rajendra, V. A. Wilmot i Willard K. Wynn. "Visualization, adhesiveness, and cytochemistry of the extracellular matrix produced by urediniospore germ tubes of Puccinia sorghi". Canadian Journal of Botany 69, nr 9 (1.09.1991): 2044–54. http://dx.doi.org/10.1139/b91-257.
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Adherence of germinating urediniospores of the common maize rust fungus (Puccinia sorghi Schw.) to substrata was studied by ultrastructural and cytochemical examination of extracellular matrix produced by germ tubes in conjunction with measurements of adhesion to plastic and glass surfaces. Copious amounts of extracellular matrix on germ tubes could consistently be visualized by scanning and transmission electron microscopy only when (i) a cationic detergent (cetylpyridinium chloride, polydiallyldimethylammonium chloride) or a cationic stain (ruthenium red, alcian blue, cuprolinic blue) was added to the fixation solutions, (ii) germ tubes were fixed by rapid-freezing and freeze-substitution and observed with a scanning electron microscope, or when (iii) germ tubes were observed in a frozen-hydrated state by low-temperature scanning electron microscopy. Incubation of germinated spores with dilute alkalies (NaOH, KOH), pronase E (nonspecific protease), and laminarinase (β-1,3 (1,3; 1,4-glucanase) removed the extracellular matrix and detached germ tubes from surfaces. Treatments with water, dilute acids, ionic and neutral detergents, organic solvents, hydrocarbons, and several polysaccharide-degrading enzymes did not remove the extracellular matrix and also did not detach germ tubes. These results, together with staining patterns obtained with lectins and other polysaccharide-specific reagents, indicate that the extracellular matrix is composed mainly of glycoproteins rich in acidic amino acids and β-1,3-glucan polymers, and that it is probably responsible for the adhesion of the rust germ tubes to the host leaf surfaces. Key words: Puccinia sorghi, germ tube adhesion, extracellular matrix, cytochemistry.
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Garrido, M. Dolores, Jamal El Haskouri, María D. Marcos, Francisco Pérez-Pla, José Vicente Ros-Lis i Pedro Amorós. "One-Pot Synthesis of MnOx-SiO2 Porous Composites as Nanozymes with ROS-Scavenging Properties". Nanomaterials 12, nr 19 (7.10.2022): 3503. http://dx.doi.org/10.3390/nano12193503.
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The development of nanomaterials that mimic the activity of enzymes is a topic of interest, for the decomposition of reactive oxygen species (ROS). We report the preparation of a novel nanocomposite of MnOx needles covered with SiO2 porous material. The material was prepared in one pot with a two-step procedure. The material was characterized by EDX, SEM, TEM, XRD, nitrogen adsorption–desorption isotherms, and XPS. The synthesis protocol took advantage of the atrane method, favoring the nucleation and initial growth of manganese oxide needles that remained embedded and homogeneously dispersed in a mesoporous silica matrix. The final composite had a high concentration of Mn (Si/Mn molar ratio of ca. 1). The nanozyme presented bimodal porosity: intraparticle and interparticle association with the surfactant micelles and the gaps between silica particles and MnOx needles, respectively. The porosity favored the migration of the reagent to the surface of the catalytic MnOx. The nanozyme showed very efficient SOD and catalase activities, thus improving other materials previously described. The kinetics were studied in detail, and the reaction mechanisms were proposed. It was shown that silica does not play an innocent role in the case of catalase activity, increasing the reaction rate.
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Bar-Nun, Nurit, Annie L'Hyvernay, Bernard Donèche i Alfred M. Mayer. "Changes in mycelial structure of Botrytis cinerea induced by removal of the glucan matrix". OENO One 41, nr 3 (30.09.2007): 149. http://dx.doi.org/10.20870/oeno-one.2007.41.3.843.
