Serum Glycome as a Diagnostic and Prognostic Factor in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a risk factor for both mother and fetus/neonate during and after the pregnancy. Inconsistent protocols and cumbersome screening procedures warrant the search for new and easily accessible biomarkers. We investigated a potential of serum N-glycome to differentiate between healthy pregnant women (n = 49) and women with GDM (n = 53) using a lectin-based microarray and studied the correlation between the obtained data and parameters of glucose and lipid metabolism. Four out of 15 lectins used were able to detect the differences between the control and GDM groups in fucosylation, terminal galactose/N-acetylglucosamine (Gal/GlcNAc), presence of Galα1,4Galß1,4Glc (Gb3 antigen), and terminal α2,3-sialylation with AUC values above 60%. An increase in the Gb3 antigen and α2,3-sialylation correlated positively with GDM, whereas the amount of fucosylated glycans correlated negatively with the content of terminal Gal/GlcNAc. The content of GlcNAc oligomers correlated with the highest number of blood analytes, indices, and demographic characteristics, but failed to discriminate between the groups. The presence of terminal Gal residues correlated positively with the glucose levels and negatively with the LDL levels in the non-GDM group only. The results suggest fucosylation, terminal galactosylation, and the presence of Gb3 antigen as prediction markers of GDM.

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

A Holistic 4D Approach to Optimize Intrinsic and Extrinsic Factors Contributing to Variability in Microarray Biosensing in Glycomics.

Protein-carbohydrate interactions happen to be a crucial facet of biology, discharging a myriad of functions. Microarrays have become a premier choice to discern the selectivity, sensitivity and breadth of these interactions in a high-throughput manner. The precise recognition of target glycan ligands among the plethora of others is central for any glycan-targeting probe being tested by microarray analyses. Ever since the introduction of the microarray as an elemental tool for high-throughput glycoprofiling, numerous distinct array platforms possessing different customizations and assemblies have been developed. Accompanying these customizations are various factors ushering variances across array platforms. In this primer, we investigate the influence of various extrinsic factors, namely printing parameters, incubation procedures, analyses and array storage conditions on the protein-carbohydrate interactions and evaluate these factors for the optimal performance of microarray glycomics analysis. We hereby propose a 4D approach (Design-Dispense-Detect-Deduce) to minimize the effect of these extrinsic factors on glycomics microarray analyses and thereby streamline cross-platform analyses and comparisons. This work will aid in optimizing microarray analyses for glycomics, minimize cross-platform disparities and bolster the further development of this technology.

Alterations in the Glycan Composition of Serum Glycoproteins in Attention-Deficit Hyperactivity Disorder.

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD.

Prediction of Mortality in Patients on Peritoneal Dialysis Based on the Fibrinogen Mannosylation.

As we already reported, fibrinogen fucosylation emerged as a prognostic marker of peritoneal membrane function in end-stage renal disease (ESRD) patients on peritoneal dialysis. After a follow-up period of 18 months, we estimated the ability of employed lectins, as well as other biochemical parameters, to serve as mortality predictors in these patients. Following a univariate Cox regression analysis, ferritin, urea clearance, residual diuresis, hyperglycemia, and an increase in the signal intensity obtained with Galanthus nivalis lectin (GNL) emerged as potential mortality predictors, but additional multivariate Cox regression analysis pointed only to glucose concentration and GNL as mortality predictors. Higher signal intensity obtained with GNL in patients that died suggested the importance of paucimannosidic/highly mannosidic N-glycan structures on fibrinogen as factors that are related to unwanted cardiovascular events and all-cause mortality and can possibly be seen as a prediction tool. Altered glycan structures composed of mannose residues are expected to affect the reactivity of mannosylated glycoproteins with mannose-binding lectin and possibly the entire cascade of events linked to this lectin. Since patients with ESRD are prone to cardiovascular complications and the formation of atherosclerotic plaques, one can hypothesize that fibrinogen with increasingly exposed mannose residues may contribute to the unwanted events.

Significance of 1,25-Dihydroxyvitamin D3 on Overall Mortality in Peritoneal Dialysis Patients with COVID-19.

In previous publications, we pointed out the importance of mannosylation of fibrinogen for the development of cardiovascular complications and fucosylation as a predictor of peritoneal membrane dysfunction in patients on peritoneal dialysis (PD). After a follow-up period of 30 months from the onset of the COVID-19 pandemic, we evaluated the significance of 1,25-dihydroxyvitamin D3 (calcitriol) therapy, primary disease, biochemical and hematologic analyzes, and previously performed glycan analysis by lectin-based microarray as predictors of mortality in this patient group. After univariate Cox regression analysis, diabetes mellitus (DM) and calcitriol therapy were found to be potential predictors of mortality. Additional multivariate Cox regression analysis confirmed that only DM was a predictor of mortality. Nevertheless, the use of calcitriol in therapy significantly reduced mortality in this patient group, as shown by the Kaplan-Meier survival curve. The presence of DM as a concomitant disease proved to be a strong predictor of fatal outcome in PD patients infected with SARS-CoV-2. This is the first study to indicate the importance and beneficial effect of calcitriol therapy on survival in PD patients with COVID-19 infection. In addition, this study points to the possibility that adverse thrombogenic events observed in PD patients during the pandemic may be caused by aberrant fibrinogen glycosylation.

Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases.

Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin - glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC50 values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC50 = 10.1 µM for V. cholerae, 13.4 µM for C. perfringens and 402.9 µM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC50 = 101.9 µM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS.

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins.

Fibrinogen Fucosylation as a Prognostic Marker of End-Stage Renal Disease in Patients on Peritoneal Dialysis.

Glycosylation may strongly affect protein structure and functions. A high risk of cardiovascular complications seen in patients with end-stage renal disease (ESRD) is, at least partly associated with delayed clot formation, increased clot strength, and delayed cloth lysis. Taking into consideration that fibrinogen mediates these processes, we isolated fibrinogen from the plasma from patients with ESRD on peritoneal dialysis (ESRD-PD), and examined glycosylation of native fibrinogen and its subunits by lectin-based microarray and lectin blotting. Compared to healthy controls, fibrinogen from patients had increased levels of A2BG2 and decreased levels of FA2 glycan. The distribution of glycans on individual chains was also affected, with the γ chain, responsible for physiological functions of fibrinogen (such as coagulation and platelet aggregation), being most prone to these alterations. Increased levels of multi-antennary N-glycans in ESRD-PD patients were also associated with the type of dialysis solutions, whereas an increase in the fucosylation levels was strongly related to the peritoneal membrane damage. Consequently, investigation of fibrinogen glycans can offer better insight into fibrinogen-related complications observed in ESRD-PD patients and, additionally, contribute to prognosis, choice of personalised therapy, determination of peritoneal membrane damage, and the length of utilization of peritoneum for dialysis.