Biochemical and immunological blood parameters in patients with inflammatory complications after spine screw fixation

As a result of screw fixation at the surgical treatment of patients with spinal diseases, certain complications may occur, of which soft tissues inflammation is frequent. Therefore, it is especially important to study the metabolic status of patients before surgery to determine the increased possibility of complications. Objective. To study the biochemical and immunological blood serum parameters and determine their diagnostic sensitivity in patients with the thoracic and lumbar spine diseases after screw fixation with postoperative inflammatory response. Methods. Comparisons of blood parame­ters were carried out in two groups of 20 patients in each group: the first one — patients at whom the postoperative period passed without complications, the second group — a soft tissues inflammation around the metal structure was observed in the postoperative period. Blood for the study was taken before the surgery on an empty stomach for determination of: C-reactive protein (CRP), sialic acid, alkaline phosphatase (ALP) activity, haptoglobin, content of total chondroitin sulfate (ChS), glycoproteins, circulating immune complexes (CIC), the rate of spontaneous lymphocyte mig­ration, the level of lymphocyte migration with antibodies to bone and cartilage antigens. For quantitative characterization of diagnostic reliability of laboratory test was used the criterion of diagnostic sensitivity. Results. In patients with postoperative soft tissue inflammation around the metal device before surgery, the following indicators were the most informative: the content of ChS (95 %), CRP (80 %), glycoproteins (95 %), haptoglobin (92 %), ALP activi­ty (80 %), sialic acid content (90 %), CIC concentration (70 %), the rate of spontaneous lymphocyte migration (65 %). Conclusions. The laboratory parameters complex with the highest diagnostic sensitivity can be recommended for the selection of preventive measures in the preoperative period, which will improve the results of surgical treatment, reduce its duration and costs.

Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared to adults. Despite these differences, adult fibrinogen is used for treatment of bleeding in neonates, with mixed efficacy. In order to determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compare to those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin like enzymes (SVTLEs) that preferentially cleave fibrinopeptide A or B. We determined that sialic acid content of neonatal fibrinogen is a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal specific treatments of bleeding.

Characterization and Extraction Influence Protein Profiling of Edible Bird’s Nest

The edible bird nest (EBN) from Aerodramus fuciphagus has been consumed as a Chinese traditional food for health and medicinal purposes due to its elevated nutritional value. The present study focused on the influence of characterization and extraction methods on protein profiling, which could be a guideline for grading the EBN. The proposed extraction method is similar to the common food preparation methods of consumers and thus can accurately establish the bioactive protein available upon human consumption. The characterization includes physicochemical analysis (physical, morphology, elemental composition, and microbial content) and chemical analysis (crude protein and amino acid). The morphology of half-cup EBN was found to be uniformly shaped and rich in calcium as compared to rough surface of stripe-shaped EBN, and there was no significant microbial growth in both types of EBN. The crude protein and amino acid content in half-cup EBN were significantly higher than stripe-shaped EBN. The full stew (FS) and stew (SE) extraction methods produced a maximal yield of soluble protein. Sialic acid content in SE extract (8.47%, w/w) and FS extract (7.91%, w/w) were recorded. About seven parent proteins (39.15 to 181.68 kDa) were identified by LC-MS/MS Q-TOF, namely 78 kDa glucose-regulated protein, lysyl oxidase-3, Mucin-5AC-like, acidic mammalian chitinase-like, 45 kDa calcium-binding protein, nucleobindin-2, and ovoinhibitor-like. In conclusion, the characteristics and extraction methods influence the availability of bioactive protein and peptides, demonstrating the potential usage of EBN in improving its biological activities and nutritional properties.