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<p style="text-align: justify;"><strong>Aims</strong>: b-1,3-glucanase is one of the main pathogenesis related proteins of plants, involved in plant-pathogen interactions. Its effect on fungal pathogens is not entirely known. The hyphae of Botrytis cinerea are covered by an extra cellular matrix, mainly composed of a b-1,3-D-glucan. This matrix also contains a variety of enzymes, lipids and melanin which may play a role in fungal virulence.</p><p style="text-align: justify;"><strong>Methods and results</strong>: Cultures of Botrytis cinerea are made in presence of b-1,3-glucanase. The structure of the mycelium of Botrytis cinerea after exposure to b-1,3-glucanase during growth was examined by staining with Schiff's reagent and using the electron microscope. Without glucanase, hyphae have a normal diameter and were surrounded by a glucan matrix. Cytoplasm is dense and contains little vacuoles. The glucanase treatment removed most of the glucan sheath, but did not kill the fungus. The structure of the hyphae was changed by the treatment and their diameter increased. Membrane structure showed marked changes, the cytoplasm of the cells was less dense, but more inclusions were observed, including an increase in what appeared to be lipids.</p><p style="text-align: justify;"><strong>Conclusion</strong>: The appearance of the mycelium, whose glucan sheath has been removed, was that of cells under stress. The possible implications of the function of the glucan sheath during the interaction of Botrytis cinerea with its host during pathogenesis are discussed.</p><p style="text-align: justify;"><strong>Significance and impact of study</strong>: These changes following glucanase treatment would lead to a fungal mycelium which will be more sensitive to antifungal agents and might suggest ways of combating Botrytis infections by preventing the formation of the extra-cellular matrix.</p>
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Watkins, Nicholas A., Lily M. Du, J. Paul Scott, Willem H. Ouwehand i Cheryl A. Hillery. "Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion". Blood 102, nr 2 (15.07.2003): 718–24. http://dx.doi.org/10.1182/blood-2002-11-3497.
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AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.
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Gaspar, Andrew H., Luciano A. Marraffini, Elizabeth M. Glass, Kristin L. DeBord, Hung Ton-That i Olaf Schneewind. "Bacillus anthracis Sortase A (SrtA) Anchors LPXTG Motif-Containing Surface Proteins to the Cell Wall Envelope". Journal of Bacteriology 187, nr 13 (1.07.2005): 4646–55. http://dx.doi.org/10.1128/jb.187.13.4646-4655.2005.
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ABSTRACT Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F2, but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.
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Nevezhkina, Tatiana A., Maria A. Chernikova, Elena V. Markelova, Marina S. Tulupova, Anna V. Kostyshko, Lydmila N. Fedyanina i Natalia Yu Markova. "Dynamics of matrix metalloproteinases and their tissue inhibitors in patients with herpesvirus and papillomavirus infection". Russian Journal of Immunology 26, nr 3 (11.08.2023): 355–62. http://dx.doi.org/10.46235/1028-7221-9409-dom.
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Sexually transmitted infections are of great importance for the proper reproductive function in women. Chronic inflammatory process leads to reproductive disorders. A special role in the chronic inflammatory process is attributed to papillomavirus (PVI) and herpetic infection. MMP-2 and MMP-9 enzymes cleave type 4 collagen which makes the scaffold of basement membranes and contributes to the separation of endothelial cells from the membranes, followed by their further migration and direct participation in angiogenesis thus affecting the growth of tumors, in particular cervical cancer. Tissue inhibitors of matrix metalloproteinases are known to limit the collagen breakdown. However, the imbalance of MMP and TIMP is accompanied by accumulation of extracellular matrix and increased risk for reproductive disorders. The aim of our study was to evaluate the dynamics of acute-phase proteins affecting the state of intercellular matrix (MMP-2, MMP-9 and MMP tissue inhibitors (type 1, 2) in blood serum of patients with PVI or coinfection of PVI and HSV before and after therapy with drugs exhibiting antiviral and immunomodulatory effects, i.e., a synthetic compound (Inosine pranobex) and vegetable substance (Solanum tuberosum). We have examined 141 patients with papillomavirus and herpetic infections treated with Inosine pranobex and Solanum tuderosum. Determination of MMP-2, MMP-9 and TIMP-1, TIMP-2 levels of in blood serum was carried out using specific reagents from RD Diagnostics Inc. (USA). The drug therapy with active substances of Inosine pranobex and Solanum tuberosum was associated with positive dynamics of the level of MMP-2, MMP-9 and tissue inhibitors of types 1 and 2 in all groups under studies. However, Inosine Pranobex exerts more pronounced changes, especially in subgroups with viral coinfections.
