Synthesis of oligo-α-(1→2)-4,6-dideoxy-4-formamido-d-mannopyranosides related to the A epitope of the Brucella O-polysaccharide and their use for assaying of serum immunoglobulins

Pathogenic bacteria of the genus Brucella cause a severe threat for public health and agricultural economics. The World Health Organization considers brucellosis to be one of the most serious and also neglected zoonotic diseases. The use of traditional whole-cell brucellosis vaccines complicates the differentiation between infected and vaccinated animals (DIVA). Moreover, diagnostics based on lipopolysaccharide of Brucella are susceptible to false positive results. Structural features of Brucella O-antigens make synthetic oligosaccharides promising agents for the development of diagnostic tools and vaccines against brucellosis. Here we report the synthesis of spacer-armed di-, tri-, tetra- and penta-4,6-dideoxy-4-formamido-α-(1→2)-d-mannopyranosides which are related to the A-epitope of Brucella O-antigen. The key α-(1→2)-linked disaccharide thioglycoside donor was synthesized by employing the strategy of orthogonal glycosylation of thioglycoside acceptor with trichloroacetimidate donor. Sequential block-wise assembly yielded a series of desired compounds, which were subsequently deprotected and converted into target molecules and then into their fluorescein-labeled conjugates. The obtained conjugates were employed as tracers in a fluorescence polarization assay (FPA) to detect anti-Brucella immunoglobulins. Among the studied compounds, the trisaccharide conjugate showed the greatest difference in median FP signals between Brucella-positive and Brucella-negative sera samples making it a promising candidate for developing FP diagnostic assays. The decreased FP signal in the cases of tetra- and pentasaccharide tracers can be associated with the known “propeller-effect” due to the rotational mobility of the part bearing the fluorescent label and of the fluorescein itself and/or the enlarging of the distance between the fluorescein part and the antibody-oligosaccharide complex. This observation demonstrates the advantages of using synthetic relatively small synthetic tracers with well-defined structure in comparison with heterogeneous fluorescein-labelled O-polysaccharides which are in use today in spite of the fact that they contain poorly characterized amounts of label attached along the polysaccharide chains

Application of the Chitooligosaccharides and Fluorescence Polarization Technique for the Assay of Active Lysozyme in Hen Egg White

This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control

Second asymptomatic carotid surgery trial (ACST-2) : a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86-1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91-1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable