Total Glycoprotein Research Articles

Blood metabolites mediate effects of breakfast skipping on heart failure via Mendelian randomization analysis

Numerous observational studies have suggested a potential causal relationship between skipping breakfast and cardiovascular diseases, including heart failure (HF). However, these studies are susceptible to inherent confounders and the challenge of reverse causation, and the underlying metabolic factors are not yet clear. Therefore, our aim is to assess the causal impact of breakfast skipping on HF and the role of potential mediating metabolic products from a genetic perspective, by conducting Mendelian Randomization (MR) studies and mediation analysis. We leveraged summary data from the most extensive genome-wide association studies to date on breakfast skipping (with 193,860 participants), blood metabolites (with 118,461 participants), and HF (involving 47,309 cases and 930,014 controls). To explore the causal relationship between breakfast skipping and HF, as well as the role of 249 potential blood metabolite mediators, we conducted bidirectional MR and mediation MR analyses. We primarily employed the Inverse Variance Weighted (IVW) method, complemented by various other techniques to ensure the comprehensiveness and reliability of our analysis. Our research confirms a causal association between breakfast skipping and an increased risk of HF (odds ratio [OR]: 1.378, 95% confidence interval [CI]: 1.047–1.813; p = 0.022). Furthermore, our research findings demonstrate that breakfast skipping is positively correlated with 6 blood metabolites and negatively correlated with 2 others. Notably, our mediation MR analysis further reveals that three blood metabolites act as mediators in the relationship between breakfast skipping and the risk of HF. Specifically, the mediating effects are attributed to the ratio of docosahexaenoic acid (DHA) to total fatty acids (proportion mediated = 9.41%, 95% CI: 2.10–28.61%), glucose (proportion mediated = 6.17%, 95% CI: 0.97–28.53%), and glycoprotein acetyls (GlycA) (proportion mediated = 5.68%, 95% CI: 0.94–21.62%). The combined mediating effects of these three factors total 20.53% (95%CI: 8.59–91.06%). Our research confirms the causal relationship between genetically instrumented breakfast skipping and HF, underscoring the potential mediating roles played by three key blood metabolites: ratio of DHA to total fatty acids, glucose and GlycA. This discovery offers valuable perspectives for clinical strategies targeting HF.

THE USE OF LABORATORY CRITERIA IN THE RISK ASSESSMENT OF POSTOPERATIVE COMPLICATIONS AFTER TRANSPEDICULAR FIXATION OF THE LUMBAR SPINE

Objective. To investigate the peculiarities of the development of postoperative complications after surgical treatment of degenerative diseases of the lumbar spine with transpedicular fixation of the vertebrae and their relationship with changes in preoperative laboratory parameters. Methods. A retrospective analysis of the biochemical examination data of 35 patients with degenerative diseases of the lumbar spine who underwent discectomy with transpedicular fixation was performed. In four groups of patients with postoperative complications: 10 patients — inflammation of the what fabrics around metal structures and 5 patients — instability of the metal structure. The results of the examination of 20 people without specified complications served as a comparison group. Comparison was made with a control group (20 practically healthy people). Results. Patients have a significant increase in serum ALT activity by 42.29 and AST by 63.55 %, by 38.37 % of the level of CS content, by 37.21 % of total glycoproteins compared to those in the control group. In patients with soft tissue inflammation around the serum instrument zone, the content of LDLP was recorded by 29.27 %, by 23.53 % of the UA, by 12.34 % of TP results higher by 32.01 % ALT activity and 83 %, 49 % AST, 54.96 % AlP activity, 31.40 % — AlP/AcP, 60.47 % — concentration of CS, 95.57 % — total glycoproteins than in persons in the control group. In the serum of patients with the development of instability of metal structures, 28.55 % was observed by activity of the ALT, AlP — by 38.47 %, AcP — by 48.84 %, less by 58.35 % of AlP/AcP, the CHOL content of 13 88 %, higher by 25.43 % of the LDLP content than such persons in the control group. Content of the P was lower by 31.51 % and the Ca/P ratio was higher by 38.95 %, the content of CS by 83.72 %, total glycoproteins — by 48.83 %. Conclusions. Comparative analysis of the results of preoperative clinical and laboratory examination of patients with degenerative spinal diseases with transpedicular fixation which is reflected in the deviations of their preoperative laboratory parameters from such persons with uncomplicated course of the postoperative period after similar surgery.