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Begovac, P. C., i B. D. Shur. "Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin." Journal of Cell Biology 110, nr 2 (1.02.1990): 461–70. http://dx.doi.org/10.1083/jcb.110.2.461.
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Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.
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Grigoriev, A. M., Yu B. Basok, A. D. Kirillova, V. A. Surguchenko, N. P. Shmerko, V. K. Kulakova, R. V. Ivanov, V. I. Lozinsky, A. M. Subbot i V. I. Sevastianov. "Cryogenically structured gelatin-based hydrogel as a resorbable macroporous matrix for biomedical technologies". Russian Journal of Transplantology and Artificial Organs 24, nr 2 (13.05.2022): 83–93. http://dx.doi.org/10.15825/1995-1191-2022-2-83-93.
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Objective: to investigate the biological properties of a matrix made of cryogenically structured hydrogel in the form of a macroporous gelatin sponge, as well as the possibility of creating cell-engineered constructs (CECs) on its basis. Materials and methods. The main components of the cryogenically structured hydrogel were gelatin (type A) obtained from porcine skin collagen, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide, (EDC) and urea (all from Sigma-Aldrich, USA). Surface morphology was examined using scanning electron microscopy (SEM). The degree of swelling in water of the samples was determined by gravimetric method. Cytotoxicity was studied on NIH3T3, a fibroblast cell line isolated from a mouse, and on human adipose-derived mesenchymal stem/stromal cells (hAMSCs) using IncuCyte ZOOM (EssenBioscience, USA). The metabolic activity of hAMSCs was assessed using PrestoBlue™ reagents (Invitrogen™, USA). To create CECs, we used hAMSCs, human hepatocellular carcinoma cell line HepG2 or human umbilical vein endothelial cell lines EA.hy926. Albumin content in the culture medium was determined by enzyme immunoassay. Ammonia metabolism rate was assessed after 90 minutes of incubation with 1 mM ammonium chloride (Sigma-Aldrich, USA) diluted in a culture medium on day 15 of the experiment. Results. Obtaining a cryogenically structured hydrogel scaffold in the form of macroporous gelatin sponge included freezing an aqueous solution of a gelatin+urea mixture, removal of polycrystals of frozen solvent by lyophilization, extraction of urea with ethanol and treatment of the cryostructurate with an ethanol solution of EDC. Scanning electron microscopy identified three types of pores on the carrier surface: large (109 ± 17 μm), medium (39 ± 10 μm), and small (16 ± 6 μm). The degree of swelling in water of the matrix samples was 3.8 ± 0.2 g H2O per 1 g of dry polymer. The macroporous gelatin sponge as a part of CEC was found to have the ability to support adhesion and proliferation of hAMSCs, EA.hy926 and HepG2 for 28, 15 and 9 days, respectively. Albumin secretion and ammonia metabolism when HepG2 cells were cultured on the gelatin sponge were detected. Conclusion. The use of a matrix made from macroporous cryogenically structured gelatin-based hydrogel for tissue engineering products is shown to be promising using a cell-engineered liver construct as a case.
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Smith, Darci R., Cynthia A. Rossi, Todd M. Kijek, Erik A. Henchal i George V. Ludwig. "Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus". Clinical Diagnostic Laboratory Immunology 8, nr 6 (1.11.2001): 1070–75. http://dx.doi.org/10.1128/cdli.8.6.1070-1075.2001.
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ABSTRACT The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories.
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Liao, Wen-Chieh, Marissa Adame, Jess Webb, Dean Nguyen i Jessie Ni. "Development of Quantitative Spike and Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assays (ELISAs) for Measurement of Antibodies against SARS-CoV-2". Journal of Immunology 208, nr 1_Supplement (1.05.2022): 125.23. http://dx.doi.org/10.4049/jimmunol.208.supp.125.23.