Content of sialic acids, ATP and superoxide anion-radical in loach embryos under the influence of flurenizyd

It is known that flurenizide is a newly synthesized drug with pronounced antimicrobial, immunomodulatory functions. However, its effect on the functional properties of cells, in particular germ cells, is unknown. The aim of this study was to determine the presence of sialic acids as the final components of glycoconjugates, the main macroergic compound - ATP, to evaluate the generation of O2¯ in embryos of lobster (Misgurnus fossilis L.) under the influence of the antibiotic flurenizide. Studies have been performed on embryos of Misgurnus fossilis L. After fertilization, the zygotes were placed in Petri dishes with solutions of flurenizide in concentrations of 0.01; 0.05; 0.15; 1.0; 5.0; 15.0 mM, where allowed to develop. At the development stages, 2 blastomeres, 16 blastomeres, 64 blastomeres, VIII (256 blastomeres) and X divisions (1024 blastomeres) were sampled. In parallel, control studies were performed, where flureniside was not added to the samples. The content of sialic acids, ATP, superoxide anion radical was determined in the selected samples. We found that flureniside at the lowest concentration of 0.01 mM does not cause changes in the content of superoxide anion radical during early embryogenesis. The antibiotic in the maximum concentration causes changes in the content of free radical from the stage of development of 16 blastomeres to 1024 blastomeres, and from the stage of 64 blastomeres there is an increase in its number. Probably flurenizide at a concentration of 15.0 mM is the most reactive. It is known that flurenizide has antioxidant properties, but in its structure there are groups that have a toxic effect, which is most pronounced when exposed to high concentrations. In general, at the stage of development of 16 blastomeres there is a decrease in the amount of О2¯ under the influence of the studied antibiotic. At this time, the content of this free radical in the control increases, compared with other stages of development. Flureniside leads to an increase in the content of superoxide anion radical at development stage 2, 64 blastomeres and is particularly pronounced at concentrations from 0.05 to 15.0 mM at development stages 256 and 1024 blastomeres. 1024 blastomeres are the 10th stage of separation, where desynchronization occurs and the mitotic index decreases. Probably, the changes that take place at this stage of development are related to this. The antibiotic causes an increase in the content of sialic acids in the first stage of crushing (2 blastomeres). In step 16 of the blastomere, flureniside in low concentrations leads to a decrease in the content of sialic acids. However, already at the stage of separation of 64 and 256 blastomeres, the test substance in all concentrations causes a predominant decrease in the amount of sialic acids. At the last stage of synchronous crushing (1024 blastomeres) flurenizide in the maximum investigated concentration (15.0 mM) causes a significant accumulation of sialic acid content. Flureniside at concentrations of 0.15, 5.0 and 15.0 mМ causes a decrease in ATP content in loach embryos at the stage of development of 256 blastomeres by 28, 67 and 38 %, respectively. An increase in ATP content by 33 % occurs under the influence of flurenizide at a concentration of 1.0 mM. The ATP content also increases at the stage of development of embryos of 1024 blastomeres under the influence of flurenizide of all studied concentrations. Analysis of variance has shown that a significant contribution to the growth of ATP, sialic acid and superoxide anion radical, during the early embryogenesis of lobster embryos, is made by the factor of embryo development, while the factor of flureniside has a smaller contribution. Therefore, studies have shown that flurenizide causes increased generation of superoxide anion radical, decreased sialic acid content, as well as changes in ATP content in embryonic cells during early embryogenesis.

N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)–containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel–mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

Differential Sialic Acid Content in Adult and Neonatal Fibrinogen Mediate Differences in Clot Polymerization