Impact of estrogen on IgG glycosylation and serum protein glycosylation in a murine model of healthy postmenopause.

The glycosylation of immunoglobulin (Ig) G regulates IgG interaction capability with Fc gamma receptors found in all immune cells. In pathogenic conditions, estrogen can impact IgG levels and glycosylation. Following menopause, when estrogen levels decline affecting the immune system and potentially leading to a heightened susceptibility of immune activation. In this study, we aim to determine if estrogen levels can regulate IgG glycosylation in postmenopausal healthy situations. Mice were ovariectomized to simulate an estrogen-deficient postmenopausal status and then treated with 17-beta-estradiol (E2) at different doses and different administration strategies. Using a highly sensitive liquid chromatography-tandem mass spectrometry (MS/MS) glycoproteomic method, we demonstrated that E2 treatment increased the degree of glycosylation on IgG-Fc with both galactosylation and sialylation in the position required for interaction with Fc gamma receptors. We also observed that only long-term estrogen deficiency reduces IgG levels and that estrogen status had no impact on total IgG sialylation on both Fab and Fc domains or general glycoprotein sialylation evaluated by ELISA. Furthermore, E2 status did not affect the total sialic acid content of total cells in lymphoid organs and neither B cells nor plasma cells. The study concluded that E2 treatment does not affect total serum glycoprotein sialylation but alters IgG glycosylation, including IgG sialylation, implying that estrogen functions as an intrinsic modulator of IgG sialylation and could thereby be one pathway by which estrogen modulates immunity.

Novel Insight into the Etiology of Haff Disease by Mapping the N-Glycome with Orthogonal Mass Spectrometry

Consumption of boiled crayfish may lead to Haff disease (HD), which is considered to result from an unidentified toxin, although the etiology is still obscure. Profiling of the N -glycome in HD would assist in deciphering the underlying molecular mechanism of the disease, whereas HD-associated glycosylation has never been explored. Herein, we enrolled 90 serum samples with HD patients and healthy controls from the Wuhan Center for Disease Control & Prevention between 2019 and 2020. N -glycome profiles of both serum and serum-derived immunoglobulin G (IgG) in HD were characterized by means of high-throughput-based orthogonal mass spectrometry. It was observed that HD is associated with an increase in the core fucosylation and mono-galactosylation of total serum glycoproteins. The serum level of IgG was found to serve as a good indicator for HD patients. In addition, differential galactosylation and sialylation of IgG were strongly correlated with HD. It was notable that the changes in the galactosylation and sialylation of IgG1 and IgG2 were subclass specific. Interestingly, altered sialylation and galactosylation of IgG2 or IgG3/4 strongly correlated with clinical markers for HD. Our study reveals the association of differential IgG N-glycosylation with HD, providing new insight into the etiology of this rare disease.

N-glycans from serum IgG and total serum glycoproteins specific for endometriosis

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005–0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients.

Differential recognition of influenza A virus H1N1 neuraminidase by DNA vaccine-induced antibodies in pigs and ferrets.