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Abstract In the COVID-19 pandemic, serological assays will have important public health and clinical uses to monitor and respond to the pandemic. Assays to detect and quantify virus-specific antibodies are important to complement the RNA-based tests, improve our understanding of the pathogenesis of COVID-19, and inform vaccine development. The majority of commercial assays for measuring IgG to SARS-CoV-2 have been limited by a lack of quantitative information about antiviral antibodies in standardized units. Here, we have developed and commercialized several fully validated, quantitative ELISA kits using purified receptor-binding domain (RBD) or full-length of Spike S1, or Nucleocapsid proteins developed in BioLegend as the capture reagents. The SARS-CoV-2 Spike Protein antibody ELISA kits have both LEGEND MAXTM and RAPID MAXTM formats and are optimized to mitigate matrix effects and avoid false-positive results, and have a readout in standardized units (mg/mL). We have applied these sensitive and specific SARS-CoV-2 Spike Protein antibody ELISAs in an employee cohort study to monitor antibody immune response after first and second doses of 2 mRNA SARS-CoV-2 vaccines – BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). We found that although all participants in this study are responsive to the first dose of immunization by both mRNA vaccines as evidenced by the elevation of anti-Spike S1 and RBD IgG levels above the pre-immunization baseline levels, more robust antibody responses to Spike S1 and RBD are observed after the second dose. In summary, our assays have good performance characteristics, have a quantitative readout in standardized units, and could be used by research and clinical laboratories that routinely use ELISA.
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Koch, Dietmar, Christian Soltmann i Georg Grathwohl. "Bioactive Ceramics - New Processing Technologies for Immobilization of Microorganisms for Filtration and Bioreactor Applications". Key Engineering Materials 336-338 (kwiecień 2007): 1683–87. http://dx.doi.org/10.4028/www.scientific.net/kem.336-338.1683.
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The metabolism of biocomponents and microorganisms is widely used for the production of medical products as well as for biotechnological processes. The productivity of the related reactions can be improved by the immobilization of the living species in granules or in a gel matrix. Since these structures can suffer during operation due to their weakness a new type of porous ceramic composites was developed where the biological phase is immobilized in a rigid inorganic matrix of high permeability for fluids. During the so-called freeze gelation process (FGP) both, a highly efficient immobilization of the biocomponents and an ideal permeability is achieved. A high versatility is then offered due to the particular advantages of the freezing step necessary for the sol-gel transition and the preservation of embedded microorganisms. While the freezing conditions are decisive for the resulting porosity of the biocer, they are also crucial for the survival rate of the embedded biocomponents. The porosity can be adjusted over a wide range by controlling the composition and the freezing conditions. By the directional ice crystal growth large pore channels can be achieved inside the biocers. Thus, the embedded biocomponents are easily accessible by external reagents and biochemical reactions can proceed with a high rate. Furthermore, cell division is conceivable inside the biocers by safe immobilization at the same time. These biocers allow a wide field of applications depending on the class of immobilized biospecies. Biocatalysis with enzymes can also be applied as bioaccumulation and absorption/desorption of metal ions for separation processes of contaminated water or highly selective filters for metallic complexes in solution.
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Runyan, R. B., G. D. Maxwell i B. D. Shur. "Evidence for a novel enzymatic mechanism of neural crest cell migration on extracellular glycoconjugate matrices." Journal of Cell Biology 102, nr 2 (1.02.1986): 432–41. http://dx.doi.org/10.1083/jcb.102.2.432.