Recent studies have identified several major qualitative and quantitative differences in important hemostatic proteins between adult and neonatal humans, including the primary coagulation protein fibrinogen.Despite these differences, neonates with post-operative bleeding from procedures requiring cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) are treated with transfusions of adult blood products, namely adult fibrinogen. The effectiveness of such transfusions is inconsistent in neonates and often results in a deficient fibrin matrix structure which may not be sufficient for mitigating bleeding. Our recent studies have also identified differences at the bulk clot level between purified neonatal and adult fibrin clots, including major structural and functional distinctions, which could contribute to these outcomes. Notably, adult fibrinogen degrades slower than neonatal clots, therefore transfusion of adult blood products to neonatal patients could contribute to thrombotic complications. Given the inconsistent results and potential complications from the transfusion of adult blood products to neonatal patients, there is a critical need to better understand the mechanistic differences in hemostatic processes between adults and neonates. Neonates possess a molecular variant of fibrinogen known as fetal fibrinogen. Increased sialic acid concentration compared to adults has been identified in many neonatal glycoproteins across physiological systems, including fetal fibrinogen. Studies have shown that fibrin clot properties are influenced by fibrin polymerization mechanisms and post translational modifications (e.g. sialic acid). For example, an increased sialic acid content in the dysfibrinogenemia associated with liver disease has been associated with an altered fibrin clot structure. Additionally, recent work from our group has found that the increased sialic acid in neonatal fibrin networks results in significantly greater fibroblast attachment than adult networks. Therefore, we hypothesized that differences in neonatal and adult fibrin clot properties are due to mechanistic differences in fibrin polymerization between neonates and adults owing to altered sialic acid concentrations. The activation of fibrinogen and conversion into fibrin by the proteolytic enzyme thrombin is essential for the formation of a stable blood clot and the cessation of bleeding. Thrombin converts soluble fibrinogen to insoluble fibrin via cleavage of fibrinopeptides A and B, exposing fibrin knobs A and B. Fibrin protofibrils are then formed from the noncovalent binding of fibrin knobs to complementary fibrin holes a and b on adjacent proteins. In adults, fibrin A:a knob:hole interactions are critical for polymerization. However, these mechanisms have not been explored in neonates. Therefore, we characterized the influence of sialic acid on the knob:hole interactions in neonatal fibrin polymerization. We first investigated the influence of sialic acid concentration on neonatal fibrin polymerization by removing sialic acid via enzymatic digestion and performing structural and functional analysis on desialylated fibrinogen. Desialylated adult and neonatal fibrinogen had roughly equivalent structure, polymerization kinetics, and clottability results. These results indicate that differential sialylation may at least partially explain functional differences been adult and neonatal clots. Additionally, we investigated the role that sialylation plays on neonatal fibrin polymerization dynamics by comparing neonatal or adult fibrin clots formed with snake venom thrombin-like enzymes (svTLE) that preferentially cleave either A or B fibrinogen fibrinopeptides or thrombin. Structural, mechanical, fibrinolytic, and polymerization assays were conducted. Quantitative release of fibrinopeptides was determined via ELISA. Results indicate neonatal fibrin polymerization mechanisms are more dependent on B:b knob:hole interactions than adult fibrin. Results from this study provide insight into the mechanism of the neonatal clotting process and are a critical contribution for the development of neonatal-specific treatments for bleeding and thrombosis. Disclosures Brown: Selsym Biotech, Inc.: Other: Founder and CEO.

Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.

The effect of sialic acid content in glycans on thrombolytic activity of tissue plasminogen activators

Purpose: to investigate the dependence of the thrombolytic activity of tissue plasminogen activators (tPA) alteplase and tenecteplase on the degree of sialylation of the oligosaccharide component of molecules. Materials and methods: tPA specimens with an average degree of sialylation were obtained by cultivating CHO clones-producers in the fed-batch mode; desialylated forms of tPA were obtained by processing with neuraminidase, and hypersialylated forms of tPA were obtained by cultivating clones-producers in a medium with the addition of butyrate. The content of sialic acids was determined by resorcinol method, and the activity of tPA was determined by fibrin clot lysis. Results: the dependence of tenecteplase activity on the content of sialic acids in the molecule is demonstrated. The activity of tenecteplase falls below the limits of the target range when the content of sialic acids is more than 5 residues per tenecteplase molecule. No such relation was found for alteplase. Conclusion: the content of sialic acid residues affects the biological activity of tenecteplase; the activity of alteplase does not depend on the degree of sialylation of the molecule.