Neuraminidase (NA) accounts for approximately 10-20% of the total glycoproteins on the surface of influenza viruses. It cleaves sialic acids on glycoproteins, which facilitates virus entry into the airways by cleaving heavily glycosylated mucins in mucus and the release of progeny virus from the surface of infected cells. These functions make NA an attractive vaccine target. To inform rational vaccine design, we define the functionality of influenza DNA vaccine-induced NA-specific antibodies relative to antigenic sites in pigs and ferrets challenged with a vaccine-homologous A/California/7/2009(H1N1)pdm09 strain. Sera collected pre-vaccination, post-vaccination and post-challenge were analyzed for antibody-mediated inhibition of NA activity using a recombinant H7N1CA09 virus. Antigenic sites were further identified with linear and conformational peptide microarrays spanning the full NA of A/California/04/2009(H1N1)pdm09. Vaccine-induced NA-specific antibodies inhibited the enzymatic function of NA in both animal models. The antibodies target critical sites of NA such as the enzymatic site, second sialic binding site and framework residues, shown here by high-resolution epitope mapping. New possible antigenic sites were identified that potentially block the catalytic activity of NA, including an epitope recognized solely in pigs and ferrets with neuraminidase inhibition, which could be a key antigenic site affecting NA function. These findings show that our influenza DNA vaccine candidate induces NA-specific antibodies that target known critical sites, and new potential antigenic sites of NA, inhibiting the catalytic activity of NA.

Human Milk Oligosaccharides And Site-Specific Glycosylation Heterogeneity Of Filipino Human Milk Glycoproteins Exhibit Dynamic Variations During Lactation As Influenced By Secretor Status And Coastal Environmental Condition – A Preliminary Study

Human Milk Oligosaccharides (HMOs) have a role in the immune system development of infants. HMOs and the N-glycans of human milk glycoproteins, especially highly fucosylated ones, function as prebiotics to the bifidogenic gut microbiota. This study reports the first longitudinal profile of HMOs and human milk glycoproteins from coastal, noncoastal, secretor and nonsecretor Filipino mothers analyzed and annotated using QTOF LC/MS and QqQ LC/MS - MRM, respectively. A milk glycan profile from coastal mothers revealed higher (P<0.05) presence of fucosylated branched HMOs, such as DFLNHa, LDFT, LNDFH I, and LNFP II. Moreover, secretor mothers expressed the highest 2’FL from colostrum (Day 5) until the end of the study (Month 6). Interestingly, the predominance of secretory IgA (64%) on the total milk glycoproteins from Filipino mothers supported their biological adaptation in conferring immune protection to their breastfeeding infant. Glycoform variations on milk glycoproteins, sIgA, IgG, Lactoferrin, and a1-Antitrypsin, in coastal mothers were observed. Moreover, lactoferrin was found to be highly glycosylated with 20 fucosylated and sialylated glycoforms. These results suggest that the coastal mother’s unique long-term seafood diet may have influenced their milk glycan profile and the potential incorporation of L-fucose derived from seafood in the biosynthesis of their milk glycans.

Solanum nigram - A Potential Medicinal Herb

This plant commonly referred to as "Black nightshade," Solanum nigrum Linn. (Solanaceae) it is widely used in traditional medicine in India and other parts of the world to treat liver conditions, chronic skin conditions (such as psoriasis and ringworm), inflammatory conditions, painful periods, fevers, diarrhoea, eye diseases, hydrophobia, etc. It has been discovered that Solanum nigrum contains compounds with anti-tumor action, such as total alkaloids, steroid alkaloids, steroidal saponins, and glycoprotein. The herb is utilised as a hepatoprotective in Indian traditional medicine. This plant have potential role as a multipurpose medicinal agent, we have investigated the phyto-pharmacological properties of the plant and compiled its comprehensive pharmacological uses in this review paper.