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Migrating embryonic cells have high levels of cell surface galactosyltransferase (GalTase) activity. It has been proposed that GalTase participates during migration by recognizing and binding to terminal N-acetylglucosamine (GlcNAc) residues on glycoconjugates within the extracellular matrix (Shur, B. D., 1982, Dev. Biol. 91:149-162). We tested this hypothesis using migrating neural crest cells as an in vitro model system. Cell surface GalTase activity was perturbed using three independent sets of reagents, and the effects on cell migration were analyzed by time-lapse microphotography. The GalTase modifier protein, alpha-lactalbumin (alpha-LA), was used to inhibit surface GalTase binding to terminal GlcNAc residues in the underlying substrate. alpha-LA inhibited neural crest cell migration on basal lamina-like matrices in a dose-dependent manner, while under identical conditions, alpha-LA had no effect on cell migration on fibronectin. Control proteins, such as lysozyme (structurally homologous to alpha-LA) and bovine serum albumin, did not effect migration on either matrix. Second, the addition of competitive GalTase substrates significantly inhibited neural crest cell migration on basal lamina-like matrices, but as above, had no effect on migration on fibronectin. Comparable concentrations of inappropriate sugars also had no effect on cell migration. Third, addition of the GalTase catalytic substrate, UDPgalactose, produced a dose-dependent increase in the rate of cell migration. Under identical conditions, the inappropriate sugar nucleotide, UDPglucose, had no effect. Quantitative enzyme assays confirmed the presence of GalTase substrates in basal lamina matrices, their absence in fibronectin matrices, and the ability of alpha-LA to inhibit GalTase activity towards basal lamina substrates. Laminin was found to be a principle GalTase substrate in the basal lamina, and when tested in vitro, alpha-LA inhibited cell migration on laminin. Together, these experiments show that neural crest cells have at least two distinct mechanisms for interacting with the substrate during migration, one that is fibronectin-dependent and one that uses GalTase recognition of basal lamina glycoconjugates.
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Oldak, Lukasz, Anna Leśniewska, Beata Zelazowska-Rutkowska, Eryk Latoch, Zenon Lukaszewski, Maryna Krawczuk-Rybak i Ewa Gorodkiewicz. "An Array SPRi Biosensor for Simultaneous VEGF-A and FGF-2 Determination in Biological Samples". Applied Sciences 12, nr 24 (11.12.2022): 12699. http://dx.doi.org/10.3390/app122412699.
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A new method was developed for the simultaneous determination of vascular endothelial growth factor (VEGF-A) and fibroblast growth factor-2 (FGF-2) in blood serum, using biosensors with array Surface Plasmon Resonance imaging (SPRi) detection. It can be applied as a single method for simultaneous VEGF-A and FGF-2 determination or as two separate methods for testing only one selected protein in each case. Validation was carried out for each method. Limit of detection (LOD) and limit of quantification (LOQ) values were determined and were found not to differ significantly from the parameters obtained in comparisons with commercial enzyme-linked immunosorbent assay (ELISA) tests. Tests were carried out to check the robustness of the method. The results indicate a lack of robustness of the analytical method to elevated temperature and pH values other than those recommended by the manufacturers of the reagents (recommended pH = 7.40). The values of recoveries were determined and confirmed the reliability of the results obtained with the use of the newly developed method. The selectivity studies showed no negative influence of other proteins present in the matrix of the tested samples on the results of the VEGF-A and FGF-2 concentration measurements. The developed method is also characterized by high reproducibility of the results obtained and agreement with the VEGF-A and FGF-2 concentration values obtained with commercial ELISA tests. The proposed method offers fast, reproducible, and accurate simultaneous quantification of VEGF-A and FGF-2 in human body fluids. Only 4 µL of test sample are required for simultaneous analysis. The total time for simultaneous analysis of both biomarkers does not exceed 20 min. The developed analytical method is superior to ELISA in terms of analysis time and sample volume for analysis, and it offers lower LOD and LOQ values and allows for the simultaneous analysis of two biomarkers. There is also no need to collect a large number of samples. Standard ELISAs usually have 96 reaction wells. The proposed biosensor can be used to analyse only one sample, without the need to waste reagents on unused reaction sites. In addition, it is possible to regenerate the biosensor and reuse it.
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Chen, Shuxiong, Natalie A. Parlane, Jason Lee, D. Neil Wedlock, Bryce M. Buddle i Bernd H. A. Rehm. "New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli". Applied and Environmental Microbiology 80, nr 8 (14.02.2014): 2526–35. http://dx.doi.org/10.1128/aem.04168-13.
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ABSTRACTThe tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared fromMycobacterium bovisare present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenicMycobacterium tuberculosiscomplex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinantEscherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected withM. boviswith no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.
