Immunological and biochemical characterization of extracellular polysaccharides of mucoralean moulds

In this thesis the characterization is described of the antigenic determinants (epitopes) of the extracellular polysaccharides (EPSs) from moulds belonging to the order of Mucorales. Detailed knowledge of the structure of these epitopes allows for further development of a new generation of methods for reliable detection of moulds in food. These immunoassays, such as the ELISA (Enzyme-linked Immunosorbent Assay), the latex agglutination assay and the dot- blot assay, are based on the specific recognition of antigenic EPSs by IgG antibodies raised against these polysaccharides.As described in Chapter 2, the water-soluble extracellular polysaccharides which are excreted under various conditions of growth by the mucoralean moulds tested, including the genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum and Thamnidium, consist mainly of carbohydrate residues and some protein. The polyclonal IgG antibodies raised in rabbits against EPS of Mucor racemosus were very specific for species of Mucorales; as no cross-reactivity with other moulds was observed. In Chapter 10, the production and characterization of mouse-monoclonal IgG antibodies against the same EPS is described. These antibodies are also very specific for mucoralean moulds but based on different epitopes. However, as shown in Chapter 10, monoclonal antibodies are not necessarily more specific and may be less sensitive than polyclonal antibodies. The immunogenic specificity provides a taxonomic value to EPS as was shown by analysis of EPS preparations of species belonging to the Mortierella isabellina group, which pointed to a classification of this group into the Mucoraceae and not to the Mortierellaceae (Chapter 9).A new method was developed (Chapter 4) for accurate sugar analysis of complex carbohydrate structures with acid sugar residues. This method includes the successive use of methanolysis and TFA hydrolysis followed by high-performance anion-exchange chromatography (HPAEC) analysis of the monosaccharides. This method provided a rapid, accurate and sensitive assay to determine the exact carbohydrate composition of the uronic-acid containing mucoralean EPSs on the microgram scale without any derivatisation. With this method the presence of fucose, mannose, glucose, galactose, and glucuronic acid residues in the mucoralean EPSs was established as well as their relative amounts.A rapid method was developed in which high-performance size-exclusion chromatography (HPSEC) was combined with ELISA detection (Chapter 3). This method allowed the rapid screening and determination of fractions for the presence of antigenic polysaccharides, and enabled an optimal choice for column material to be used for isolation of the antigenic fractions. With this method a common rabbit antigenic fraction, characteristic for moulds of Mucorales, was found. It had an apparent molecular mass of approx. 30 kDa. This fraction accounted for only a minor part of the total EPSs and was mainly composed of mannose residues. The major part, a fraction containing approx.50% glucuronic acid which is known in the literature as mucoran, was found to be antigenic in mice (monoclonal IgG) but not antigenic in rabbits (polyclonal IgG). A β(1-4)-linked D- glucuronan polymer isolated from the mucoralean EPS preparations did not react with the antibodies raised in mice and rabbits (Chapter 6). The antigenic polysaccharides could also be separated from the non-antigenic polysaccharides with immobilized antibodies. As demonstrated for polyclonal rabbit IgG tile antigenic polysaccharides could be separated in one step with an immunoaffinity column with covalently linked IgG antibodies (Chapter 5).A valuable approach to reveal the structure of fungal carbohydrate epitopes, is the use of purified enzymes which are able to degrade the epitopes, which can then be screened with ELISA. In Chapter 7, such an enzyme was purified from a commercial enzyme preparation from Trichoderma harzianum. It appeared to be an exo-α-D-mannanase. EPSs were treated with this enzyme and the products were analysed by highperformance anion-exchange chromatography and by gas-liquid chromatography/mass spectrometry after derivatisation to alditol acetates. The exo-α-D-mannanase removed the ELISA activity of polysaccharides from Mucorales by hydrolysing the terminal α-D-mannose residues from α(1-2)-linked chains of D-mannose and terminal 2- O -methylmannose residues. This 2- O -methyl-mannose residue was identified in all mucoralean EPS preparations. It represents less than 0.5% (w/w) in all samples tested, and was initially overlooked with all other methods. In particular, determination of glycosidic linkages of carbohydrate residues is often performed by methylation analysis, a method which leaves 2- O -methyl-mannose residues undetected. To our knowledge, this compound has never been reported to occur in fungi. It was proved that the epitopes reactive with rabbit-IgG of Mucorales carry this 2- O -methyl-mannose residue at the non- reducing terminal.As shown in Chapter 8, the antigenic activity of the mucoralean moulds with polyclonal IgG antibodies is mainly based on the 2- O -methyl-mannose residues. Therefore, it can be assumed that the results of the methylation analyses on antigenic mucoralean oligosaccharides performed by Miyazaki and coworkers were erroneously interpreted as proof of α(1-6)-linked mannose residues. It is most likely, that the oligomers they isolated indeed carried 2- O -methyl- mannose residues at the non-reducing end which were not recognised. Finally, the structure of the epitopes reactive with rabbit-IgG raised against mucoralean EPSs was consolidated by hapten-inhibition experiments with synthetic oligosaccharides, which unequivocally proved that 2- O -methyl-mannose residues play a vital role in this immunochemical reaction (Chapter 8).This thesis is a contribution to the basic knowledge of the structure of extracellular polysaccharides from moulds. This knowledge can be used in the development of immunochemical methods for moulds, particularly in food products. A protocol was developed to elucidate the antigenic determinant of fungal polysaccharides. This approach appeared fruitful in discovering that the immunochemical reactivity of EPSs from Mucorales moulds reside mainly in non-reducing terminal 2- O -methyl-mannose residues.Furthermore, this thesis can be considered as a contribution to the development of the knowledge of immunochemistry of sugars in general. This approach may be used to investigate structural features of any polysaccharide or glycoconjugate which is or can be made immunologically active. The methodology is particularly suitable in detecting minor sugars with unusual structure which occur at non-reducing terminals of biopolymers and may have specific biological functions. Specific enzymatic removal of such sugars may uncover structures of which the biological function or specificity (e.g. immunochemical reactivity) may be studied subsequently