ЦІЛІСНІСТЬ КИШКОВОГО БАР'ЄРА ПІД ВПЛИВОМ ТВЕРДИХ ЧАСТИНОК, ОТРИМАНИХ ВІД ЗГОРЯННЯ ДЕРЕВИНИ ТОПОЛІ

The epidemiological data pointed out that exposure to ambient air pollution particulate matter (PM) may increase the risk of irritable bowel syndrome and ulcerative colitis. Air pollutants might affect the intestine through blood, mucociliary clearance of PM from the lungs, by polluted food or water intake. In the present study, we tested the hypothesis that water-suspended smoke preparations (SP) derived from the combustion of cottonwood might affect the integrity of the intestinal mucosa, induce an inflammatory response, and increase the susceptibility to chemically-induced colonic inflammation. Smoke from cottonwood was collected under laboratory conditions during the entire flaming, smouldering, and mixed combustion phases. Male Wistar rats (180-200 g) were injected with SP in two ways: 1) into the tail vein at a dose of 500 µg/100 g; 2) by gavage at a dose of 180 µg/100 g. The experimental colitis was induced 1 hour after SP injection by a single rectal injection of 0.1 ml 6% iodoacetamide (7 cm from the anus). Rats were euthanized in 2 h after colitis induction. We examined colonic vascular permeability by Evans blue extravasation (mg/g wet colon), myeloperoxidase level, total glycoprotein level at surface mucus by PAS-staining, and intestinal permeability by the translocation of bacteria into the blood of the portal vein. This study demonstrates that intravenous administration of the SP decreases the amount of surface mucus in the large intestine and increases myeloperoxidase activity in the rat colon. It did not increase the negative effect of iodoacetamide-induced colitis on changes in the state of the endothelial barrier. But, intravenous administration of SP led to the appearance of blood in the urine, which may be due to the damage to the fenestrated capillaries of the glomerulus. At the same time, oral administration of SP increased the translocation of bacteria into the blood of the portal vein which represents the increased gut permeability accompanied by a reduced level of total glycoprotein of colonic surface mucus. However, oral administration of SP did not have a significant effect on colonic vascular permeability and the activity of myeloperoxidase vs. the placebo group with colitis. The airborne PM obtained from the combustion of natural precursors such as cottonwood can cause primary pro-inflammatory changes in the intestines in a very short time after penetration into the organism through the blood and oral ingestion. Moreover, they can contribute to the violation of the integrity of the intestinal barrier.

Polymer-tethered glycosylated gold nanoparticles recruit sialylated glycoproteins into their protein corona, leading to off-target lectin binding.

Upon exposure to biological fluids, the fouling of nanomaterial surfaces results in non-specific capture of proteins, which is particularly important when in contact with blood for in vivo and ex vivo applications. It is crucial to evaluate not just the protein components but also the glycans attached to those proteins. Polymer-tethered glycosylated gold nanoparticles have shown promise for use in biosensing/diagnostics, but the impact of the glycoprotein corona has not been established. Here we investigate how polymer-tethered glycosylated gold nanoparticles interact with serum proteins and demonstrate that the protein corona introduces new glycans and hence off-specific targeting capability. Using a panel of RAFT-derived polymers grafted to the gold surface, we show that the extent of corona formation is not dependent on the type of polymer. In lectin-binding assays, a glycan (galactose) installed on the chain-end of the polymer was available for binding even after protein corona formation. However, using sialic-acid binding lectins, it was found that there was significant off-target binding due to the large density of sialic acids introduced in the corona, confirmed by western blotting. To demonstrate the importance, we show that the nanoparticles can bind Siglec-2, an immune-relevant lectin post-corona formation. Pre-coating with (non-glycosylated) bovine serum albumin led to a significant reduction in the total glycoprotein corona. However, sufficient sialic acids were still present in the residual corona to lead to off-target binding. These results demonstrate the importance of the glycans when considering the protein corona and how 'retention of the desired function' does not rule out 'installation of undesired function' when considering the performance of glyco-nanomaterials.

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVNLec1. We found that Sf-RVN and Sf-RVNLec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVNLec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVNLec1 cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.