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Avdeeva, A. S., V. V. Rybakova, O. G. Alekseeva i E. L. Nasonov. "The role of monitoring the level of matrix metalloproteinase 3 in patients with rheumatoid arthritis on anti-B-cell therapy". Rheumatology Science and Practice 60, nr 4 (7.09.2022): 473–80. http://dx.doi.org/10.47360/1995-4484-2022-473-480.
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Objective: to evaluate the role of monitoring the level of matrix metalloproteinase 3 (MMP-3) in patients with rheumatoid arthritis (RA) during anti-B-cell therapy.Material and methods. The study included 54 patients with a reliable diagnosis of RA. Depending on the therapy, all patients were divided into two groups: 34 patients received the original RTM (group 1) and 20 patients – biosimilar (group 2) in a total dose of 1200 mg according to the standard scheme. The concentration of MMP-3 in serum was measured by enzyme immunoassay using a kit of reagents from Invitrogen (USA).Results. The level of MMP-3 in patients with RA was significantly higher than in healthy donors, its median was 42.9 [10.0; 110.7] and 7.8 [5.5; 11.8] ng/ml, respectively (p<0.05). 12 and 24 weeks after the first infusion of the original RTM, there was a statistically significant decrease in the concentration of MMP-3, amounting to 80% of the initial level. Against the background of the use of the RTM biosimilar, after 12 and 24 weeks, a statistically significant decrease in the concentration of MMP-3 was observed, which was 46.8 and 59% of the basal level, respectively. According to the ROC analysis, it was found that the basal level of IL-6 more than 100.0 pg/ ml and the level of MMP-3 more than 78.6 ng/ml were associated with the preservation of inflammatory activity by the 24th week of therapy with the RTM biosimilar with a sensitivity of 85% and 57% and a specificity of 62% and 61.5%, respectively. Conclusion. Determining the level of MMP-3 in patients receiving anti-B-cell therapy is important for a more objective assessment of disease activity and predicting the effectiveness of treatment. Key words: rheumatoid arthritis, matrix metalloproteinase 3, anti-B-cell therapy, rituximab biosimilar>˂ 0.05). 12 and 24 weeks after the first infusion of the original RTM, there was a statistically significant decrease in the concentration of MMP-3, amounting to 80% of the initial level. Against the background of the use of the RTM biosimilar, after 12 and 24 weeks, a statistically significant decrease in the concentration of MMP-3 was observed, which was 46.8 and 59% of the basal level, respectively. According to the ROC analysis, it was found that the basal level of IL-6 more than 100.0 pg/ ml and the level of MMP-3 more than 78.6 ng/ml were associated with the preservation of inflammatory activity by the 24th week of therapy with the RTM biosimilar with a sensitivity of 85% and 57% and a specificity of 62% and 61.5%, respectively.Conclusion. Determining the level of MMP-3 in patients receiving anti-B-cell therapy is important for a more objective assessment of disease activity and predicting the effectiveness of treatment.
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Stasinopoulos, Ioannis, Shazia Khan, Logan C. MacKay, Roger W. Brown, Matthew J. Bailey i Ruth Andrew. "Mapping of Corticosteroids in Murine Kidneys Using Mass Spectrometry Imaging". Journal of the Endocrine Society 5, Supplement_1 (1.05.2021): A822—A823. http://dx.doi.org/10.1210/jendso/bvab048.1676.