Evaluation of some important physicochemical properties of starch free grewia gum

Gums obtained by extraction from the inner bark of stems can be found in association with starch, which must be digested in order to obtain a refined polysaccharide isolate. In the present study, grewia gum obtained from the inner bark of the stems of Grewia mollis was shown to co-exist with starch and the effect of starch digestion on the physicochemical properties of the resultant polysaccharide was evaluated. The gum was extracted by maceration of the inner bark in deionized water and isolated by a combination of filtration, centrifugation and finally precipitation with absolute ethanol to produce the crude grewia gum extract (GG). The presence and content of starch in the gum sample was determined followed by enzymatic digestion of the starch using α-amylase (Termamyl 120L) to give a starch-free extract (GGDS). Physicochemical properties of the extracts such as total carbohydrates, total protein, differential sugar composition, NMR, intrinsic viscosity and rheological behaviour of the samples were evaluated. The GG extract had total carbohydrate content of ∼ 60 % out of which 11.8 % was starch, and a protein content of 2.3 %. Samples also contained galacturonic and glucuronic acid which were highly acetylated. Both samples had a higher proportion of galacturonic acid than glucuronic acid and contained rhamnose, arabinose, galactose, glucose and xylose as neutral sugars in varying proportions. Rheological measurements on 2 %w/w dispersions of the extracts show minor differences between both the original extract and the de-starched material but were influenced by changes in pH

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Normering van meetnauwkeurigheden van klimaatmetingen in praktijkkassen : [eindrapport]

In het project 'Normering van meetnauwkeurigheden van klimaatmetingen in praktijkkassen' zijn eisen opgesteld waaraan meetsystemen voor de kasklimaatregeling in praktijkkassen, zowel uit technisch als uit teelttechnisch oogpunt, zouden moeten voldoen. Op basis van de geformuleerde eisen, is vervolgens een protocol met normen opgesteld voor de nauwkeurigheid van het totale meetsysteem (sensor, bekabeling, computer) en de positie van de meetsensor. Er zijn normen opgesteld voor de volgende metingen in de kas: - luchttemperatuur; - luchtvochtigheid; - koolzuurgehalte (C02) en - buistemperatuur. Ook is een procedure opgesteld waarin beschreven staat, hoe controlemetingen dienen te worden uitgevoerd. Fabrikanten van klimaatcomputers, installateurs, gebruikers en controlerende instanties zijn hierdoor in staat te controleren of aan de in het protocol gestelde normen wordt voldaan. De normen en de controleprocedures, getest op een achttal praktijkbedrijven met in totaal vier verschillende klimaatcomputers, bleken goed realiseerbaar. Het protocol met normen is gepubliceerd als PBG-rapport 101, "Normering van meetnauwkeurigheden van klimaatmetingen in kassen", met als ondertitel: 'Protocol met normen voor meetsystemen en procedure voor controlemeting'