Агрессивные и протективные факторы желудочного сока у больных со стенозом пилородуоденальной зоны язвенного генеза

Цель: оценить агрессивные и протективные свойства желудочного сока у больных со стенозом пилородуоденальной зоны язвенного генеза в зависимости от его степени. Материалы и методы. В отделении хирургии органов пищеварения ГУ «Институт гастроэнтерологии НАМН У­краины» за 2014–2019 годы обследовано 157 больных с различной степенью стеноза пилородуоденальной зоны язвенного генеза. Из них для изучения желудочной кислото- и слизепродукции было отобрано 92 пациента с компенсированным и субкомпенсированным стенозом, среди которых 71 мужчина, 21 женщина в возрасте от 38 до 56 лет, средний возраст (45,3 ± 5,2) года. Больные были разделены на 2 группы в зависимости от степени стеноза: 1 группа — 57 больных с компенсированным стенозом, 2 группа — 35 больных с субкомпенсированным стенозом. Контрольную группу составили 20 практически здоровых лиц. В желудочном содержимом определяли: уровень pH, концентрацию пепсина, гликопротеинов, гексозаминов, фукозы, сиаловых кислот. Результаты. Анализ изучения уровней кислото- и слизепродукции у больных со стенозом пилородуоденальной зоны различной выраженности показал, что у пациентов с субкомпенсированным стенозом отмечалось увеличение частоты гиперацидной секреции на 21,1 % (р < 0,05), существенное повышение уровня пепсина (р < 0,05), уменьшение концентрации общих гликопротеинов (р < 0,05) (за счет существенного уменьшения концентрации фукозы и гексозаминов) по сравнению с пациентами с компенсированным стенозом, которым была более свойственна нормацидная секреция. Полученные нами данные о дисбалансе факторов агрессии и защиты желудочного содержимого позволили нам сформировать различные адаптационно-компенсаторные варианты их соотношения: 1 (гиперреактивный) — одно­временное повышение факторов агрессии и защиты; 2 (компенсаторный) — повышение уровня агрессивных факторов, нормальная концентрация защитных факторов; 3 (декомпенсаторный) — повышение уровня агрессивных факторов, снижение уровня факторов защиты. Выводы. Выявлено, что прогрессирование степени стеноза пилородуоденальной зоны язвенного генеза из компенсированного в субкомпенсированный сопровождается достоверным преобладанием факторов агрессии над факторами защиты желудочного сока и истощением функциональных возможностей слизепродуцирующего аппарата желудка. В результате выделения трех вариантов соотношения факторов агрессии и защиты желудочного сока (гиперреактивный, компенсаторный, декомпенсаторный) установлено, что декомпенсаторный вариант достоверно чаще встречается у пациентов с субкомпенсированным стенозом пилородуоденальной зоны, а значит, риск прогрессирования степени стеноза у таких больных значительно выше по сравнению с другими вариантами соотношений. Обнаруженные варианты соотношений факторов агрессии и защиты у пациентов со стенозом дают возможность дифференцированного подхода к выбору тактики лечения (мини-инвазивного эндоскопического или комбинированного эндо- и лапароскопического метода).

P–340 Novel non-invasive diagnostic options for endometriosis - based on glycome analysis