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Abstract Renal sodium reabsorption is important for blood pressure homeostasis and is physiologically regulated by aldosterone; glucocorticoids may also contribute. Abnormal steroid hormone activity within the kidney contributes to hypertension but the mechanisms are not fully defined. Molecular profiling of receptors and metabolising enzymes indicates that steroid hormone action is compartmentalised within the kidney. Ambient steroid concentrations are a critical factor governing bioactivity at a cellular level, but this is largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) was applied recently to localise steroids in brain and testes, and here is applied to kidney. Image reconstruction permits characterisation and co-registration of kidney histological regions based on regional markers detectable by MSI. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of murine kidneys, using an optimised MSI method. This approach has the potential to map steroids within functional zones of the kidney, providing fundamental new information relevant to hormone action in health and in disease. Cryosections of male C57BL6 mouse kidneys (age 12 weeks, n=6) were subject to MSI following derivatisation using Girard T reagent and α-cyano-4-hydroxycinnamic acid matrix application. Images were reconstructed, and methods optimised to enhance signal and limit diffusion of analytes of interest. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry. Ions with m/z 458.3010, 460.3166 and 474.2957 were detected, using MALDI, in renal sections, close to the predicted masses of 458.3013 (Δppm=0.65), 460.3169 (Δppm=0.65), and 474.2962 (Δppm=1.05), for derivatives of 11-dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. The Heat maps generated indicated that corticosterone intensity was higher in the inner cortex area close to the corticomedullary junction than the rest of the kidney. In contrast 11-dehydrocorticosterone was detected mainly in medulla and aldosterone signal was equally strong in medulla and outer cortex. Thus, MSI can be used map the sites where glucocorticoid and mineralocorticoids are most active in regulating renal tubular function. Co-localisation of steroids of interest with zonal markers by MSI permits steroid mapping in functional renal zones of the kidney. This approach provides fundamental new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure. The project was supported and funded by Kidney Research UK.
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Kushlinskii, N. E., O. V. Kovaleva, Yu B. Kuzmin, E. V. Samoilova, P. L. Prishchep, E. S. Gershtein, S. R. Varfolomeeva i in. "The content of sEMMPRIN/CD147 in the blood serum of patients with bone tumors and its relationship with the clinical and morphological characteristics of the disease". Advances in Molecular Oncology 10, nr 2 (10.07.2023): 100–107. http://dx.doi.org/10.17650/2313-805x-2023-10-2-100-107.
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Introduction. Enzyme-linked immunosorbent assay of biochemical markers is one of the most important methods for diagnosing tumors. One of these markers is an inducer of expression of matrix metalloproteases EMMPRIN/CD147. Changes in its expression are associated with the progression of some tumors. This study is the first work devoted to the study of the content of the soluble form of the transmembrane glycoprotein EMMPRIN (sEMMPRIN) in the blood serum of patients with various bone tumors.Aim. To study the content of sEMMPRIN in the blood serum of patients with malignant bone tumors, its relationship with the clinical and morphological characteristics of neoplasms and prognosis.Materials and methods. The study included 88 patients with malignant tumors (osteosarcoma – 37 cases, chondrosarcoma – 39, chordoma – 5, Ewing’s sarcoma – 7) and borderline (11 cases) bone neoplasms, of which 14 patients were under the age of 18 years. The control group consisted of 29 healthy donors, 8 of which were under the age of 18 years. The concentration of EMMPRIN was determined in the serum of patients and donors with reagents for direct enzyme immunoassay “Human EMMPRIN” (R&D, USA) in accordance with the manufacturer’s instructions and expressed in nanograms (ng) per 1 ml of blood serum. The obtained data were processed using the GraphPad Prizm 9.4 program. When comparing indicators and analyzing their relationships, we used the nonparametric Mann–Whitney and Kruskal–Wallis tests. Overall survival was analyzed using the Kaplan–Meier method.Results. Our analysis of the sEMMPRIN content in the blood serum of patients with bone tumors did not reveal statistically significant differences between the control group and patients with borderline and malignant tumors, both in adults and in children. At the same time, a trend towards a decrease in the level of sEMMPRIN in the blood serum was noted in the presence of a malignant neoplasm of the bone compared with the corresponding control group. Additionally, we found that the content of sEMMPRIN is associated with age and higher in the group of patients under 18 years of age, both among healthy donors and oncological patients. An analysis of the association of sEMMPRIN content with clinical and morphological characteristics did not reveal statistically significant patterns, however, a trend towards an increase in the level of the marker with disease progression in both studied age groups was observed, which is consistent with other studies conducted on other solid tumors.Conclusion. ELISA revealed the marker sEMMPRIN in the blood serum of all examined children and adults with borderline malignant bone tumors and healthy donors. At the same time, the levels of sEMMPRIN did not differ between the above groups, however, there was a tendency for a decrease in the concentration of the marker in patients with bone sarcomas compared with the control group, regardless of age.