Periodic acid–Schiff’s reagent assay for carbohydrates in a microtiter plate format

Kilcoyne, Michelle; Gerlach, Jared Q.; Farrell, Mark; Bhavanandan, V. P.; Joshi, Lokesh

doi:10.1016/j.ab.2011.05.006

Cited by 65 publications

(52 citation statements)

References 23 publications

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“…The ePS evaluated on the three 48-h biofilms with the periodic acid-Schiff reaction (Kilcoyne et al 2011) showed concentrations much lower than the interference limit established in the first part of this study. Carbohydrate amounts of 12.6± 2.1, 13.5±1.5 and 10.4±0.7 μg eq dextran per well were measured for biofilms B. licheniformis, P. aeruginosa and W. confusa, respectively.…”

Section: In Situ Quantification Of Epns Within Bacterial Biofilmscontrasting

confidence: 52%

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms

Randrianjatovo

Girbal-Neuhauser

Marcato-Romain

2015

Appl Microbiol Biotechnol

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Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml(-1)) showed little or no sugar interference up to 2000 μg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

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Section: In Situ Quantification Of Epns Within Bacterial Biofilmscontrasting

confidence: 52%

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms

Randrianjatovo

Girbal-Neuhauser

Marcato-Romain

2015

Appl Microbiol Biotechnol

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“…The microtiter PAS assay was adapted from procedures described previously (41). Prior to the procedure, 1 ml of a 50% (wt/vol) periodic acid (Sigma-Aldrich) stock solution was made.…”

Section: Methodsmentioning

confidence: 99%

Quantifying Vibrio cholerae Enterotoxicity in a Zebrafish Infection Model

Mitchell

Breen

Britton

et al. 2017

Appl Environ Microbiol

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Vibrio cholerae is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment. Vibrio cholerae is an aquatic bacterium that inhabits coastal and estuarine areas, and it is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization, pathogenesis, and transmission. As early as 6 h after exposure to V. cholerae, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of V. cholerae in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish and, consequently, which toxins may play a role in environmental transmission.IMPORTANCE Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for V. cholerae. It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of V. cholerae back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving V. cholerae an advantage when environmental conditions are not optimal for survival in the water. Studies such as this one are critical because fish could be an overlooked source of cholera transmission in the environment.KEYWORDS cholera, enteric pathogens, Vibrio cholerae, zebrafish V ibrio cholerae is a Gram-negative aquatic bacterium that is responsible for causing cholera, an acute intestinal infection in humans characterized by profuse watery diarrhea. It is typically found in brackish estuaries and along coastal areas. In the environment, it has been found in fish and bird intestinal tracts (1-3) and is known to form biofilms on chitinous substrates (4).The primary virulence factors are cholera toxin (CTX) and toxin-coregulated pilus (TCP), both of which are required for colonization of the human small intestine (5). It is the action of cholera toxin that is responsible for the voluminous secretory diarrhea, the hallmark of cholera (6). However, V. cholerae also has accessory toxins that are able to cause diarrhea even in the absence of cholera toxin (5, 7, 8). There are at ...

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“…[ 48 ] The relative glycan content of native mucins, partially deglycosylated mucins, fully deglycosylated mucins, and bovine serum albumin solutions as a negative control were assessed by a periodic acid-Schiff colorimetric assay following a previously published protocol. [ 49 ] The decrease in sugar content of the coatings was revealed by an enzyme-linked lectin assay (ELLA) using fl uorescently labeled WGA. In short, mucin, deglycosylated mucin, or bovine serum albumin coatings were generated in the wells of 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Modulating Mucin Hydration and Lubrication by Deglycosylation and Polyethylene Glycol Binding

Crouzier

Boettcher

Geonnotti

et al. 2015

Adv Materials Inter

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glycoproteins, a family of high molecular weight and densely glycosylated biopolymers. Those mucins can act as an infection shield by trapping various viruses in the biopolymer matrix [ 1 ] and they reduce bacterial adhesion to surfaces. [ 2,3 ] Mucins also form highly hydrated and lubricative layers that protect the underlying epithelium from dehydration and shear stress that emerge during eye blinking or when swallowing food. Water typically accounts for up to 95% of the total mass in the mucus gel. [ 4,5 ] Shifts in the mucus water content correlate with substantial changes of the mucus barrier function [6][7][8] and result in important pathological disorders such as dry mouth [ 9 ] and dry eye. [ 10 ] Mucin-associated glycans contribute up to 80% of the molecular weight of mucins [ 11 ] and contain highly hydrated hydroxyl groups. Differences in the amount and composition of glycans from mucins extracted from pig stomachs and mucins from bovine submaxillary glands correlate with their capacity to adsorb water. [ 12 ] However, other properties such as protein composition also differ between these two mucin species, precluding the establishment of a defi nitive link between the presence of mucin glycans and hydration.Mucins, and other well-hydrated molecules such as zwitterionic polymers, polysaccharides, and polyelectrolytes are generally good boundary lubricants when adsorbed [ 13,14 ] or assembled in brush structures [15][16][17][18] on surfaces. Hence, mucin-associated glycans could also be essential for the lubricative function of mucus. In a related system, the mucin-like macromolecule lubricin showed a signifi cant decrease in lubricity when its associated glycans were removed. [ 19 ] Moreover, the lubricity of salivary fi lms, which contain mucins, has also been correlated with the total amount of glycosylation present in the fi lm. However, also here, the complexity of saliva composition precluded the identifi cation of the specifi c role of mucin glycosylation. [ 20 ] Hence, there is a need for a more defi ned experimental system to determine if mucin glycosylation is essential to the hydration and lubrication properties of mucins.This study explores the role of glycosylation in mucin hydration and lubricity, using mucin coatings as a simplifi ed model system. Such mucin coatings can reconstitute levels of surface hydration [ 21,22 ] and lubrication [23][24][25] similar to those found in native mucus. Our data show that the removal of mucin-bound glycans results in a signifi cant reduction in hydration and lubricity of the mucin-coated surface. We show that the hydration and lubricity of deglycosylated mucins can be partially A key property of mucin glycoproteins is their exceptional capacity to hydrate and lubricate surfaces. In vivo, mucins assemble into mucus hydrogels that cover the epithelium and protect it from dehydration and shear stress. A better understanding of the origin of these properties could lead to new treatment strategies for patients with poor mucus coverage, defective mucus produ...

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Periodic acid–Schiff’s reagent assay for carbohydrates in a microtiter plate format

Cited by 65 publications

References 23 publications

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms

Quantifying Vibrio cholerae Enterotoxicity in a Zebrafish Infection Model

Modulating Mucin Hydration and Lubrication by Deglycosylation and Polyethylene Glycol Binding

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