Abstract Study question Could glycosylation changes on serum and/or urine glycoproteins be suitable biomarkers for the non-invasive diagnosis of endometriosis? Summary answer The glycosylation pattern on serum and urine glycoproteins differed significantly in endometriosis patients compared to controls, suggesting a novel role as biomarkers of the disease. What is known already There is little published on endometriosis and glycosylation, and most of the studies are conducted with tissue or peritoneal fluid samples, collected by invasive means. An Iraqi study draws attention to the importance of serum sialylation, which is dramatically changed in endometriosis patients after zoladex therapy, indicating that changes in serum sialylation may be a new biomarker of the disease. While glycosylation of urine in endometriosis has not been studied so far, in a study of endometrial cancer, the urinary level of two glycoproteins was significantly increased in the patients compared to the control group. Study design, size, duration This was a prospective study. In this basic research project, serum and urine samples were collected for glycome analysis in women with and without endometriosis, as diagnosed at laparoscopy. Glycated haemoglobin (HbA1c), fasting glucose levels as well as hormone levels were also collected from the patients to link our glycomic findings with metabolic and hormone profiles. The study was approved by the Research and Ethics Committee of the National Maternity Hospital, Dublin (EC19.2018). Participants/materials, setting, methods Samples from 24 cases of endometriosis (patients without previous anti-inflammatory or hormonal therapy, endometriosis was confirmed by laparoscopy) and 27 control patients (patients without endometriosis) were processed to analyse N-glycans (total serum), urine glycoproteins, and IgG. The pre-processed, PNGase F-digested serum and urine samples were labelled with fluorescent tag and then analysed by mass spectrometry, ultra-performance liquid chromatography (UPLC) in combination with exoglycosidase digestions and Glycostore (https://glycostore.org/). Main results and the role of chance Glycosylation on total serum and urine glycoproteins and IgG was investigated and differed in endometriosis compared to controls. The N-glycome from the total glycoproteins in serum and urine was also different. The proportion of the galactosylation and sialylation differed between urine and serum IgG and these alterations have an impact on the IgG function. Our preliminary data indicate, that there is an increase in alpha 2–3 sialylation, galactosylation, and fucosylation on urine glycans from endometriosis patients compared to the control pool. Urine is a good source of biomarkers as it can be collected non-invasively. Our group is the first to have developed a protocol for the recovery of N-glycans in urine and to have identified the total N-glycome in urine. The urine N-glycome contains mostly complex N-glycans and also some oligomannosylated and hybrid glycans. Our results may lead to non-invasive biomarkers for the diagnosis of endometriosis and the monitoring of the disease. Limitations, reasons for caution The number of participants involved in this basic research is low but this is a pilot study. A larger, validation study, is warranted in the future. Furthermore, the follow-up of treated patients also would be an interesting field of research. Wider implications of the findings: Glycomics may be a potent source of biomarkers of endometriosis, with a number of glyco-biomarkers already approved by the FDA. Endometriosis-associated glycomic profiles from serum and/or urine glycoproteins may represent viable targets for development of innovative non-invasive or minimally invasive diagnostics in this debilitating disease. Trial registration number Not applicable

The effects of edaravone, a free-radical scavenger in lung injury induced by valproic acid demonstrated via different biochemical parameters.

In this study, we aimed to evaluate whether edaravone (EDA) has a protective role against valproic acid (VPA)-induced lung damage via its antioxidative activity. Male Sprague-Dawley rats were split into four groups. Control (n = 8) rats; rats given EDA (30 mg kg-1 day-1 ; n = 10); rats given only (VPA, 500 mg kg-1 day-1 ; n = 10); rats given VPA + EDA (in the same dose and time) for 7 days. EDA and VPA were applied intraperitoneally. After 8 days, lung tissues were immediately taken from the rats. In lung homogenates, reduced glutathione, total antioxidant status levels, and superoxide dismutase, glutathione peroxidase, sodium/potassium ATPase, paraoxonase1, and carbonic anhydrase activities significantly abated, whereas catalase, glutathione reductase, glutathione-S-transferase activities insignificantly decreased in the VPA-treated group. In contrast, lipid peroxidation, reactive oxygen species, and total oxidant status levels, glycoprotein and protein carbonyl contents, nitric oxide, hydroxyproline levels, and xanthine oxidase, lactate dehydrogenase, arginase, and prolidase activities significantly increased in the VPA-given group. Administration of EDA caused the reverse effects. As a consequence, EDA prevented oxidative stress-mediated lung injury via its robust antioxidant effects.

High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration

ABSTRACT: Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p<0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and α1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.

Endotoxemia is associated with an adverse metabolic profile.

Our aim was to analyze whether endotoxemia, i.e. translocation of LPS to circulation, is reflected in the serum metabolic profile in a general population and in participants with cardiometabolic disorders. We investigated three Finnish cohorts separately and in a meta-analysis (n = 7178), namely population-based FINRISK97, FinnTwin16 consisting of young adult twins, and Parogene, a random cohort of cardiac patients. Endotoxemia was determined as serum LPS activity and metabolome by an NMR platform. Potential effects of body mass index (BMI), smoking, metabolic syndrome (MetS), and coronary heart disease (CHD) status were considered. Endotoxemia was directly associated with concentrations of VLDL, IDL, LDL, and small HDL lipoproteins, VLDL particle diameter, total fatty acids (FA), glycoprotein acetyls (GlycA), aromatic and branched-chain amino acids, and Glc, and inversely associated with concentration of large HDL, diameters of LDL and HDL, as well as unsaturation degree of FAs. Some of these disadvantageous associations were significantly stronger in smokers and subjects with high BMI, but did not differ between participants with different CHD status. In participants with MetS, however, the associations of endotoxemia with FA parameters and GlycA were particularly strong. The metabolic profile in endotoxemia appears highly adverse, involving several inflammatory characters and risk factors for cardiometabolic disorders.

HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in Congenital Disorders Glycosylation and human diseases.

N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

Evaluation of different PNGase F enzymes in immunoglobulin G and total plasma N-glycans analysis

Glycoproteins, proteins that are co- and posttranslationally modified by sugars (glycans), have significant roles in pathophysiology of many different diseases. One of the main steps in sample preparation for free N-glycan analysis is deglycosylation or glycan removal. The aim of this study was to compare different peptide N-glycosidase F (PNGase F) enzymes (Rapid PNGase F and two recombinant versions) for deglycosylation of total human plasma glycoproteins and different amounts of human immunoglobulin G (IgG). Deglycosylation with different PNGase F enzymes resulted in different IgG and plasma N-glycosylation hydrophilic interaction liquid chromatography ultra-performance liquid chromatography profiles. Additionally, one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared with Rapid PNGase F and recombinant version of PNGase F from a different manufacturer. In terms of chromatographic peak intensities and coefficient of variation %Area values, all tested versions of PNGase F enzymes were very reproducible and on the similar level when used in optimal conditions. However, care should be taken in terms of which enzyme is used with which protocol, particularly when scaling up.

Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of concanavalin A- and wheat germ agglutinin-reactive glycans mark seminal prostasome populations from normozoospermic and oligozoospermic men

Background: Human seminal prostasomes are intrinsically heterogeneous extracellular vesicles (EVs) whose composition is, additionally, influenced by different physiological conditions. Aiming at the molecular properties of the prostasomal surface exemplified by glycan compositions as a possible distinction factor, we applied lectin-affinity chromatography (LAC) as a new tool for their separation. Since glycans, generally, exhibit various biological activities, introduction of glyco-parameters as reference could upgrade standardization of EVs isolated by different methods and intended for use in biomedicine.Methods: Preparations of seminal prostasomes from normozoospermic (sPro-N) and oligozoospermic (sPro-O) men were subjected to LAC on concanavalin A (Con A) and wheat germ agglutinin (WGA) columns. Prostasomes recovered in LAC-separated fractions were characterized according to the distribution of selected markers: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), tetraspanin CD63, and total protein/glycoprotein composition.Results: Two CD63-immunoreactive populations exhibiting prostasome signature bands but differing in GGT activity and surface glycans were separated on the WGA column. Additional populations having distinct profiles of total glycoproteins and which can be tracked down by ALP activity were enriched on the Con A column. WGA-separated populations were similar in sPro-N and sPro-O, whereas Con A-separated ones were strikingly different.Conclusions: Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of Con A- and WGA-reactive glycans mark seminal prostasomes populations from normozoospermic and oligozoospermic men.